141 results on '"James K. Chen"'
Search Results
2. Basal constriction during midbrain–hindbrain boundary morphogenesis is mediated by Wnt5b and focal adhesion kinase
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Jennifer H. Gutzman, Ellie Graeden, Isabel Brachmann, Sayumi Yamazoe, James K. Chen, and Hazel Sive
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Basal constriction ,Cell shape ,Morphogenesis ,Zebrafish ,Wnt5b ,Focal adhesion kinase ,Midbrain–hindbrain boundary ,Science ,Biology (General) ,QH301-705.5 - Abstract
Basal constriction occurs at the zebrafish midbrain–hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3β overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.
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- 2018
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3. HIPK4 is essential for murine spermiogenesis
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J Aaron Crapster, Paul G Rack, Zane J Hellmann, Austen D Le, Christopher M Adams, Ryan D Leib, Joshua E Elias, John Perrino, Barry Behr, Yanfeng Li, Jennifer Lin, Hong Zeng, and James K Chen
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spermiogenesis ,cytoskeleton ,fertility ,HIPK4 ,kinase ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Optical and electron microscopy of HIPK4-null male germ cells reveals defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts and that HIPK4 deficiency alters the subcellular distribution of an F-actin capping protein in the testis, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.
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- 2020
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4. Bicyclic Caged Morpholino Oligonucleotides for Optical Gene Silencing**
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Sankha Pattanayak, Bhagyesh R. Sarode, Alexander Deiters, and James K. Chen
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Organic Chemistry ,Animals ,Molecular Medicine ,Gene Silencing ,Molecular Biology ,Biochemistry ,Zebrafish ,Morpholinos - Abstract
Caged morpholino oligonucleotides (cMOs) are synthetic tools that allow light-inducible gene silencing in live organisms. Previously reported cMOs have utilized hairpin, duplex, and cyclic structures, as well as caged nucleobases. While these antisense technologies enable efficient optical control of RNA splicing and translation, they can have limited dynamic range. A new caging strategy was developed where the two MO termini are conjugated to an internal position through a self-immolative trifunctional linker, thereby generating a bicyclic cMO that is conformationally resistant to RNA binding. The efficacy of this alternative cMO design has been demonstrated in zebrafish embryos and compared to linear MOs and monocyclic constructs.
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- 2022
5. Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1
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Zhiping Feng, Marisa E. Hom, Thomas E. Bearrood, Zachary C. Rosenthal, Daniel Fernández, Alison E. Ondrus, Yuchao Gu, Aaron K. McCormick, Madeline G. Tomaske, Cody R. Marshall, Toni Kline, Che-Hong Chen, Daria Mochly-Rosen, Calvin J. Kuo, and James K. Chen
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Aldehydes ,Proteome ,Aldehyde Dehydrogenase, Mitochondrial ,Molecular Probes ,Colonic Neoplasms ,Humans ,Cell Biology ,Aldehyde Dehydrogenase ,Colorectal Neoplasms ,Molecular Biology ,Guanidines ,Article ,Aldehyde Dehydrogenase 1 Family - Abstract
Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered the first selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs (IGUANAs) exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions, and yield leads for developing ALDH1B1-targeting therapies.
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- 2022
6. Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action
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Jonathan B Steinman, Cristina C Santarossa, Rand M Miller, Lola S Yu, Anna S Serpinskaya, Hideki Furukawa, Sachie Morimoto, Yuta Tanaka, Mitsuyoshi Nishitani, Moriteru Asano, Ruta Zalyte, Alison E Ondrus, Alex G Johnson, Fan Ye, Maxence V Nachury, Yoshiyuki Fukase, Kazuyoshi Aso, Michael A Foley, Vladimir I Gelfand, James K Chen, Andrew P Carter, and Tarun M Kapoor
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biochemistry ,chemical biology ,Hedgehog pathway ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.
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- 2017
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7. Discovery of novel determinants of endothelial lineage using chimeric heterokaryons
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Wing Tak Wong, Gianfranco Matrone, XiaoYu Tian, Simion Alin Tomoiaga, Kin Fai Au, Shu Meng, Sayumi Yamazoe, Daniel Sieveking, Kaifu Chen, David M Burns, James K Chen, Helen M Blau, and John P Cooke
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Heterokaryons ,Endothelial lineage ,nuclear reprogramming ,zebrafish ,pou3f2 ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.
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- 2017
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8. Hyaluronic acid synthesis is required for zebrafish tail fin regeneration.
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Xiaohu Ouyang, Nicholas J Panetta, Maya D Talbott, Alexander Y Payumo, Caroline Halluin, Michael T Longaker, and James K Chen
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Medicine ,Science - Abstract
Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. has3 expression is induced in the wound epithelium within hours after tail amputation, and its onset and maintenance requires fibroblast growth factor, phosphoinositide 3-kinase, and transforming growth factor-ß signaling. Inhibition of hyaluronic acid (HA) synthesis by the small molecule 4-methylumbelliferone (4-MU) impairs tail regeneration in zebrafish larvae by preventing injury-induced cell proliferation. In addition, 4-MU reduces the expression of genes associated with wound epithelium and blastema function. Treatment with glycogen synthase kinase 3 inhibitors rescues 4-MU-induced defects in cell proliferation and tail regeneration, while restoring a subset of wound epithelium and blastema markers. Our findings demonstrate a role for HA biosynthesis in zebrafish tail regeneration and delineate its epistatic relationships with other regenerative processes.
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- 2017
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9. Design Method for Tunable Planar Bandpass Filters With Single-Bias Control and Wide Tunable Frequency Range
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Yongshik Lee, Taejun Lim, Akash Anand, Xiaoguang Liu, and James K. Chen
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Physics ,business.industry ,Bandwidth (signal processing) ,020206 networking & telecommunications ,02 engineering and technology ,Lambda ,01 natural sciences ,Capacitance ,Electric power transmission ,Planar ,Band-pass filter ,Filter (video) ,0103 physical sciences ,0202 electrical engineering, electronic engineering, information engineering ,Optoelectronics ,Electrical and Electronic Engineering ,Center frequency ,010306 general physics ,business - Abstract
A design method for a frequency-tunable planar bandpass filter is demonstrated. With the method, all reasonators can be designed with identical capacitances, allowing the filter to be tuned with identical varactors using a single bias control. This allows not only low-cost fabrication, but also a simple structure and design procedure. Moreover, the filter is based on miniaturized transmission lines. Therefore, a wide tuning range is achieved with a small size and outstanding out-of-band response. Most importantly, the method facilitates the design of a filter to vary the fractional bandwidth in a predetermined way as the center frequency is tuned. Thus, a tunable filter can be designed not only to maintain the fractional bandwidth, but also to expand or to reduce the bandwidth as the frequency is tuned. The experimental results of a compact third-order bandpass filter based on CPW lines with a $0.11\lambda _{g}\times 0.8\lambda _{g}$ size show a very wide frequency tuning range of 62.8% with a 3-dB fraction bandwidth that remains within 28.65± 1.55% and an outstanding spurious suppression of under −40 dB up to as high as $3f_{0}$ .
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- 2021
10. Organic wastewater treatment by a single-atom catalyst and electrolytically produced H2O2
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Zhiyi Lu, Jinwei Xu, William Y. C. Huang, Yanbin Li, Guangxu Chen, Djordje Vuckovic, Hansen Wang, Kecheng Wang, Zewen Zhang, Zhiping Feng, Yuanqing Li, James K. Chen, Sheng Dai, Yi Cui, William A. Mitch, and Xueli Zheng
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Global and Planetary Change ,Electrolysis ,Materials science ,Ecology ,Renewable Energy, Sustainability and the Environment ,business.industry ,Radical ,Geography, Planning and Development ,Graphitic carbon nitride ,Management, Monitoring, Policy and Law ,law.invention ,Renewable energy ,Catalysis ,Urban Studies ,chemistry.chemical_compound ,chemistry ,Wastewater ,Chemical engineering ,law ,Sewage treatment ,business ,Filtration ,Nature and Landscape Conservation ,Food Science - Abstract
The presence of organic contaminants in wastewater poses considerable risks to the health of both humans and ecosystems. Although advanced oxidation processes that rely on highly reactive radicals to destroy organic contaminants are appealing treatment options, substantial energy and chemical inputs limit their practical applications. Here we demonstrate that Cu single atoms incorporated in graphitic carbon nitride can catalytically activate H2O2 to generate hydroxyl radicals at pH 7.0 without energy input, and show robust stability within a filtration device. We further design an electrolysis reactor for the on-site generation of H2O2 from air, water and renewable energy. Coupling the single-atom catalytic filter and the H2O2 electrolytic generator in tandem delivers a wastewater treatment system. These findings provide a promising path toward reducing the energy and chemical demands of advanced oxidation processes, as well as enabling their implementation in remote areas and isolated communities.
