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1.  Formin-like2 regulates Rho/ROCK pathway to promote actin assembly and cell invasion of colorectal cancer 
Cancer Science  2015;106(10):1385-1393.
Formin-like2 (FMNL2) is a member of the diaphanous-related formins family, which act as effectors and upstream modulators of Rho GTPases signaling and control the actin-dependent processes, such as cell motility or invasion. FMNL2 has been identified as promoting the motility and metastasis in colorectal carcinoma (CRC). However, whether FMNL2 regulates Rho signaling to promote cancer cell invasion remains unclear. In this study, we demonstrated an essential role for FMNL2 in the activations of Rho/ROCK pathway, SRF transcription or actin assembly, and subsequent CRC cell invasion. FMNL2 could activate Rho/ROCK pathway, and required ROCK to promote CRC cell invasion. Moreover, FMNL2 promoted the formation of filopodia and stress fiber, and activated the SRF transcription in a Rho-dependent manner. We also demonstrated that FMNL2 was necessary for LPA-induced invasion, RhoA/ROCK activation, actin assembly and SRF activation. FMNL2 was an essential component of LPA signal transduction toward RhoA by directly interacting with LARG. LARG silence inhibited RhoA/ROCK pathway and CRC cell invasion. Collectively, these data indicate that FMNL2, acting as upstream of RhoA by interacting with LARG, can promote actin assembly and CRC cell invasion through a Rho/ROCK-dependent mechanism.
PMCID: PMC4638017  PMID: 26258642
Colorectal cancer; FMNL2; invasion; LARG; Rho/ROCK signaling
2.  The positive feedback between Snail and DAB2IP regulates EMT, invasion and metastasis in colorectal cancer 
Oncotarget  2015;6(29):27427-27439.
DAB2IP has been identified as a tumor suppressor in several cancers but its oncogenic role and transcriptionally regulatory mechanisms in the progression of colorectal carcinoma (CRC) remain unknown. In this study, DAB2IP was down-regulated in CRC tissues and a valuable prognostic marker for survival of CRC patients, especially in the late stage. Moreover, DAB2IP was sufficient to suppress proliferation, epithelial-mesenchymal transition (EMT), invasion and metastasis in CRC. Mechanically, the linear complex of EZH2/HDAC1/Snail contributed to DAB2IP silencing in CRC cells. The study further proved that the positive feedback loop between Snail and DAB2IP existed in CRC cells and DAB2IP was required for Snail-induced aggressive cell behaviors. Finally, DAB2IP correlated negatively with Snail and EZH2 expressions in CRC tissues. Our findings reveal the suppressive role and a novel regulatory mechanism of DAB2IP expression in the progression of CRC. DAB2IP may be a potential, novel therapeutic and prognostic target for clinical CRC patients.
PMCID: PMC4695000  PMID: 26336990
DAB2IP; Ezh2; Snail; colorectal carcinoma; metastasis
3.  Analysis of the serum reproductive system related autoantibodies of infertility patients in Tianjin region of China 
Object: Reproductive system related autoantibodies have been proposed to be associated with natural infertility. However, large scale systematic analysis of these of antibodies has not been conducted. The aim of this study is to analyze the positive rate of antisperm antibody (ASAb), anti-endometrium antibody (EMAb), anti-ovary antibody (AOAb), anti-zona pellucida antibody (AZP) and anticardiolipin antibody (ACA) in infertility patients in Tianjin region of China. Methods: 1305 male and 1711 female primary infertility patients and 1100 female secondary infertility patients were included in this study, as well as 627 healthy female controls. The above autoantibodies were tested and the positive rates in each group were calculated. Results: the positive rate of ASAb were significantly higher in primary infertility female than that in male, further analysis revealed that primary infertility population all exhibit significant higher positive rate of EMAb, AOAb, AZP and ACA compared with control group. Furthermore, the positive rates of all the antibodies in primary infertility female were significantly higher than those in secondary infertility female. Conclusions: Our study thus indicates that these autoantibodies might be associated with immunological related primary infertility and may have clinical significance in its diagnosis and treatment.
PMCID: PMC4613051  PMID: 26550366
Immune; autoantibody; infertility; EMAb; AOAb; AZP; ACA
4.  The SOX17/miR-371-5p/SOX2 axis inhibits EMT, stem cell properties and metastasis in colorectal cancer 
Oncotarget  2015;6(11):9099-9112.
