Publications by authors named "Wenlin Yuan"

21 Publications

  • Page 1 of 1

Chemical profiling of Honghua Xiaoyao tablet and simultaneous determination of its quality markers by liquid chromatography-tandem mass spectrometry combined with chemometrics methods.

J Sep Sci 2020 Dec 20;43(23):4263-4280. Epub 2020 Oct 20.

School of Pharmacy, The Second Military Medical University, Shanghai, P. R. China.

Discovering marker components of traditional Chinese medicine formulas is challenging because of the hundreds of components they inherently contain. This study first proposed a reliable and validated method for the comprehensive profiling of chemical constituents in Honghua Xiaoyao tablet by using high-performance liquid chromatography coupled with mass spectrometry. After searching within the in-house library, a total of 55 constituents were unambiguously characterized or tentatively identified through reference standards and by comparing mass spectrometry data with literature values. Quantitative analysis of 14 compounds, which were selected as the quality marker components based on a serum pharmacochemistry study, has been performed by triple-quardrupole mass spectrometry technique. Multiple chemometric methods, including principal components analysis and hierarchical cluster analysis, were subsequently used to analyze the quantitative results, classify samples from three manufacturers, and distinguish the analytical markers. In method validation results, 14 quality marker compounds have shown good linearity (R ≥ 0.9965) with a relative wide concentration range and acceptable recovery at 98.39-102.46%. The proposed approach provides the chemical evidence for revealing the material basis of Honghua Xiaoyao tablet, and establishes a reliable statistical analysis-based strategy of quality marker investigation for controlling its quality.
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http://dx.doi.org/10.1002/jssc.202000689DOI Listing
December 2020

Dexmedetomidine alleviated sepsis‑induced myocardial ferroptosis and septic heart injury.

Mol Med Rep 2020 Jul 4;22(1):175-184. Epub 2020 May 4.

Department of Anesthesiology, Shenzhen People's Hospital and Shenzhen Anesthesiology Engineering Center, The Second Clinical Medical College of Jinan University, Shenzhen, Guangdong 518000, P.R. China.

Cardiac dysfunction resulting from sepsis may cause significant morbidity and mortality, and ferroptosis plays a role in this pathology. Dexmedetomidine (Dex), a α2‑adrenergic receptor (α2‑AR) agonist exerts cardioprotective effects against septic heart dysfunction, but the exact mechanism is unknown. In the present study, sepsis was induced by cecal ligation and puncture (CLP) in male C57BL/6 mice. Dex and yohimbine hydrochloride (YOH), an α2‑AR inhibitor, were administered before inducing CLP. Then, 24 h after CLP, serum and heart tissue were collected to detect changes of troponin‑I (TN‑I), interleukin 6 (IL‑6), superoxide dismutase (SOD), malonaldehyde (MDA) and glutathione (GSH) levels, and iron release. Ferroptosis‑targeting proteins, apoptosis and inflammatory factors were assessed by western blotting or ELISA. It was found that, 24 h after CLP, TN‑I, a biomarker of myocardial injury, was significantly increased compared with the control group. Furthermore, the levels of MDA, 8‑hydroxy‑2'‑deoxyguanosine and the inflammatory factors IL‑6 and monocyte chemoattractant protein‑1 were also significantly increased. It was demonstrated that treatment with Dex reverted or attenuated these changes (CLP + Dex vs. CLP; P<0.05), but these protective effects of Dex were reversed by YOH. Moreover, CLP significantly decreased the protein expression levels of glutathione peroxidase 4 (GPX4), SOD and GSH. However, CLP increased expression levels of heme oxygenase‑1 (HO‑1), transferrin receptor, cleaved caspase 3, inducible nitric oxide synthase and gasdermin D, and iron concentrations. It was found that Dex reversed these changes, but YOH abrogated the protective effects of Dex (CLP + Dex + YOH vs. CLP + Dex; P<0.05). Therefore, the present results suggested that the attenuation of sepsis‑induced HO‑1 overexpression and iron concentration, and the reduction of ferroptosis via enhancing GPX4, may be the major mechanisms via which Dex alleviates sepsis‑induced myocardial cellular injury.
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http://dx.doi.org/10.3892/mmr.2020.11114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248514PMC
July 2020

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2-related programmed cell death in an AMPK-dependent manner.