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- 2020
11. Addressing personal protective equipment (PPE) decontamination : methylene blue and light inactivates severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on N95 respirators and medical masks with maintenance of integrity and fit
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Etienne Thiry, F. Selcen Kilinc-Balci, Cyrus J. Mackie, Hans Nauwynck, John Conly, Kareem B. Kabra, Florine E. M. Scholte, Mark Mayo, Alpa N. Patel, Thor A. Wagner, Thomas S. Lendvay, David H. Evans, Lei Liao, Yi Chan Lin, Mervin Zhao, May C. Chu, Lorène Dams, Rebecca J. Malott, Rod Parker, Sarah R. Tritsch, Christopher N. Mores, Ying Ling Lin, Jean Luc Lemyre, Steven Chu, Peter Faris, Tanner Clark, Simon de Jaeger, Vanessa Molloy-Simard, Belinda Heyne, Constance Wielick, Sarah J. Smit, Yi Cui, Brian H. Harcourt, Jaya Sahni, Jean Francois Willaert, James K. Chen, Tom Gallagher, Olivier Jolois, Sarah Simmons, Kamonthip Homdayjanakul, Larry F. Chu, Ken Page, Jan M. Davies, Susan Reader, Louisa F. Ludwig-Begall, Emily Timm, Eric Haubruge, Amy Price, Molly M. Lamb, Jan Laperre, Nicolas Macia, and Karen Hope
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Microbiology (medical) ,business.product_category ,N95 Respirators ,Epidemiology ,IMPACT ,030204 cardiovascular system & hematology ,medicine.disease_cause ,Virus ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Equipment Reuse ,Medicine and Health Sciences ,Humans ,Medicine ,030212 general & internal medicine ,Respiratory system ,Respirator ,Personal Protective Equipment ,Personal protective equipment ,Decontamination ,Coronavirus ,PLASMA ,SARS-CoV-2 ,business.industry ,EXTENDED USE ,Masks ,COVID-19 ,Human decontamination ,Methylene Blue ,Infectious Diseases ,chemistry ,Virus Diseases ,Original Article ,Vaporized hydrogen peroxide ,business ,Methylene blue - Abstract
Objective:The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus.Design:The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance.Methods:MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method.Results:Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O3.Conclusions:MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.
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- 2022
12. Small Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger Reduction
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Alexander Deiters, Joshua S. Wesalo, Bradley Lukasak, Kristie Darrah, James K. Chen, and Michael Tsang
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Embryo, Nonmammalian ,Morpholino ,Oligonucleotides ,Biochemistry ,Catalysis ,Article ,Colloid and Surface Chemistry ,Gene expression ,Animals ,Genes, Developmental ,Gene ,Zebrafish ,Fluorescent Dyes ,Gene knockdown ,Chemistry ,Oligonucleotide ,Rhodamines ,General Chemistry ,Oligonucleotides, Antisense ,Thionucleotides ,Small molecule ,Cell biology ,Gene Knockdown Techniques ,Nucleic Acid Conformation ,Bioorthogonal chemistry ,Linker - Abstract
Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small molecule-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger reduction. This is enabled by a highly flexible linker design that offers opportunities for the installation of chemically activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small molecule-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.
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- 2021
13. Organic wastewater treatment by a single-atom catalyst and electrolytically produced H
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Jinwei, Xu, Xueli, Zheng, Zhiping, Feng, Zhiyi, Lu, Zewen, Zhang, William, Huang, Yanbin, Li, Djordje, Vuckovic, Yuanqing, Li, Sheng, Dai, Guangxu, Chen, Kecheng, Wang, Hansen, Wang, James K, Chen, William, Mitch, and Yi, Cui
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Article - Abstract
The presence of organic contaminants in wastewater poses considerable risks to the health of both humans and ecosystems. Although advanced oxidation processes that rely on highly reactive radicals to destroy organic contaminants are appealing treatment options, substantial energy and chemical inputs limit their practical applications. Here we demonstrate that Cu single atoms incorporated in graphitic carbon nitride can catalytically activate H(2)O(2) to generate hydroxyl radicals at pH 7.0 without energy input, and show robust stability within a filtration device. We further design an electrolysis reactor for the on-site generation of H(2)O(2) from air, water and renewable energy. Coupling the single-atom catalytic filter and the H(2)O(2) electrolytic generator in tandem delivers a wastewater treatment system. These findings provide a promising path toward reducing the energy and chemical demands of advanced oxidation processes, as well as enabling their implementation in remote areas and isolated communities.
- Published
- 2021
14. Probing tissue transglutaminase mediated vascular smooth muscle cell aging using a novel transamidation-deficient Tgm2-C277S mouse model
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James K. Chen, Sandeep Jandu, William J. Savage, Kavitha Nandakumar, Alan Poe, Jochen Steppan, Maria Bauer, Sebastian F. Barreto-Ortiz, Sean Melucci, Huilei Wang, Shivam Rastogi, Lakshmi Santhanam, and Sara K. Kang
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0301 basic medicine ,Cancer Research ,Vascular smooth muscle ,Tissue transglutaminase ,Immunology ,medicine.disease_cause ,Article ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,In vivo ,medicine ,Vascular diseases ,RC254-282 ,Integrin binding ,Mutation ,QH573-671 ,biology ,Chemistry ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Cell Biology ,In vitro ,Cell biology ,Fibronectin ,Ageing ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,Cytology ,Cell aging - Abstract
Tissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.