Cancer stem cells (CSCs) and EMT-type cells, which share molecular characteristics with CSCs, have been believed to play critical roles in tumor metastasis. Although much progress has been garnered in elucidating the molecular pathways that trigger EMT, stemness and metastasis, a number of key mechanistic gaps remain elusive. In the study, miR-371-5p was obviously down-regulated in primary CRC tissues compared with matched adjacent normal mucosa and correlated significantly with differentiation, tumor size, lymphatic and liver metastases. MiR-371-5p could attenuate proliferation, invasion in vitro and metastasis in vivo in CRC cells. It also suppressed EMT by regulating Wnt/β-catenin signaling and strongly decreased the CRC stemness phenotypes. Moreover, demethylation of SOX17 induced miR-371-5p expression and consequently suppressed its direct target SOX2 in CRC cells. MiR-371-5p was necessary for SOX17 mediated cancer-related traits and SOX2 was a functional target of miR-371-5p. A positive relationship between SOX17 and miR-371-5p expression and a negative one between miR-371-5p and SOX2 expression were observed in CRC cell lines and tissues. In conclusion, we identified miR-371-5p as an important “oncosuppressor” in CRC progression and elucidated a novel mechanism of the SOX17/miR-371-5p/SOX2 axis in the regulation of EMT, stemness and metastasis, which may be a potential therapeutic target.
PMCID: PMC4496205  PMID: 25868860
miR-371-5p; SOX17; SOX2; metastasis; colorectal cancer
5.  Investigation on the Gas-Phase Decomposition of Trichlorfon by GC-MS and Theoretical Calculation 
PLoS ONE  2015;10(4):e0121389.
The gas phase pyrolysis of trichlorfon was investigated by the on-line gas chromatography – mass spectrometry (GC-MS) pyrolysis and theoretical calculations. Two reaction channels were proposed in the pyrolytic reaction, by analyzing the detected pyrolytic products in the total ion chromatography, including 2,2,2-trichloroacetaldehyde, dimethyl phosphite, and dichlorvos. Theoretical calculations showed that there is an intramolecular hydrogen bond between the hydroxyl group and the phosphate O atom in trichlorfon, through which the hydroxyl H atom can be easily transferred to phosphate O atom to trigger two pyrolytic channels. In path-a, migration of H atom results in direct decomposition of trichlorfon to give 2,2,2-trichloroacetaldehyde and dimethyl phosphite in one step. In path-b, migration of H atom in trichlorfon is combined with formation of the O-P bond to give an intermediate, followed by HCl elimination to afford dichlorvos. Path-a is kinetically more favorable than path-b, which is consistent with the GC-MS results.
PMCID: PMC4391870  PMID: 25856549
6.  Arabidopsis ABA Receptor RCAR1/PYL9 Interacts with an R2R3-Type MYB Transcription Factor, AtMYB44 
Abscisic acid (ABA) signaling plays important roles in plant growth, development and adaptation to various stresses. RCAR1/PYL9 has been known as a cytoplasm and nuclear ABA receptor in Arabidopsis. To obtain further insight into the regulatory mechanism of RCAR1/PYL9, a yeast two-hybrid approach was performed to screen for RCAR1/PYL9-interacting proteins and an R2R3-type MYB transcription factor, AtMYB44, was identified. The interaction between RCAR1/PYL9 and AtMYB44 was further confirmed by glutathione S-transferase (GST) pull-down and bimolecular fluorescence complementation (BiFC) assays. Gene expression analysis showed that AtMYB44 negatively regulated the expression of ABA-responsive gene RAB18, in contrast to the opposite role reported for RCAR1/PYL9. Competitive GST pull-down assay and analysis of phosphatase activity demonstrated that AtMYB44 and ABI1 competed for binding to RCAR1/PYL9 and thereby reduced the inhibitory effect of RCAR1/PYL9 on ABI1 phosphatase activity in the presence of ABA in vitro. Furthermore, transient activation assay in protoplasts revealed AtMYB44 probably also decreased RCAR1/PYL9-mediated inhibition of ABI1 activity in vivo. Taken together, our work provides a reasonable molecular mechanism of AtMYB44 in ABA signaling.