J Cell Mol Med 2020 06 29;24(12):6670-6679. Epub 2020 Apr 29.

Department of Anesthesiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N-acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2-siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2-associated oxidative stress in an AMPK-dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.
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http://dx.doi.org/10.1111/jcmm.15318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299688PMC
June 2020

Dexmedetomidine alleviates HO-induced oxidative stress and cell necroptosis through activating of α2-adrenoceptor in H9C2 cells.

Mol Biol Rep 2020 May 27;47(5):3629-3639. Epub 2020 Apr 27.

Department of Anesthesiology, First Affiliated Hospital of Kunming Medical University, Kunming, 650032, Yunnan, China.

Oxidative stress induced necroptosis is important in myocardial ischemia/reperfusion injury. Dexmedetomidine (Dex), an α2-adrenoceptor (α2-AR) agonist, has protective effect on oxidative stress induced cell apoptosis, but effects of Dex and Dex-mediated α2-AR activation on oxidant induced necroptosis was unclear. H9C2 cardiomyocytes were pre-treated with or without Dex and α2-AR antagonist yohimbine hydrochloride (YOH) before being exposed to HO to induce oxidative cellular damage. Cell viability and lactate dehydrogenase (LDH) were detected by ELISA kits, protein expressions of Heme Oxygenase 1(HO-1), receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) were observed by WB, and TUNEL was used to detected cell apoptosis. HO significantly decreased cell viability and increased LDH release and necroptotic and apoptotic cell deaths (all p < 0.05, HO vs. Control). Dex preconditioning alleviated these injuries induced by HO. Dex preconditioning significantly increased expression of protein HO-1 and decreased expressions of proteins RIPK1 and RIPK3 induced by HO, while all these protective effects of Dex were reversed by YOH (all p < 0.05, Dex + HO vs. HO; and YOH + Dex + HO vs. Dex + HO). However, YOH did not prevent this protective effect of Dex against HO induced apoptosis (YOH + Dex + HO vs. Dex + HO, p > 0.05). These findings indicated that Dex attenuates HO induced cardiomyocyte necroptotic and apoptotic cell death respectively dependently and independently of α2-AR activation.
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http://dx.doi.org/10.1007/s11033-020-05456-wDOI Listing
May 2020

S100a4 upregulation in Pik3caH1047R;Trp53R270H;MMTV-Cre-driven mammary tumors promotes metastasis.

Breast Cancer Res 2019 12 27;21(1):152. Epub 2019 Dec 27.

Department of Molecular Biology, Genentech Inc, 1 DNA Way, South San Francisco, CA, 94080, USA.

Background: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations.

Methods: A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis.

Results: We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis.

Conclusions: Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.
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http://dx.doi.org/10.1186/s13058-019-1238-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935129PMC
December 2019

Low-Dose Arsenic Trioxide Modulates the Differentiation of Mouse Embryonic Stem Cells.

Chem Res Toxicol 2018 06 24;31(6):472-481. Epub 2018 May 24.

Department of Biotechnology, School of Life Science and Technology , Jinan University , Guangzhou 510632 , Guangdong Province , China.