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- 2021
15. HIF2A gain-of-function mutation modulates the stiffness of smooth muscle cells and compromises vascular mechanics
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Rayyan Gorashi, Lilly Fang, Morgan B. Elliott, Joon Eoh, Josef T. Prchal, Jing Wang, Brian L. Lin, James K. Chen, Rebecca Black, Jihyun Song, Sharon Gerecht, Lakshmi Santhanam, Eugenia Volkova, Frank S. Lee, Linzhao Cheng, Xin Yi Chan, and Sebastian F. Barreto-Ortiz
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0301 basic medicine ,Science ,Biophysics ,02 engineering and technology ,Pathophysiology ,Article ,03 medical and health sciences ,Myosin ,medicine ,Biomechanics ,Induced pluripotent stem cell ,Gene knockdown ,Multidisciplinary ,biology ,business.industry ,Hypoxia (medical) ,musculoskeletal system ,021001 nanoscience & nanotechnology ,medicine.disease ,Pulmonary hypertension ,Endothelin 1 ,030104 developmental biology ,Cancer research ,biology.protein ,medicine.symptom ,0210 nano-technology ,business ,Fibrillin ,Elastin - Abstract
Summary Heterozygous gain-of-function (GOF) mutations of hypoxia-inducible factor 2α (HIF2A), a key hypoxia-sensing regulator, are associated with erythrocytosis, thrombosis, and vascular complications that account for morbidity and mortality of patients. We demonstrated that the vascular pathology of HIF2A GOF mutations is independent of erythrocytosis. We generated HIF2A GOF-induced pluripotent stem cells (iPSCs) and differentiated them into endothelial cells (ECs) and smooth muscle cells (SMCs). Unexpectedly, HIF2A-SMCs, but not HIF2A-ECs, were phenotypically aberrant, more contractile, stiffer, and overexpressed endothelin 1 (EDN1), myosin heavy chain, elastin, and fibrillin. EDN1 inhibition and knockdown of EDN1-receptors both reduced HIF2-SMC stiffness. Hif2A GOF heterozygous mice displayed pulmonary hypertension, had SMCs with more disorganized stress fibers and higher stiffness in their pulmonary arterial smooth muscle cells, and had more deformable pulmonary arteries compared with wild-type mice. Our findings suggest that targeting these vascular aberrations could benefit patients with HIF2A GOF and conditions of augmented hypoxia signaling., Graphical abstract, Highlights • HIF2-SMCs are stiffer than WT-SMCs and differ in contractile SMC marker expression • HIF2-SMCs and WT-SMCs differ in EDN1 production and ECM composition • HIF- 2α induces EDN1; EDNI subsequently induces SMC stiffening • Hif2A GOF mouse arterial SMCs have more disorganized stress fibers and are stiffer, Pathophysiology; Biophysics; Biomechanics
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- 2021
16. Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator
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Che-Hong Chen, Daria Mochly-Rosen, Soichi Wakatsuki, Lisa E. Dorn, James K. Chen, Andrew G. Raub, Simin Rahighi, Karen Mruk, Naoki Horikoshi, and Sunhee Hwang
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0301 basic medicine ,Hemolytic anemia ,Indoles ,Science ,Mutant ,Drug Evaluation, Preclinical ,Mutation, Missense ,General Physics and Astronomy ,Pharmacology ,Glucosephosphate Dehydrogenase ,medicine.disease_cause ,Hemolysis ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Enzyme activator ,0302 clinical medicine ,Chloroquine ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,lcsh:Science ,Zebrafish ,Multidisciplinary ,Chemistry ,Activator (genetics) ,Protein Stability ,Point mutation ,General Chemistry ,medicine.disease ,3. Good health ,Enzyme Activation ,Oxidative Stress ,030104 developmental biology ,Glucosephosphate Dehydrogenase Deficiency ,lcsh:Q ,030217 neurology & neurosurgery ,Oxidative stress ,medicine.drug ,Glucose-6-phosphate dehydrogenase deficiency - Abstract
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency., Glucose-6-phosphate dehydrogenase (G6PD) deficiency provides insufficient protection from oxidative stress, contributing to diverse human pathologies. Here, the authors identify a small molecule that increases the activity and/or stability of mutant G6PD and show that it reduces oxidative stress in zebrafish and hemolysis in isolated human erythrocytes.
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- 2018
17. Hunger or thirst state uncertainty is resolved by outcome evaluation in medial prefrontal cortex to guide decision-making
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Jon Arnold, Susu Chen, Scott M. Sternson, Tahnbee Kim, Marius Pachitariu, Anne-Kathrin Eiselt, and James K. Chen
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0301 basic medicine ,Male ,Hunger ,media_common.quotation_subject ,Decision Making ,Prefrontal Cortex ,Outcome (game theory) ,Water consumption ,Article ,Thirst ,Interoception ,03 medical and health sciences ,Mice ,0302 clinical medicine ,State (polity) ,medicine ,Food seeking ,Animals ,Prefrontal cortex ,media_common ,Two-alternative forced choice ,General Neuroscience ,digestive, oral, and skin physiology ,030104 developmental biology ,Female ,medicine.symptom ,Psychology ,Neuroscience ,030217 neurology & neurosurgery ,Cognitive psychology - Abstract
Physiological need states direct decision-making toward re-establishing homeostasis. Using a two-alternative forced choice task for mice that models elements of human decisions, we found that varying hunger and thirst states caused need-inappropriate choices, such as food seeking when thirsty. These results show limits on interoceptive knowledge of hunger and thirst states to guide decision-making. Instead, need states were identified after food and water consumption by outcome evaluation, which depended on the medial prefrontal cortex.
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- 2021
18. trLRET microscopy: Ultrasensitive imaging of lanthanide luminophores
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Paulina, Ciepla, Ukrae, Cho, and James K, Chen
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Microscopy, Fluorescence ,Fluorescence Resonance Energy Transfer ,Animals ,Lanthanoid Series Elements ,Zebrafish ,Chelating Agents - Abstract
In principle, the long emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. However, most lanthanide-imaging systems cannot achieve sensitivities that exceed those of conventional fluorescence microscopes, since they are limited by inefficient lanthanide excitation, the low photon flux of excited lanthanide luminophores, and optics-derived background photoluminescence. We recently reported a new lanthanide-imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which overcomes each of these constraints. Here we provide a detailed procedure for visualizing endogenous protein expression in zebrafish embryos, using lanthanide-labeled antibodies, Q-switched laser illumination, and trLRET microscopy. These methods allow ultrasensitive molecular imaging in cells and organisms, establishing a new paradigm for biological exploration and discovery.
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- 2020
19. Bicyclic imidazolium inhibitors of Gli transcription factor activity
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Marisa E. Hom, James K. Chen, Alison E. Ondrus, Tomoyo Sakata-Kato, and Paul G. Rack
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Scaffold protein ,01 natural sciences ,Biochemistry ,Heterocyclic Compounds, 2-Ring ,Zinc Finger Protein GLI1 ,Article ,Oxidative Phosphorylation ,law.invention ,Mice ,Structure-Activity Relationship ,Transcription (biology) ,law ,Drug Discovery ,Animals ,General Pharmacology, Toxicology and Pharmaceutics ,Transcription factor ,Hedgehog ,Pharmacology ,Membrane Potential, Mitochondrial ,Molecular Structure ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Imidazoles ,Hedgehog signaling pathway ,0104 chemical sciences ,Cell biology ,Mitochondria ,010404 medicinal & biomolecular chemistry ,NIH 3T3 Cells ,Molecular Medicine ,Suppressor ,Signal transduction ,Smoothened ,Heterocyclic Compounds, 3-Ring ,Signal Transduction - Abstract
Gli transcription factors within the Hedgehog (Hh) signaling pathway direct key events in mammalian development and promote a number of human cancers. Current therapies for Gli-driven tumors target Smoothened (SMO), a G protein-coupled receptor-like protein that functions upstream in the Hh pathway. Although these drugs can have remarkable clinical efficacy, mutations in SMO and downstream Hh pathway components frequently lead to chemoresistance. In principle, therapies that act at the level of Gli proteins, through direct or indirect mechanisms, would be more efficacious. We therefore conducted a screen of 325,120 compounds for their ability to block the constitutive Gli activity induced by loss of Suppressor of Fused (SUFU), a scaffolding protein that directly inhibits Gli function. Our studies reveal a family of bicyclic imidazolium derivatives that can inhibit Gli-dependent transcription without affecting the ciliary trafficking or proteolytic processing of these transcription factors. We anticipate that these chemical antagonists will be valuable tools for investigating the mechanisms of Gli regulation and developing new strategies for targeting Gli-driven cancers.
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- 2020
20. Multiple domains in ARHGAP36 regulate PKA degradation and Gli activation
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Jun Ni, Peter K. Jackson, James K. Chen, Patricia R. Nano, Takamasa Kudo, Nancie Mooney, and Janos Demeter
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biology ,Chemistry ,Cilium ,Protein subunit ,Plasma protein binding ,Subcellular localization ,medicine.disease_cause ,Ubiquitin ligase ,Cell biology ,medicine ,biology.protein ,Carcinogenesis ,Protein kinase A ,Transcription factor - Abstract
ARHGAP36 is a Rho GTPase-activating protein (GAP) family member that contributes to spinal cord development and tumorigenesis. This multidomain protein is composed of N-terminal sequences that vary between splice isoforms, the GAP-like region, and a unique C-terminal domain, and previous studies have revealed an N-terminal arginine-rich region that promotes protein kinase A catalytic subunit (PKAcat) degradation and Gli transcription factor activation. Although these signaling mechanisms can account for at least some aspects of ARHGAP36-dependent physiology, the functional roles of other structural elements in ARHGAP36 are not well understood. To address this question, we have mapped the ARHGAP36 structure-activity landscape with both domain- and amino acid-level resolution. We observe that ARHGAP36-mediated Gli activation in cultured mammalian cells can be autoinhibited by isoform-specific N-terminal sequences, which also regulate ARHGAP36 trafficking and the subcellular targeting of PKAcat pools. ARHGAP36 autoinhibition is counteracted by the GAP-like and C-terminal domains, which promote protein trafficking to the plasma membrane and primary cilium, respectively. In addition, our studies provide evidence that the GAP-like domain conditionally suppresses the N-terminal arginine-rich region and regulates ARHGAP36 binding to several interactors, including the prolyl oligopeptidase-like protein PREPL and the E3 ubiquitin ligase PRAJA2. Thus, multiple domains within ARHGAP36 can modulate its activity state, subcellular localization, and protein binding, potentially providing a means for achieving tissue-specific ARHGAP36 functions.