PMCID: PMC4057743  PMID: 24828206
ABA; signaling; RCAR1/PYL9; receptor; AtMYB44; ABI1
7.  The Suppressive Role and Aberrent Promoter Methylation of BTG3 in the Progression of Hepatocellular Carcinoma 
PLoS ONE  2013;8(10):e77473.
BTG3 (B-cell translocation gene 3) has been identified as a tumor suppressor and hypermethylation contributes to its down-regulation in some tumors, but its role in hepatocellular carcinoma (HCC) remain unknown. This study aimed to detect the expression and methylation status of BTG3 in HCC cell lines or tissues, and determine its function in HCC progression.
The expression of BTG3 was detected in HCC cell lines and HCC tissue by real-time RT-PCR, Western blot or immunohistochemistry. The promoter methylation status of BTG3 was measured by using methylation-specific PCR in HCC cell lines. A series of assays were performed to evaluate the effect of BTG3 on proliferation, invasion and cell cycle transition in vitro.
BTG3 expression was lower in HCC cell lines than in hepatocyte cell line LO2 (P<0.05). BTG3 was also down-regulated in HCC tissues. Its expression was positively correlated with differentiation and distant metastasis (P<0.05). Patients with lower BTG3 expression had shorter overall survival time (P=0.029). DNA methylation directed repression of BTG3 mRNA expression in HCC cell lines. BTG3 suppressed proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.
Down-regulation of BTG3 due to the promoter hypermethylation is closely associated with proliferation, invasion and cell cycle arrest of HCC cells. It may be a novel prognostic biomarker for HCC patients.
PMCID: PMC3798399  PMID: 24147003
8.  IL-6 pathway-driven investigation of response to IL-6 receptor inhibition in rheumatoid arthritis 
BMJ Open  2013;3(8):e003199.
To determine whether heterogeneity in interleukin-6 (IL-6), IL-6 receptor and other components of the IL-6 signalling pathway/network, at the gene, transcript and protein levels, correlate with disease activity in patients with rheumatoid arthritis (RA) and with clinical response to tocilizumab.
Biomarker samples and clinical data for five phase 3 trials of tocilizumab were analysed using serum (3751 samples), genotype (927 samples) and transcript (217 samples) analyses. Linear regression was then used to assess the association between these markers and either baseline disease activity or treatment response.
Higher baseline serum IL-6 levels were significantly associated (p<0.0001) with higher baseline DAS28, erythrocyte sedimentation rate, C reactive protein and Health Assessment Questionnaire in patients whose responses to disease-modifying antirheumatic drugs (DMARD-IR) and to antitumour necrosis factor (aTNF-IR) were inadequate and patients who were naive/responders to methotrexate (MTX). Higher baseline serum IL-6 levels were also significantly associated with better clinical response to tocilizumab (versus placebo) measured by cDAS28 in the pooled DMARD-IR (p<0.0001) and MTX-naive populations (p=0.04). However, the association with treatment response was weak. A threefold difference in baseline IL-6 level corresponded to only a 0.17-unit difference in DAS28 at week 16. IL-6 pathway single nucleotide polymorphisms and RNA levels also were not strongly associated with treatment response.
Our analyses illustrate that the biological activity of a disease-associated molecular pathway may impact the benefit of a therapy targeting that pathway. However, the variation in pathway activity, as measured in blood, may not be a strong predictor. These data suggest that the major contribution to variability in clinical responsiveness to therapeutics in RA remains unknown.
PMCID: PMC3753518  PMID: 23959753
Rheumatology; Immunology
9.  FBX8 Acts as an Invasion and Metastasis Suppressor and Correlates with Poor Survival in Hepatocellular Carcinoma 
PLoS ONE  2013;8(6):e65495.
F-box only protein 8 (FBX8), a novel component of F-box proteins, is lost in several cancers and has been associated with invasiveness of cancer cells. However, its expression pattern and role in the progression of hepatocellular carcinoma remain unclear. This study investigated the prognostic significance of FBX8 in hepatocellular carcinoma samples and analyzed FBX8 function in hepatocellular carcinoma cells by gene manipulation.
The expression of FBX8 was detected in 120 cases of clinical paraffin-embedded hepatocellular carcinoma tissues, 20 matched pairs of fresh tissues and five hepatocellular carcinoma cell lines by immunohistochemistry with clinicopathological analyses, real-time RT-PCR or Western blot. The correlation of FBX8 expression with cell proliferation and invasion in five HCC cell lines was analyzed. Moreover, loss of function and gain of function assays were performed to evaluate the effect of FBX8 on cell proliferation, motility, invasion in vitro and metastasis in vivo.