Arsenic (As) is a well-known environmental pollutant, while arsenic trioxide (ATO) has been proven to be an effective treatment for acute promyelocytic leukemia, however, the mechanism underlying its dual effects is not fully understood. Embryonic stem cells (ESCs) exhibit properties of stemness and serve as a popular model to investigate epigenetic modifiers including environmental pollutants. Herein, the effects of low-dose ATO on differentiation were evaluated in vitro using a mouse ESCs (mESCs) cell line, CGR8. Cells treated with 0.2-0.5 μM ATO for 3-4 days had slight inhibition of proliferation with elevation of apoptosis, but obvious alterations of differentiation by morphological checking and alkaline phosphatase (AP) staining. Moreover, ATO exposure significantly decreased the mRNA expression of the stemness maintenance genes including Oct4, Nanog, and Rex-1 ( P < 0.01), whereas obviously increased some tissue-specific differentiation marker genes such as Gata4, Gata-6, AFP, and IHH. These alterations were consistent with the differentiation phenotype induced by retinoic acid (RA) and the expression patterns of distinct pluripotency markers such as SSEA-1 and Oct4. Furthermore, low-dose ATO led to a quantitative increase in Caspase 3 (CASP3) activation and subsequent cleavage of Nanog around 27 kDa, which corresponded with the mouse Nanog cleaved by CASP3 in a tube cleavage assay. Taken together, we suggest that low-dose ATO exposure will induce differentiation, other than apoptosis, of ESCs, such effects might be tuned partially by ATO-induced CASP3 activation and Nanog cleavage coupling with other differentiation related genes involved. The present findings provide a preliminary action mechanism of arsenic on the cell fate determination.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00027DOI Listing
June 2018

Massively parallel nanowell-based single-cell gene expression profiling.

BMC Genomics 2017 07 7;18(1):519. Epub 2017 Jul 7.

Molecular Biology Department, Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.

Background: Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable.

Results: Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells.

Conclusions: Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.
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http://dx.doi.org/10.1186/s12864-017-3893-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501953PMC
July 2017

Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer.

Nat Commun 2014 May 8;5:3830. Epub 2014 May 8.

Department of Bioinformatics and Computational Biology, Genentech Inc., South San Francisco, California 94080, USA.

Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.
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http://dx.doi.org/10.1038/ncomms4830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024760PMC
May 2014

Oncogenic ERBB3 mutations in human cancers.

Cancer Cell 2013 May;23(5):603-17

Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.
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http://dx.doi.org/10.1016/j.ccr.2013.04.012DOI Listing
May 2013

Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

Proc Natl Acad Sci U S A 2012 Nov 7;109(47):19368-73. Epub 2012 Nov 7.

Department of Molecular Biology, Genentech Inc., South San Francisco, CA 94080, USA.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.
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http://dx.doi.org/10.1073/pnas.1204384109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511101PMC
November 2012

Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer.

Nat Genet 2012 Oct 2;44(10):1111-6. Epub 2012 Sep 2.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA.

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein-coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.
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http://dx.doi.org/10.1038/ng.2405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557461PMC
October 2012

PML-RARA can increase hematopoietic self-renewal without causing a myeloproliferative disease in mice.

J Clin Invest 2011 Apr;121(4):1636-45

Section of Stem Cell Biology, Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63119, USA.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation that generates the fusion protein promyelocytic leukemia-retinoic acid receptor α (PML-RARA) in nearly all cases. Multiple prior mouse models of APL constitutively express PML-RARA from a variety of non-Pml loci. Typically, all animals develop a myeloproliferative disease, followed by leukemia in a subset of animals after a long latent period. In contrast, human APL is not associated with an antecedent stage of myeloproliferation. To address this discrepancy, we have generated a system whereby PML-RARA expression is somatically acquired from the mouse Pml locus in the context of Pml haploinsufficiency. We found that physiologic PML-RARA expression was sufficient to direct a hematopoietic progenitor self-renewal program in vitro and in vivo. However, this expansion was not associated with evidence of myeloproliferation, more accurately reflecting the clinical presentation of human APL. Thus, at physiologic doses, PML-RARA primarily acts to increase hematopoietic progenitor self-renewal, expanding a population of cells that are susceptible to acquiring secondary mutations that cause progression to leukemia. This mouse model provides a platform for more accurately dissecting the early events in APL pathogenesis.
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http://dx.doi.org/10.1172/JCI42953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068978PMC
April 2011

Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples.