- Published
- 2020
21. Lanthanide-Based Optical Probes of Biological Systems
- Author
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James K. Chen and Ukrae Cho
- Subjects
Lanthanide ,Ytterbium ,Lanthanide probes ,Metal ions in aqueous solution ,Clinical Biochemistry ,chemistry.chemical_element ,Nanotechnology ,Terbium ,01 natural sciences ,Biochemistry ,Lanthanoid Series Elements ,Article ,Coordination Complexes ,Drug Discovery ,Fluorescence Resonance Energy Transfer ,Animals ,Molecular Biology ,Fluorescent Dyes ,Pharmacology ,010405 organic chemistry ,Chemistry ,Optical Imaging ,Proteins ,Fluorescence ,Photon upconversion ,0104 chemical sciences ,Microscopy, Fluorescence ,Molecular Medicine ,Nanoparticles ,Europium - Abstract
The unique photophysical properties of lanthanides, such as europium, terbium, and ytterbium, make them versatile molecular probes of biological systems. In particular, their long-lived photoluminescence, narrow bandwidth emissions, and large Stokes shifts enable experiments that are infeasible with organic fluorophores and fluorescent proteins. The ability of these metal ions to undergo luminescence resonance energy transfer, and photon upconversion further expands the capabilities of lanthanide probes. In this review, we describe recent advances in the design of lanthanide luminophores and their application in biological research. We also summarize the latest detection systems that have been developed to fully exploit the optical properties of lanthanide luminophores. We conclude with a discussion of remaining challenges and new frontiers in lanthanide technologies. The unprecedented levels of sensitivity and multiplexing afforded by rare-earth elements illustrate how chemistry can enable new approaches in biology.
- Published
- 2020
22. HIPK4 is essential for murine spermiogenesis
- Author
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Barry Behr, Ryan D. Leib, Austen D Le, Jennifer Lin, John Perrino, Paul G. Rack, Hong Zeng, James K. Chen, Christopher M. Adams, Yanfeng Li, Joshua E. Elias, Zane J Hellmann, and J. Aaron Crapster
- Subjects
Male ,Mouse ,Spermiogenesis ,medicine.medical_treatment ,Fluorescent Antibody Technique ,Intracytoplasmic sperm injection ,Mice ,0302 clinical medicine ,Biology (General) ,Cytoskeleton ,Mice, Knockout ,fertility ,0303 health sciences ,General Neuroscience ,Gene Expression Regulation, Developmental ,cytoskeleton ,General Medicine ,Spermatids ,Spermatozoa ,Cell biology ,Phenotype ,medicine.anatomical_structure ,Medicine ,Acrosome ,Protein Binding ,Signal Transduction ,Research Article ,endocrine system ,kinase ,QH301-705.5 ,Science ,HIPK4 ,Protein Serine-Threonine Kinases ,Biology ,Models, Biological ,Filamentous actin ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,medicine ,Animals ,Spermatogenesis ,Protein kinase A ,030304 developmental biology ,General Immunology and Microbiology ,Spermatid ,urogenital system ,Cell Biology ,Oocyte ,Sperm ,Actins ,spermiogenesis ,Mutation ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Optical and electron microscopy of HIPK4-null male germ cells reveals defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts and that HIPK4 deficiency alters the subcellular distribution of an F-actin capping protein in the testis, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.
- Published
- 2020
23. Author response: HIPK4 is essential for murine spermiogenesis
- Author
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Joshua E. Elias, John Perrino, Austen D Le, Jennifer Lin, James K. Chen, Zane J Hellmann, Ryan D. Leib, Christopher M. Adams, Paul G. Rack, Hong Zeng, Yanfeng Li, Barry Behr, and J. Aaron Crapster
- Subjects
Biology ,Cell biology - Published
- 2020
24. Afabicin, a First-in-Class Antistaphylococcal Antibiotic, in the Treatment of Acute Bacterial Skin and Skin Structure Infections: Clinical Noninferiority to Vancomycin/Linezolid
- Author
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Catherine Vincent, James K. Chen, François Leylavergne, Heidi Kabler, Frederick Wittke, Guennaëlle Dieppois, Barry Heller, and J Scott Overcash
- Subjects
Methicillin-Resistant Staphylococcus aureus ,medicine.medical_specialty ,medicine.drug_class ,Antibiotics ,Population ,Phases of clinical research ,medicine.disease_cause ,Staphylococcal infections ,Gastroenterology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Double-Blind Method ,Vancomycin ,Internal medicine ,medicine ,Humans ,Pharmacology (medical) ,030212 general & internal medicine ,Naphthyridines ,education ,Benzofurans ,Pharmacology ,0303 health sciences ,education.field_of_study ,030306 microbiology ,business.industry ,Linezolid ,Skin Diseases, Bacterial ,medicine.disease ,Anti-Bacterial Agents ,Infectious Diseases ,Treatment Outcome ,chemistry ,Tolerability ,Staphylococcus aureus ,business ,medicine.drug - Abstract
Afabicin (formerly Debio 1450, AFN-1720) is a prodrug of afabicin desphosphono, an enoyl-acyl carrier protein reductase (FabI) inhibitor, and is a first-in-class antibiotic with a novel mode of action to specifically target fatty acid synthesis in Staphylococcus spp. The efficacy, safety, and tolerability of afabicin were compared with those of vancomycin/linezolid in the treatment of acute bacterial skin and skin structure infections (ABSSSI) due to staphylococci in this multicenter, parallel-group, double-blind, and double-dummy phase 2 study. Randomized patients (1:1:1) received either low-dose (LD) afabicin (intravenous [i.v.] 80 mg, followed by oral 120 mg, twice a day [BID]), high-dose (HD) afabicin (i.v. 160 mg, followed by oral 240 mg, BID), or vancomycin/linezolid (i.v. vancomycin 1 g or 15 mg/kg, followed by oral linezolid 600 mg, BID). The most frequent baseline pathogen was Staphylococcus aureus (97.5% of microbiological intent-to-treat [mITT] population), and 50.4% of patients had methicillin-resistant S. aureus Clinical response rates at 48 to 72 h postrandomization in the mITT population were comparable among treatment groups (94.6%, 90.1%, and 91.1%, respectively). Both LD and HD afabicin were noninferior to vancomycin/linezolid (differences, -3.5% [95% confidence interval {CI}, -10.8%, 3.9%] and 1.0% [95% CI, -7.3%, 9.2%], respectively). Most common treatment-emergent adverse events were mild and were headache (9.1% and 16.8%) and nausea (6.4% and 8.4%) with LD and HD afabicin, respectively. Afabicin was efficacious and well tolerated in the treatment of ABSSSI due to staphylococci, and these data support further development of afabicin for the treatment of ABSSSI and potentially other types of staphylococcal infections. (This study has been registered at ClinicalTrials.gov under identifier NCT02426918.).
- Published
- 2020
25. trLRET microscopy: Ultrasensitive imaging of lanthanide luminophores
- Author
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Paulina Ciepla, Ukrae Cho, and James K. Chen
- Subjects
Lanthanide ,0303 health sciences ,Microscope ,Materials science ,Photoluminescence ,030303 biophysics ,Nanotechnology ,Fluorescence ,law.invention ,03 medical and health sciences ,Autofluorescence ,Optical microscope ,law ,Microscopy ,Molecular imaging - Abstract
In principle, the long emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. However, most lanthanide-imaging systems cannot achieve sensitivities that exceed those of conventional fluorescence microscopes, since they are limited by inefficient lanthanide excitation, the low photon flux of excited lanthanide luminophores, and optics-derived background photoluminescence. We recently reported a new lanthanide-imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which overcomes each of these constraints. Here we provide a detailed procedure for visualizing endogenous protein expression in zebrafish embryos, using lanthanide-labeled antibodies, Q-switched laser illumination, and trLRET microscopy. These methods allow ultrasensitive molecular imaging in cells and organisms, establishing a new paradigm for biological exploration and discovery.