We found that FBX8 was obviously down-regulated in HCC tissues and cell lines (P<0.05). The FBX8 down-regulation correlated significantly with poor prognosis, and FBX8 status was identified as an independent significant prognostic factor. Over-expression of FBX8 decreased proliferation, migration and invasion in HepG2 and 97H cells, while knock-down of FBX8 in 7721 cells showed the opposite effect. FBX8 negatively correlated with cell proliferation and invasion in 7701, M3, HepG2 and 97H cell lines. In vivo functional assays showed FBX8 suppressed tumor growth and pulmonary metastatic potential in mice. Our results indicate that down-regulation of FBX8 significantly correlates with invasion, metastasis and poor survival in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy and control in hepatocellular carcinoma treatment.
PMCID: PMC3694991  PMID: 23826080
10.  Cd14 SNPs Regulate the Innate Immune Response 
Molecular Immunology  2012;51(2):112-127.
CD14 is a monocytic differentiation antigen that regulates innate immune responses to pathogens. Here, we show that murine Cd14 SNPs regulate the length of Cd14 mRNA and CD14 protein translation efficiency, and consequently the basal level of soluble CD14 (sCD14) and type I IFN production by murine macrophages. This has substantial downstream consequences for the innate immune response; the level of expression of at least 40 IFN-responsive murine genes was altered by this mechanism. We also observed that there was substantial variation in the length of human CD14 mRNAs and in their translation efficiency. sCD14 increased cytokine production by human dendritic cells (DCs), and sCD14-primed DCs augmented human CD4 T cell proliferation. These findings may provide a mechanism for exploring the complex relationship between CD14 SNPs, serum sCD14 levels, and susceptibility to human infectious and allergic diseases.
PMCID: PMC3341513  PMID: 22445606
11.  Fragmentation of Deprotonated Diacylhydrazine Derivatives in Electrospray Ionization Tandem Mass Spectrometry: Generation of Acid Anions via Intramolecular Rearrangement 
PLoS ONE  2013;8(5):e63097.
The gas-phase fragmentation pathways of deprotonated diacylhydrazine derivatives (R1(C = O)-N(t-Bu)NH(C = O)R2, Compounds 1–6) were investigated by the combination of electrospray ionization tandem mass spectrometry (ESI-MS/MS) and theoretical calculations. Upon collisional activation, the deprotonated molecular ions [M – H]− dissociate in two reaction channels, both of which involve intramolecular rearrangement. The main product ion is confirmed to be an anionic acid species, [R1-CO2]−, generated through intramolecular rearrangement of [M – H]− initiated by the nucleophilic attack of the amide O6 on the carbonyl C2 (Path-1). The minor fragment channel (Path-2) involves methylpropene elimination of the precursor ion, followed by a similar nucleophilic displacement reaction to produce another acid anion [R2-CO2]−. Density functional theory calculations at the B3LYP/6-31+G(d,p) level indicate that Path-1 is more favorable than Path-2 for dissociation of the deprotonated halofenozide.
PMCID: PMC3660572  PMID: 23704891
12.  The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus) 
Ming, Ray | Hou, Shaobin | Feng, Yun | Yu, Qingyi | Dionne-Laporte, Alexandre | Saw, Jimmy H. | Senin, Pavel | Wang, Wei | Ly, Benjamin V. | Lewis, Kanako L. T. | Salzberg, Steven L. | Feng, Lu | Jones, Meghan R. | Skelton, Rachel L. | Murray, Jan E. | Chen, Cuixia | Qian, Wubin | Shen, Junguo | Du, Peng | Eustice, Moriah | Tong, Eric | Tang, Haibao | Lyons, Eric | Paull, Robert E. | Michael, Todd P. | Wall, Kerr | Rice, Danny W. | Albert, Henrik | Wang, Ming-Li | Zhu, Yun J. | Schatz, Michael | Nagarajan, Niranjan | Acob, Ricelle A. | Guan, Peizhu | Blas, Andrea | Wai, Ching Man | Ackerman, Christine M. | Ren, Yan | Liu, Chao | Wang, Jianmei | Wang, Jianping | Na, Jong-Kuk | Shakirov, Eugene V. | Haas, Brian | Thimmapuram, Jyothi | Nelson, David | Wang, Xiyin | Bowers, John E. | Gschwend, Andrea R. | Delcher, Arthur L. | Singh, Ratnesh | Suzuki, Jon Y. | Tripathi, Savarni | Neupane, Kabi | Wei, Hairong | Irikura, Beth | Paidi, Maya | Jiang, Ning | Zhang, Wenli | Presting, Gernot | Windsor, Aaron | Navajas-Pérez, Rafael | Torres, Manuel J. | Feltus, F. Alex | Porter, Brad | Li, Yingjun | Burroughs, A. Max | Luo, Ming-Cheng | Liu, Lei | Christopher, David A. | Mount, Stephen M. | Moore, Paul H. | Sugimura, Tak | Jiang, Jiming | Schuler, Mary A. | Friedman, Vikki | Mitchell-Olds, Thomas | Shippen, Dorothy E. | dePamphilis, Claude W. | Palmer, Jeffrey D. | Freeling, Michael | Paterson, Andrew H. | Gonsalves, Dennis | Wang, Lei | Alam, Maqsudul
Nature  2008;452(7190):991-996.
Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3× draft genome sequence of ‘SunUp’ papaya, the first commercial virus-resistant transgenic fruit tree1 to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far2–5, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica's distinguishing morpho-physiological, medicinal and nutritional properties.
PMCID: PMC2836516  PMID: 18432245
13.  Synthesis, Tubulin Assembly, and Antiproliferative Activity Against MCF7 and NCI/ADR-RES Cancer Cells of 10-O-Acetyl-5′-hydroxybutitaxel 
A highly efficient kinetic resolution of racemic cis-4-(2-tert-butyldimethylsilyloxy-1,1-dimethyl)ethyl-3-tert-butyldimethylsilyloxy-azetidin-2-one with 7-O-triethylsilylbaccatin III was carried out to furnish 10-O-acetyl-5′-hydroxybutitaxel after removal of the silyl protecting groups. The compound was 50% as active as paclitaxel in a tubulin assembly assay and showed significantly decreased activity against MCF7 cell proliferation compared to paclitaxel.
PMCID: PMC2636847  PMID: 18977659
14.  Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2) Is Involved in ABA-Mediated Early Seedling Development 
The voltage-dependent anion channel (VDAC) is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana.
PMCID: PMC2705501  PMID: 19582214
Arabidopsis thaliana; voltage-dependent anion channel; abscisic acid; ABA signaling
15.  A Recalibrated Molecular Clock and Independent Origins for the Cholera Pandemic Clones 
PLoS ONE  2008;3(12):e4053.
Cholera, caused by Vibrio cholerae, erupted globally from South Asia in 7 pandemics, but there were also local outbreaks between the 6th (1899–1923) and 7th (1961–present) pandemics. All the above are serotype O1, whereas environmental or invertebrate isolates are antigenically diverse. The pre 7th pandemic isolates mentioned above, and other minor pathogenic clones, are related to the 7th pandemic clone, while the 6th pandemic clone is in the same lineage but more distantly related, and non-pathogenic isolates show no clonal structure. To understand the origins and relationships of the pandemic clones, we sequenced the genomes of a 1937 prepandemic strain and a 6th pandemic isolate, and compared them with the published 7th pandemic genome. We distinguished mutational and recombinational events, and allocated these and other events, to specific branches in the evolutionary tree. There were more mutational than recombinational events, but more genes, and 44 times more base pairs, changed by recombination. We used the mutational single-nucleotide polymorphisms and known isolation dates of the prepandemic and 7th pandemic isolates to estimate the mutation rate, and found it to be 100 fold higher than usually assumed. We then used this to estimate the divergence date of the 6th and 7th pandemic clones to be about 1880. While there is a large margin of error, this is far more realistic than the 10,000–50,000 years ago estimated using the usual assumptions. We conclude that the 2 pandemic clones gained pandemic potential independently, and overall there were 29 insertions or deletions of one or more genes. There were also substantial changes in the major integron, attributed to gain of individual cassettes including copying from within, or loss of blocks of cassettes. The approaches used open up new avenues for analysing the origin and history of other important pathogens.
PMCID: PMC2605724  PMID: 19115014
16.  Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia 
Biology Direct  2008;3:26.
The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia.
We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins.