BMC Med Genomics 2011 Jan 24;4:11. Epub 2011 Jan 24.

Department of Bioinformatics and Molecular Biology, Genentech, Inc, South San Francisco, California 94080, USA.

Background: Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome.

Methods: We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays.

Results: Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%.We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer cell lines.

Conclusions: Deep transcriptional sequencing and analysis with targeted and spliced alignment methods can effectively identify TIC events across the genome in individual tissues. Prostate and reference samples exhibit a wide range of TIC events, involving more genes than estimated previously using ESTs. Tissue specificity of TIC events is correlated with expression patterns of the upstream gene. Some TIC events, such as MSMB-NCOA4, may play functional roles in cancer.
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http://dx.doi.org/10.1186/1755-8794-4-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041646PMC
January 2011

Diverse somatic mutation patterns and pathway alterations in human cancers.

Nature 2010 Aug 28;466(7308):869-73. Epub 2010 Jul 28.

Department of Molecular Biology, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA.

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.
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http://dx.doi.org/10.1038/nature09208DOI Listing
August 2010

Novel ACTG1 mutation causing autosomal dominant non-syndromic hearing impairment in a Chinese family.

J Genet Genomics 2008 Sep;35(9):553-8

Key Laboratory of Molecular Biophysics of Ministry of Education, Center for Human Genome Research, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.

The gamma-actin (ACTG1) gene is a cytoplasmic nonmuscle actin gene, which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea. Mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families, respectively. In this study, a novel missense mutation (c.364A>G; p.I122V) co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls. The alteration of residue Ile122 was predicted to damage its interaction with actin-binding proteins, which may cause disruption of hair cell organization and function. These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.
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http://dx.doi.org/10.1016/S1673-8527(08)60075-2DOI Listing
September 2008

Computational identification of the normal and perturbed genetic networks involved in myeloid differentiation and acute promyelocytic leukemia.

Genome Biol 2008 21;9(2):R38. Epub 2008 Feb 21.

Department of Biomedical Engineering, Washington University, St Louis, MO 63130, USA.

Background: Acute myeloid leukemia (AML) comprises a group of diseases characterized by the abnormal development of malignant myeloid cells. Recent studies have demonstrated an important role for aberrant transcriptional regulation in AML pathophysiology. Although several transcription factors (TFs) involved in myeloid development and leukemia have been studied extensively and independently, how these TFs coordinate with others and how their dysregulation perturbs the genetic circuitry underlying myeloid differentiation is not yet known. We propose an integrated approach for mammalian genetic network construction by combining the analysis of gene expression profiling data and the identification of TF binding sites.

Results: We utilized our approach to construct the genetic circuitries operating in normal myeloid differentiation versus acute promyelocytic leukemia (APL), a subtype of AML. In the normal and disease networks, we found that multiple transcriptional regulatory cascades converge on the TFs Rora and Rxra, respectively. Furthermore, the TFs dysregulated in APL participate in a common regulatory pathway and may perturb the normal network through Fos. Finally, a model of APL pathogenesis is proposed in which the chimeric TF PML-RARalpha activates the dysregulation in APL through six mediator TFs.

Conclusion: This report demonstrates the utility of our approach to construct mammalian genetic networks, and to obtain new insights regarding regulatory circuitries operating in complex diseases in humans.
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http://dx.doi.org/10.1186/gb-2008-9-2-r38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374711PMC
May 2008

Melanin biosynthesis by Frankia strain CeI5.

Physiol Plant 2007 Oct;131(2):180-90

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, MO 63110, USA.