- Published
- 2020
26. HIF2A Gain-of-Function Mutation Modulates the Stiffness of Smooth Muscle Cells and Compromises Vascular Mechanics
- Author
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Josef T. Prchal, Rayyan Gorashi, Linzhao Cheng, Morgan B. Elliott, Frank S. Lee, Joon Eoh, Xin Chan, Sharon Gerecht, James K. Chen, Rebecca Black, Lilly Fang, Jing Wang, Sebastian F. Barreto-Ortiz, Jihyun Song, Lakshmi Santhanam, Eugenia Volkova, and Brian L. Lin
- Subjects
Vascular smooth muscle ,biology ,business.industry ,Hypoxia (medical) ,medicine.disease ,Endothelin 1 ,Pulmonary hypertension ,Myosin ,Cancer research ,biology.protein ,Medicine ,medicine.symptom ,business ,Induced pluripotent stem cell ,Fibrillin ,Elastin - Abstract
Heterozygous gain-of-function (GOF) mutations of hypoxia-inducible factor 2α (HIF2A), a significant regulator of hypoxia sensing, are associated with erythrocytosis, thrombosis and vascular complications that account for morbidity and mortality of patients with these disorders. We demonstratethat vascular pathology of HIF2A GOF mutations is independent of erythrocytosis. We generated HIF2A GOF induced pluripotent stem cells (iPSCs) and differentiated them into endothelial cells (ECs) and smooth muscle cells (SMCs). Unexpectedly, the HIF2A-SMCs, but not HIF2A-ECs, were phenotypically aberrant, more contractile and stiffer, and overexpressed endothelin 1(EDN1), myosin heavy chain, elastin and fibrillin. EDN1 inhibition or knockdown of EDN1-receptors reduced HIF2-SMC stiffness. Hif2A GOF heterozygous mice displayed early onset of pulmonary hypertension, had SMCs with more disorganized stress fibers and higher stiffness in their pulmonary arterial vascular smooth muscle cells, and more deformable main pulmonary arteries compared with wild type mice. We propose that targeting these vascular aberrations may benefit patients with HIF2A GOF and other conditions ofaugmented hypoxia signaling.
- Published
- 2020
27. Targeted cell ablation in zebrafish using optogenetic transcriptional control
- Author
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Paulina Ciepla, Karen Mruk, Mohammad A. Alnaqib, Patrick A. Piza, and James K. Chen
- Subjects
Cell type ,Embryo, Nonmammalian ,Light ,Cell ,Gene Expression ,Optogenetics ,Gene product ,Animals, Genetically Modified ,Viral Matrix Proteins ,03 medical and health sciences ,Nitroreductase ,0302 clinical medicine ,Techniques and Resources ,Rimantadine ,Bacterial Proteins ,Genes, Reporter ,medicine ,Transcriptional regulation ,Cytotoxic T cell ,Animals ,Promoter Regions, Genetic ,Molecular Biology ,Zebrafish ,030304 developmental biology ,Neurons ,0303 health sciences ,biology ,Promoter ,Nitroreductases ,biology.organism_classification ,Axons ,Cell biology ,medicine.anatomical_structure ,Mutagenesis, Site-Directed ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have utilized the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis-regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control.
- Published
- 2019
- Full Text
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28. A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies
- Author
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Michael C. Bassik, Sascha Hoogendoorn, Brandon Vu, Gaelen T. Hess, Kyuho Han, Maxence V. Nachury, Margaret C. Kennedy, David W. Morgens, Adam R Kopp, Amy Li, James K. Chen, and David K. Breslow
- Subjects
0301 basic medicine ,Centriole ,Computational biology ,Biology ,Medical and Health Sciences ,Ciliopathies ,Article ,Mice ,03 medical and health sciences ,Heart disorder ,Genetics ,centriole ,Animals ,Humans ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Hedgehog Proteins ,Cilia ,Cilium ,HEK 293 cells ,Biological Sciences ,Hedgehog signaling pathway ,High-Throughput Screening Assays ,ciliopathy ,HEK293 Cells ,030104 developmental biology ,genetic screen ,NIH 3T3 Cells ,Signal transduction ,signaling ,functional genomics ,Hedgehog ,Developmental Biology ,primary cilium ,Signal Transduction - Abstract
Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.
- Published
- 2018
29. Ultrasensitive optical imaging with lanthanide lumiphores
- Author
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Pehr B. Harbury, Paulina Ciepla, Kiranmai S. Kocherlakota, Ukrae Cho, Daniel P. Riordan, and James K. Chen
- Subjects
0301 basic medicine ,Lanthanide ,Materials science ,Luminescent Measurements ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Lanthanoid Series Elements ,Sensitivity and Specificity ,Article ,03 medical and health sciences ,Coordination Complexes ,Microscopy ,Fluorescence microscope ,Animals ,Luminescent Agents ,Molecular Biology ,Zebrafish ,Millisecond ,business.industry ,Optical Imaging ,Cell Biology ,Zebrafish Proteins ,0104 chemical sciences ,030104 developmental biology ,Optoelectronics ,Biological imaging ,business ,Luminescence - Abstract
In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.
- Published
- 2017
30. Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains
- Author
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Jun Ni, Patricia R. Nano, Taylor K. Johnson, Takamasa Kudo, Nancie Mooney, James K. Chen, Janos Demeter, and Peter K. Jackson
- Subjects
Gene isoform ,Science ,Interaction Networks ,Cell Membranes ,Mutant ,Equipment ,Mutagenesis (molecular biology technique) ,Transfection ,Research and Analysis Methods ,Proteomics ,Homology (biology) ,Mice ,Structure-Activity Relationship ,Cell Signaling ,Protein Domains ,Genetics ,Point Mutation ,Animals ,Humans ,Protein Isoforms ,Cilia ,Molecular Biology Techniques ,Protein kinase A ,Molecular Biology ,Transcription factor ,Multidisciplinary ,Chemistry ,Lasers ,GTPase-Activating Proteins ,Biology and Life Sciences ,Membrane Proteins ,Cell Biology ,Cell biology ,Signal Filtering ,Bandpass Filters ,HEK293 Cells ,Optical Equipment ,Membrane protein ,Mutation ,Signal Processing ,Hedgehog Signaling ,NIH 3T3 Cells ,Engineering and Technology ,Medicine ,Cellular Structures and Organelles ,Research Article ,Signal Transduction - Abstract
ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.
- Published
- 2021
31. An inducible long noncoding RNA amplifies DNA damage signaling
- Author
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Laura D. Attardi, Julia T. Garcia, Ashwin Peres-da-Silva, Howard Y. Chang, Kun Qu, Rachel Baum, Adam M. Schmitt, James K. Chen, Shuling Guo, Alexander Y. Payumo, Tiffany Hung, Ying Shen, Ryan A. Flynn, and Daniela Kenzelmann Broz
- Subjects
Male ,0301 basic medicine ,DNA damage ,Biology ,Article ,Cell Line ,03 medical and health sciences ,Genetics ,Animals ,Humans ,Feedback, Physiological ,Mice, Knockout ,Regulation of gene expression ,Extramural ,RNA ,Molecular biology ,Long non-coding RNA ,Rats ,Mice, Inbred C57BL ,030104 developmental biology ,Cell culture ,P53 protein ,Female ,RNA, Long Noncoding ,Tumor Suppressor Protein p53 ,Signal transduction ,DNA Damage ,Signal Transduction - Abstract
Long noncoding RNAs (lncRNAs) are prevalent genes with frequently precise regulation but mostly unknown functions. Here we demonstrate that lncRNAs guide the organismal DNA damage response. DNA damage activated transcription of the DINO (Damage Induced Noncoding) lncRNA via p53. DINO was required for p53-dependent gene expression, cell cycle arrest and apoptosis in response to DNA damage, and DINO expression was sufficient to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO bound to p53 protein and promoted its stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampened p53 signaling and ameliorated acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks.