The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria.
This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.
PMCID: PMC2474590  PMID: 18593465
17.  A Genetic Analysis of Opioid-Induced Hyperalgesia in Mice 
Anesthesiology  2006;104(5):1054-1062.
Genetics impact the propensity of different strains of mice to display hyperalgesia after opioid administration. In these studies we demonstrate that variants of the β2 adrenergic receptor gene are linked to these differences in murine opioid-induced hyperalgesia.
Opioid-induced hyperalgesia (OIH) is a syndrome of increased sensitivity to noxious stimuli seen after both the acute and chronic administration of opioids which has been observed in humans and rodent models. This syndrome may reduce the clinical utility of opioids in treating acute and chronic pain.
In these studies we measured the propensity of 15 strains of inbred mice to develop mechanical manifestations of OIH. These data were subjected to in silico genetic analysis which resulted in the association of haplotypic blocks within or near several known genes. Both pharmacological agents and transgenic mice were used to confirm the functional association of the most strongly linked gene with OIH.
Both baseline mechanical nociceptive thresholds as well as the percentage changes in these thresholds after 4 days of morphine treatment were found to be highly strain dependent. The haplotypic block most strongly associated with the mechanical OIH data was located within the β2 adrenergic receptor gene (β2-AR). Using the selective β2-AR antagonist butoxamine, we observed a dose-dependent reversal of OIH. Furthermore, deletion of the β2-AR gene sharply reduced the mechanical allodynia present after morphine treatment in the wild type mouse strain. Analysis of the associated β2-AR haplotypic block identified single nucleotide polymorphisms potentially explaining in part the strain specific differences in OIH.
Genetic variants of the β2-AR gene appear to explain some part of the differences between various strains of mice to develop OIH. The association of this gene with OIH suggests specific pharmacological strategies for reducing the impact of OIH on patients consuming opioids.
PMCID: PMC1464476  PMID: 16645459
Genetics; Hyperalgesia; Pain; Morphine; Beta Adrenergic
18.  In Silico Pharmacogenetics: Warfarin Metabolism 
Nature biotechnology  2006;24(5):531-536.
A recently developed murine haplotype-based computational method was used to identify genetic factors regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. The amount of warfarin and 9 identified metabolites in plasma was quantitated after dosing 13 inbred mouse strains. Strain-specific differences in drug metabolism through generation of 7-hydroxywarfarin metabolites were computationally correlated with genetic variation within a chromosomal region encoding cytochrome P450 2C enzymes. This computational prediction was experimentally confirmed by showing that the rate limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by a Cyp2c isoform specific substrate (tolbutamide) and was mediated by expressed recombinant Cyp2c29. Genetic variants responsible for inter-individual pharmacokinetic differences for clinically important drugs can be identified by computational genetic analysis in mice.
PMCID: PMC1459533  PMID: 16680137
HPLC: high pressure liquid chromatography; LC: liquid chromatography; MS: mass spectrometry; Cyp2c29: cytochrome P450 2c29; PK: pharmacokinetics; IS: internal standard; NMR: nuclear magnetic resonance; IP: intraperitoneal; QC: quality control; MRM: multiple reaction monitoring; AUC 0-8: area under concentration-time curve within the first 8 h; SNPs: single nucleotide polymorphisms
19.  Understanding Our Drugs and Our Diseases 
Analysis of mouse genetic models of human disease–associated traits has provided important insight into the pathogenesis of human disease. As one example, analysis of a murine genetic model of osteoporosis demonstrated that genetic variation within the 15-lipoxygenase (Alox15) gene affected peak bone mass, and that treatment with inhibitors of this enzyme improved bone mass and quality in rodent models. However, the method that has been used to analyze mouse genetic models is very time consuming, inefficient, and costly. To overcome these limitations, a computational method for analysis of mouse genetic models was developed that markedly accelerates the pace of genetic discovery. It was used to identify a genetic factor affecting the rate of metabolism of warfarin, an anticoagulant that is commonly used to treat clotting disorders. Computational analysis of a murine genetic model of narcotic drug withdrawal suggested a potential new approach for treatment of narcotic drug addiction. Thus, the results derived from computational mouse genetic analysis can suggest new treatment strategies, and can provide new information about currently available medicines.
PMCID: PMC2658704  PMID: 16799083
computational biology; genetics; pharmacogenetics

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