Many Frankia strains are pigmented and presumed to produce melanin. However, melanin biosynthesis has yet to be rigorously characterized in Frankia. This study was initiated to determine whether or not Frankia strain CeI5 produced melanin and to identify the biochemical pathway of pigment production. Frankia strain CeI5 first produced a dark pigment in mycelial and other tissue and then in the liquid culture medium when grown in a defined medium containing l-tyrosine. The pigment resisted solvents, lightened when subjected to the action of oxidants, as well as reductants, and produced a flocculent brown precipitate with FeCl(3). Spectroscopic characteristics of the extracted pigment were those of melanin. When subjected to gradual dilution, the absorbance decreased unevenly, occurring in the near red range first, then in the visible range, and lastly in the UV range. This observation might resolve the question of why quite different descriptions of melanin UV-visible light absorption spectra exist in the literature. The tyrosinase cofactor copper greatly enhanced melanin biosynthesis at 5.3 x 10(-6) M, while 1 x 10(-8) M 3,4-dihydroxy-l-phenylalanine hastened pigmentation. The copper-chelating agent KCN and the tyrosinase inhibitor tropolone decreased melanin production at the same concentration of 1 x 10(-5) M. This evidence suggests that Frankia strain CeI5 produces melanin via the Raper and Mason pathway.
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http://dx.doi.org/10.1111/j.1399-3054.2007.00948.xDOI Listing
October 2007

Commonly dysregulated genes in murine APL cells.

Blood 2007 Feb 28;109(3):961-70. Epub 2006 Sep 28.

Department of Medicine, Siteman Cancer Center, and Department of Pathology and Immunology, Washington University, St Louis, MO 63110, USA.

To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (APL), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. Many genes were reproducibly expressed in restricted developmental "windows," suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. We compared the normal myeloid developmental transcriptome with that of APL cells derived from mice expressing PML-RARalpha under control of the murine cathepsin G locus. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples, including Fos, Jun, Egr1, Tnf, and Vcam1. However, this set of commonly dysregulated genes was expressed normally in preleukemic, early myeloid cells from the same mouse model, suggesting that dysregulation occurs as a "downstream" event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis.
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http://dx.doi.org/10.1182/blood-2006-07-036640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1785140PMC
February 2007

Conserved roles for Slit and Robo proteins in midline commissural axon guidance.

Neuron 2004 Apr;42(2):213-23

Howard Hughes Medical Institute, Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.
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http://dx.doi.org/10.1016/s0896-6273(04)00179-5DOI Listing
April 2004

Lmx1b is essential for the development of serotonergic neurons.

Nat Neurosci 2003 Sep;6(9):933-8

Department of Anesthesiology, School of Medicine Pain Center, Washington University, St. Louis, Missouri 63110, USA.

The specification and differentiation of serotonergic (5-HT) neurons require both extrinsic signaling molecules and intrinsic transcription factors to work in concert or in cascade. Here we identify the genetic cascades that control the specification and differentiation of 5-HT neurons in mice. A major determinant in the cascades is an LIM homeodomain-containing gene, Lmx1b, which is required for the development of all 5-HT neurons in the central nervous system. Our results suggest that, during development of 5-HT neurons, Lmx1b is a critical intermediate factor that couples Nkx2-2-mediated early specification with Pet1-mediated terminal differentiation. Moreover, our data indicate that genetic cascades controlling the caudal and rostral 5-HT neurons are distinct, despite their shared components.
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http://dx.doi.org/10.1038/nn1104DOI Listing
September 2003

A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3.

Proc Natl Acad Sci U S A 2003 Apr 17;100(9):5217-22. Epub 2003 Apr 17.

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Congenital diaphragmatic hernia (CDH) is a significant cause of pediatric mortality in humans with a heterogeneous and poorly understood etiology. Here we show that mice lacking Slit3 developed a central (septum transversum) CDH. Slit3 encodes a member of the Slit family of guidance molecules and is expressed predominantly in the mesothelium of the diaphragm during embryonic development. In Slit3 null mice, the central tendon region of the diaphragm fails to separate from liver tissue because of abnormalities in morphogenesis. The CDH progresses through continuous growth of the liver into the thoracic cavity. This study establishes the first genetic model for CDH and identifies a previously unsuspected role for Slit3 in regulating the development of the diaphragm.
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http://dx.doi.org/10.1073/pnas.0730709100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154325PMC
April 2003