- Published
- 2016
32. HIPK4 is essential for murine spermiogenesis
- Author
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John Perrino, Hong Zeng, Yanfeng Li, Zane J Hellmann, James K. Chen, Barry Behr, Paul G. Rack, J. Aaron Crapster, Jennifer Lin, and Joshua E. Elias
- Subjects
endocrine system ,0303 health sciences ,Spermatid ,urogenital system ,Spermiogenesis ,medicine.medical_treatment ,030302 biochemistry & molecular biology ,Biology ,Oocyte ,Filamentous actin ,Sperm ,Intracytoplasmic sperm injection ,Cell biology ,03 medical and health sciences ,medicine.anatomical_structure ,medicine ,Protein kinase A ,Cytoskeleton ,reproductive and urinary physiology ,030304 developmental biology - Abstract
Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Ultrastructural analyses of HIPK4-null male germ cells reveal defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.
- Published
- 2019
33. Lysyl Oxidase Like 2 Processing by Factor Xa Modulates its Activity and Localization
- Author
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Lakshmi Santhanam, James K. Chen, Huilei Wang, Jochen Steppan, Yurie Hong, Kavitha Nandakumar, Sandeep Jandu, and Alan Poe
- Subjects
Biochemistry ,Chemistry ,Genetics ,Lysyl oxidase ,Molecular Biology ,Biotechnology - Published
- 2019
34. Combinatorial control of gene function with wavelength-selective caged morpholinos
- Author
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Alexander Deiters, James K. Chen, Luis Angel Vázquez-Maldonado, and Sankha Pattanayak
- Subjects
Morpholino ,biology ,Light ,Microinjections ,Chemistry ,Computational biology ,Chemistry Techniques, Synthetic ,Oligonucleotides, Antisense ,biology.organism_classification ,Photochemical Processes ,Article ,Morpholinos ,Morpholino Oligonucleotides ,Antisense oligonucleotides ,Zebrafish embryo ,Gene silencing ,Animals ,Gene Silencing ,Gene ,Zebrafish ,Function (biology) - Abstract
Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental biology because they allow spatiotemporal control of gene expression in whole organisms. While cMOs are usually triggered by light of a single wavelength, the introduction of spectrally distinct chromophores can enable combinatorial regulation of multiple genes. This chapter describes the general principles and methods of wavelength-selective cMO design and synthesis from commercially available reagents. Synthetic protocols for the linkers and the two-step cMO assembly are described in detail, as well as the microinjection and photoactivation techniques. Following these protocols, spectrally separated cyclic cMOs for multiple genes of interest can be prepared, enabling their inhibition in zebrafish embryos and other animal models.
- Published
- 2019
35. Basal constriction during midbrain–hindbrain boundary morphogenesis is mediated by Wnt5b and focal adhesion kinase
- Author
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Hazel Sive, Isabel Brachmann, Jennifer H. Gutzman, James K. Chen, Ellie Graeden, and Sayumi Yamazoe
- Subjects
0301 basic medicine ,animal structures ,QH301-705.5 ,Science ,Morphogenesis ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Focal adhesion ,03 medical and health sciences ,Basal (phylogenetics) ,Basal constriction ,Biology (General) ,Zebrafish ,Midbrain–hindbrain boundary ,Gene knockdown ,Focal adhesion kinase ,Wnt5b ,biology.organism_classification ,Cell biology ,Midbrain-hindbrain boundary morphogenesis ,030104 developmental biology ,cardiovascular system ,Cell shape ,Signal transduction ,General Agricultural and Biological Sciences ,Brain morphogenesis ,Research Article ,circulatory and respiratory physiology - Abstract
Basal constriction occurs at the zebrafish midbrain–hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3β overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis., Summary: Focal adhesion kinase acts downstream of Wnt5b to mediate basal constriction of neuroepithelial cells during the formation of the midbrain–hindbrain boundary.
- Published
- 2018
36. Tbx16 regulates hox gene activation in mesodermal progenitor cells
- Author
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Alexander Y. Payumo, James K. Chen, Sayumi Yamazoe, Lindsey E. McQuade, and Whitney J. Walker
- Subjects
0301 basic medicine ,Mesoderm ,animal structures ,Danio ,Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Somitogenesis ,medicine ,Paraxial mesoderm ,Animals ,Hox gene ,Molecular Biology ,Zebrafish ,Molecular Structure ,Myogenesis ,Stem Cells ,Genes, Homeobox ,Cell Biology ,Zebrafish Proteins ,biology.organism_classification ,Molecular biology ,Cell biology ,Gastrulation ,030104 developmental biology ,medicine.anatomical_structure ,embryonic structures ,T-Box Domain Proteins ,030217 neurology & neurosurgery - Abstract
The transcription factor T-box 16 (Tbx16, or Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. However, the mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic. We describe here the use of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identified 124 Tbx16-regulated genes that were expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis and somitogenesis. Unexpectedly, we observed that a loss of Tbx16 function precociously activated posterior hox genes in MPCs, and overexpression of a single posterior hox gene was sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior-posterior fates and positions of paraxial MPCs.
- Published
- 2016
37. SIRT2 Plays Significant Roles in Lipopolysaccharides-Induced Neuroinflammation and Brain Injury in Mice
- Author
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Xunbing Wei, Ban Wang, James K. Chen, Weihai Ying, Wei Cao, and Youjun Zhang
- Subjects
Lipopolysaccharides ,Male ,0301 basic medicine ,Apoptosis ,Inflammation ,SIRT2 ,Biochemistry ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Sirtuin 2 ,0302 clinical medicine ,In vivo ,medicine ,Animals ,Neuroinflammation ,Messenger RNA ,TUNEL assay ,biology ,Tumor Necrosis Factor-alpha ,General Medicine ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,Integrin alpha M ,Brain Injuries ,Immunology ,biology.protein ,Microglia ,medicine.symptom ,030217 neurology & neurosurgery - Abstract
Several recent studies have suggested seemingly contrasting roles of SIRT2 in inflammation: Our previous cell culture study has indicated that SIRT2 siRNA-produced decrease in SIRT2 levels can lead to significant inhibition of lipopolysaccharides (LPS)-induced activation of BV2 microglia, suggesting that SIRT2 is required for LPS-induced microglial activation. In contrast, some studies have suggested that SIRT2 deficiency can lead to increased inflammation. In our current study, we used a mouse model of neuroinflammation to determine the roles of SIRT2 in LPS-induced inflammation. We found that administration of SIRT2 inhibitor AGK2 can significantly decrease LPS-induced increases in CD11b signals and the mRNA of TNF-α and IL-6. We further found that AGK2 can block LPS-induced nuclear translocation of NFκB. In addition, our study has shown that AGK2 can decrease not only LPS-induced increase in TUNEL signals-a marker of apoptosis-like damage, but also LPS-induced increases in the levels of active Caspase-3 and Bax. Collectively, our current in vivo study, together with our previous cell culture study, has suggested that SIRT2 is required for LPS-induced neuroinflammation and brain injury.
- Published
- 2016
38. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity
- Author
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Ruta Zalyte, Maxence V. Nachury, Andrew P. Carter, Rand M. Miller, Fan Ye, Tommaso Cupido, Jonathan B. Steinman, Andrew H. Chung, Tarun M. Kapoor, Sascha Hoogendoorn, Stephanie K. See, Tomoyo Sakata-Kato, and James K. Chen
- Subjects
Cytoplasmic Dyneins ,0301 basic medicine ,Dynein ,macromolecular substances ,Biology ,Biochemistry ,Substrate Specificity ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,Microtubule ,Animals ,Protein Isoforms ,Structure–activity relationship ,Hedgehog Proteins ,Letters ,Quinazolinones ,Molecular Structure ,Cilium ,General Medicine ,AAA proteins ,Cell biology ,030104 developmental biology ,NIH 3T3 Cells ,Dynactin ,Axoplasmic transport ,Molecular Medicine ,Signal Transduction - Abstract
Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.
- Published
- 2015
39. Probing the crosslinking independent roles of tissue transglutaminase in vascular aging using a novel transamidation‐deficient TGM2‐C277S mouse model
- Author
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Sebastian F. Barreto-Ortiz, Huilei Wang, Kavitha Nandakumar, Lakshmi Santhanam, Sean Melucci, Sandeep Jandu, and James K. Chen
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biology ,Chemistry ,Tissue transglutaminase ,Genetics ,biology.protein ,Vascular aging ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2020
40. De novo design of self-assembling helical protein filaments
- Author
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Lai-Sheng Wang, Bradley Parry, Christine Jacobs-Wagner, Linda Wordeman, Justin Decarreau, Jorge A. Fallas, Hao Shen, David Baker, Eric M. Lynch, Q. Dowling, William Sheffler, J. J. De Yoreo, Juan Jesus Vicente, Lance Stewart, N. Jannetty, Justin M. Kollman, Mike Wagenbach, James K. Chen, and Gustav Oberdorfer
- Subjects
0301 basic medicine ,Protein Conformation, alpha-Helical ,Protein Folding ,Materials science ,Fabrication ,Nanotechnology ,macromolecular substances ,010402 general chemistry ,Protein Engineering ,01 natural sciences ,Protein Structure, Secondary ,Article ,Protein filament ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Self assembling ,Escherichia coli ,Computational design ,Multidisciplinary ,Cryoelectron Microscopy ,Metamaterial ,Computational Biology ,Proteins ,0104 chemical sciences ,030104 developmental biology ,Monomer ,chemistry ,Protein folding - Abstract
Built to be reversible There has been some success in designing stable peptide filaments; however, mimicking the reversible assembly of many natural protein filaments is challenging. Dynamic filaments usually comprise independently folded and asymmetric proteins and using such building blocks requires the design of multiple intermonomer interfaces. Shen et al. report the design of self-assembling helical filaments based on previously designed stable repeat proteins. The filaments are micron scale, and their diameter can be tuned by varying the number of repeats in the monomer. Anchor and capping units, built from monomers that lack an interaction interface, can be used to control assembly and disassembly. Science , this issue p. 705
- Published
- 2018
41. 3164 – HOXB5 CONFERS INCREASED STRESS TOLERANCE AND MAINTENANCE OF SELF-RENEWAL TO THE HEMATOPOIETIC STEM CELLS
- Author
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Kevin S. Kao, Katsuyuki Nishi, Irving L. Weissman, Kay Sadaoka, Taro Sakamaki, Masanori Miyanishi, James K. Chen, and Momo Fujii
- Subjects
Cancer Research ,Mechanism (biology) ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Cell fate determination ,Biology ,Cell biology ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,Genetics ,medicine ,Compartment (development) ,Stem cell ,Molecular Biology ,Increased stress tolerance - Abstract
Self-renewal and multipotency are essential and defining features of the hematopoietic stem cell (HSC). It has been previously suggested that a self-renewal gradient exists within the classic immunophenotypic HSC compartment (Lin-c-Kit+Sca-1+CD150+CD34-/loFlk2-: pHSCs). However, we have yet to explain the selective pressure for this functional heterogeneity nor the mechanism for how this heterogeneity is regulated. Using our Hoxb5 reporter mouse line, we previously demonstrated the ability to distinguish between the long-term (LT) and short-term (ST) HSCs (as Hoxb5+ and Hoxb5- pHSCs respectively), with the former exhibiting a higher self-renewal capacity. In our current studies, we demonstrate that self-renewal is impacted by tolerance to varying degrees of replication stress, resulting in this measured self-renewal heterogeneity. By isolating Hoxb5+ and Hoxb5- pHSCs, we demonstrate that Hoxb5- pHSCs quickly lose self-renewal capacity and are exhausted under various high stress conditions, including transplantation. However, under low stress conditions they can maintain their capacity for self-renewal. Furthermore, we show that ectopic Hoxb5 expression is sufficient to confer stress tolerance against loss of self-renewal to Hoxb5- pHSCs and can alter the cell fate of ST-HSCs to that of LT-HSCs. Therefore, Hoxb5 expression may not simply be a marker of increased stress tolerance but may also serve as a fail-safe system to protect the HSC compartment from exhaustion over an organism's lifespan.
- Published
- 2019
42. Title: Basal constriction during midbrain-hindbrain boundary morphogenesis is mediated by Wnt5b and Focal Adhesion Kinase
- Author
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Hazel Sive, Sayumi Yamazoe, Isabel Brachmann, James K. Chen, Ellie Graeden, and Jennifer H. Gutzman
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0303 health sciences ,Gene knockdown ,animal structures ,biology ,Chemistry ,biology.organism_classification ,Constriction ,Cell biology ,Focal adhesion ,03 medical and health sciences ,Basal (phylogenetics) ,Midbrain-hindbrain boundary morphogenesis ,0302 clinical medicine ,cardiovascular system ,Signal transduction ,Zebrafish ,Brain morphogenesis ,030217 neurology & neurosurgery ,030304 developmental biology ,circulatory and respiratory physiology - Abstract
Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion.wnt5bis expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3β overcomeswnt5bknockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak is epistatic townt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.
- Published
- 2018
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43. A Novel Missense Variant in the GLI3 Zinc Finger Domain in a Family with Digital Anomalies
- Author
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Natalia Gomez-Ospina, Louanne Hudgins, James K. Chen, and J. Aaron Crapster
- Subjects
0301 basic medicine ,musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,animal structures ,Genotyping Techniques ,Mutation, Missense ,Repressor ,Nerve Tissue Proteins ,030105 genetics & heredity ,Biology ,Article ,Fingers ,03 medical and health sciences ,Mice ,Genes, Reporter ,Zinc Finger Protein Gli3 ,GLI2 ,GLI3 ,Genetics ,medicine ,Missense mutation ,Animals ,Humans ,Amino Acid Sequence ,Genetics (clinical) ,Zinc finger ,Polydactyly ,Preaxial polydactyly ,Zinc Fingers ,Sequence Analysis, DNA ,Acrocephalosyndactylia ,Fibroblasts ,Toes ,medicine.disease ,Hedgehog signaling pathway ,Pedigree ,body regions ,Repressor Proteins ,030104 developmental biology ,Phenotype ,Thumb ,Child, Preschool ,embryonic structures ,Female ,Sequence Alignment ,Signal Transduction - Abstract
Mutations in GLI3, which encodes a transcription factor of the Hedgehog signaling pathway, cause several developmental anomalies linked to inappropriate tissue patterning. Here, we report a novel missense variant in the fifth zinc finger domain of GLI3 (c.1826G>A; p.(Cys609Tyr)) initially identified in a proband with preaxial polydactyly type IV, developmental delay, sensorineural hearing loss, skeletal, and genitourinary anomalies. Additional family members exhibited various digital anomalies such as preaxial polydactyly, syndactyly, and postaxial polydactyly either in isolation or combined. Functional studies of Cys609Tyr GLI3 in cultured cells showed abnormal GLI3 processing leading to decreased GLI3 repressor production, increased basal transcriptional activity, and submaximal GLI reporter activity with Hedgehog pathway activation, thus demonstrating an intriguing molecular mechanism for this GLI3-related phenotype. Given the complexity of GLI3 post-translational processing and opposing biological functions as a transcriptional activator and repressor, our findings highlight the importance of performing functional studies of presumed GLI3 variants. This family also demonstrates how GLI3 variants are variably expressed.
- Published
- 2017
44. Optochemical Dissection of T-box Gene-Dependent Medial Floor Plate Development
- Author
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Whitney J. Walker, James K. Chen, Sayumi Yamazoe, Lindsey E. McQuade, and Alexander Y. Payumo
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Fetal Proteins ,Mesoderm ,Embryo, Nonmammalian ,animal structures ,Light ,Epiboly ,Biology ,Biochemistry ,Article ,Morpholinos ,Somitogenesis ,Morphogenesis ,medicine ,Animals ,Zebrafish ,Floor plate ,Gene Expression Regulation, Developmental ,General Medicine ,Anatomy ,Zebrafish Proteins ,Photochemical Processes ,biology.organism_classification ,Cell biology ,Gastrulation ,T-box ,medicine.anatomical_structure ,Molecular Probes ,Molecular Medicine ,T-Box Domain Proteins ,NODAL ,Signal Transduction - Abstract
In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors no tail a (ntla) and spadetail (spt/tbx16) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in ntla;spt morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior-posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors.
- Published
- 2015
45. Sequential Gene Silencing Using Wavelength-Selective Caged Morpholino Oligonucleotides
- Author
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Alexander Deiters, James K. Chen, Qingyang Liu, Sayumi Yamazoe, and Lindsey E. McQuade
- Subjects
Mesoderm ,animal structures ,Morpholino ,biology ,Oligonucleotide ,Chemistry ,General Medicine ,General Chemistry ,biology.organism_classification ,Molecular biology ,Catalysis ,Cell biology ,medicine.anatomical_structure ,embryonic structures ,Gene expression ,medicine ,Gene silencing ,Developmental biology ,Gene ,Zebrafish - Abstract
Spectrally differentiated caged morpholino oligonucleotides (cMOs) and wavelength-selective illumination have been used to sequentially inactivate organismal gene function. The efficacy of these reverse-genetic chemical probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.
- Published
- 2014
46. A comprehensive portrait of cilia and ciliopathies from a CRISPR-based screen for Hedgehog signaling
- Author
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David K. Breslow, James K. Chen, Gaelen T. Hess, Kyuho Han, Amy Li, Maxence V. Nachury, Adam R Kopp, Michael C. Bassik, Sascha Hoogendoorn, David W. Morgens, and Brandon Vu
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Genetics ,0303 health sciences ,Centriole ,Cilium ,A protein ,Computational biology ,Biology ,Ciliopathies ,Hedgehog signaling pathway ,03 medical and health sciences ,0302 clinical medicine ,CRISPR ,Signal transduction ,030217 neurology & neurosurgery ,Function (biology) ,030304 developmental biology - Abstract
The primary cilium organizes Hedgehog signaling, shapes embryonic development and is the unifying cause of the ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen robustly identifies factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovers novel components of several ciliary structures including a protein complex containing ε- and δ- tubulin that is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and reveals that many forms of congenital heart defects are ciliopathies. Collectively, this screen enables a systematic analysis of ciliary function and of ciliopathies and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.
- Published
- 2017
47. Illuminating developmental biology through photochemistry
- Author
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James K. Chen and Lukasz Kowalik
- Subjects
0301 basic medicine ,Rhodopsin ,Photoactivatable probes ,Molecular Structure ,Extramural ,Photochemistry ,Genomics ,Cell Biology ,Computational biology ,Biology ,010402 general chemistry ,01 natural sciences ,Models, Biological ,Article ,0104 chemical sciences ,Cell biology ,03 medical and health sciences ,Multicellular organism ,030104 developmental biology ,Molecular Probes ,Profiling (information science) ,Molecular Biology ,Developmental biology ,Developmental Biology - Abstract
Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.
- Published
- 2017
48. Author response: Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action
- Author
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Vladimir I. Gelfand, Maxence V. Nachury, James K. Chen, Fan Ye, Sachie Morimoto, Tarun M. Kapoor, Kazuyoshi Aso, Jonathan B. Steinman, Alison E. Ondrus, Lola S Yu, Ruta Zalyte, Michael Foley, Andrew P. Carter, Moriteru Asano, Yuta Tanaka, Yoshiyuki Fukase, Furukawa Hideki, Cristina C. Santarossa, Alex G. Johnson, Rand M. Miller, Anna S. Serpinskaya, and Mitsuyoshi Nishitani
- Subjects
medicine.anatomical_structure ,Chemistry ,Chemical structure ,Cell ,Dynein ,medicine ,Biophysics ,Mode of action - Published
- 2017
49. Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action
- Author
-
Kazuyoshi Aso, Mitsuyoshi Nishitani, Alison E. Ondrus, Tarun M. Kapoor, Yoshiyuki Fukase, Rand M. Miller, Ruta Zalyte, Andrew P. Carter, Vladimir I. Gelfand, Maxence V. Nachury, Sachie Morimoto, Alex G. Johnson, Anna S. Serpinskaya, Furukawa Hideki, James K. Chen, Michael Foley, Cristina C. Santarossa, Jonathan B. Steinman, Lola S Yu, Moriteru Asano, Fan Ye, and Yuta Tanaka
- Subjects
0301 basic medicine ,Cytoplasmic Dyneins ,QH301-705.5 ,1.1 Normal biological development and functioning ,Science ,Dynein ,Chemical biology ,chemical biology ,Biology ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Hedgehog pathway ,Motor protein ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Intraflagellar transport ,Underpinning research ,Humans ,biochemistry ,human ,Enzyme Inhibitors ,Biology (General) ,Quinazolinones ,Crystallography ,General Immunology and Microbiology ,Molecular Structure ,General Neuroscience ,Cilium ,Dyneins ,General Medicine ,Cell biology ,030104 developmental biology ,Cytoplasm ,5.1 Pharmaceuticals ,X-Ray ,Pyrazoles ,Medicine ,Biochemistry and Cell Biology ,Development of treatments and therapeutic interventions ,030217 neurology & neurosurgery ,Research Article ,Human - Abstract
Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition. DOI: http://dx.doi.org/10.7554/eLife.25174.001
- Published
- 2017
50. Discovery of novel determinants of endothelial lineage using chimeric heterokaryons
- Author
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Gianfranco Matrone, Wing Tak Wong, John P. Cooke, Xiao Yu Tian, Helen M. Blau, Simion Alin Tomoiaga, Kin Fai Au, Kaifu Chen, Sayumi Yamazoe, James K. Chen, Daniel P Sieveking, Shu Meng, and David M Burns
- Subjects
0301 basic medicine ,Cell type ,Mouse ,QH301-705.5 ,Science ,Cellular differentiation ,Heterokaryons ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Fusion ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Vasculogenesis ,nuclear reprogramming ,Animals ,Humans ,Biology (General) ,Induced pluripotent stem cell ,Embryonic Stem Cells ,Genetics ,Cell fusion ,General Immunology and Microbiology ,Gene Expression Profiling ,General Neuroscience ,Endothelial Cells ,Endothelial lineage ,Cell Differentiation ,Cell Biology ,General Medicine ,zebrafish ,Embryonic stem cell ,3. Good health ,Cell biology ,Endothelial stem cell ,Developmental Biology and Stem Cells ,030104 developmental biology ,embryonic structures ,Medicine ,pou3f2 ,Stem cell ,030217 neurology & neurosurgery ,Research Article ,Human - Abstract
We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages. DOI: http://dx.doi.org/10.7554/eLife.23588.001, eLife digest Endothelial cells form the inner surface of blood vessels, acting like a non-stick coating. In addition to making substances that keep blood from sticking to the vessel wall, endothelial cells generate compounds that relax the vessel, and prevent it from thickening. Endothelial cells also form capillaries, the smallest vessels that provide oxygen and nutrients for all tissues. A regenerating organ, or a bioengineered tissue, requires a system of capillaries and other microvessels. Thus, regenerative medicine could benefit from a knowledge of how to generate endothelial cells from pluripotent stem cells – cells that can “differentiate” to form almost any type of cell in the body. Wong, Matrone et al. have now used a cell fusion model (named heterokaryon) to track the changes in gene expression that occur as a pluripotent stem cell differentiates to ultimately become an endothelial cell. In this model, mouse embryonic stem cells (ESCs) are fused to human endothelial cells. Over time the human endothelial cells drive gene expression in the ESCs toward that of endothelial cells. Wong, Matrone et al. discovered changes in gene expression in many genes that have not previously been described as involved in the differentiation of endothelial cells. When one of these genes – named Pou3f2 – was inactivated in ESCs, they could not be differentiated into endothelial cells. The absence of Pou3f2 also drastically impaired how blood vessels developed in zebrafish embryos. Thus the heterokaryon model can generate important information regarding the dynamic changes in gene expression that occur as a pluripotent cell differentiates to become an endothelial cell. This model may also be useful for discovering other genes that control the differentiation of other cell types. DOI: http://dx.doi.org/10.7554/eLife.23588.002
- Published
- 2017
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