23 results on '"Stefan Roberts"'
Search Results
2. Complex microparticle architectures from stimuli-responsive intrinsically disordered proteins
- Author
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Stefan Roberts, Vincent Miao, Simone Costa, Joseph Simon, Garrett Kelly, Tejank Shah, Stefan Zauscher, and Ashutosh Chilkoti
- Subjects
Science - Abstract
The production of microparticles with complex geometries for biotechnological use historically requires sophisticated fabrication techniques. Here, the authors create complex particle geometries by exploiting the metastable region of the phase diagram of thermally responsive intrinsically disordered proteins within microdroplets.
- Published
- 2020
- Full Text
- View/download PDF
3. Inducible Fibril Formation of Silk–Elastin Diblocks
- Author
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Lione Willems, Stefan Roberts, Isaac Weitzhandler, Ashutosh Chilkoti, Enrico Mastrobattista, John van der Oost, and Renko de Vries
- Subjects
Chemistry ,QD1-999 - Published
- 2019
- Full Text
- View/download PDF
4. Nanoscopic Dynamics Dictate the Phase Separation Behavior of Intrinsically Disordered Proteins
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Dariush Hinderberger, Johannes Hunold, Katharina Laaß, Felipe Garcia Quiroz, Stefan Roberts, and Ashutosh Chilkoti
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Organelles ,Materials science ,Polymers and Plastics ,Polymers ,Dynamics (mechanics) ,Water ,Bioengineering ,Intrinsically disordered proteins ,Intrinsically Disordered Proteins ,Biomaterials ,Chemical physics ,Materials Chemistry ,Amino Acids ,Nanoscopic scale - Abstract
Many intrinsically disordered proteins (IDPs) in nature may undergo liquid-liquid phase separation to assemble membraneless organelles with varied liquid-like properties and stability/dynamics. While solubility changes underlie these properties, little is known about hydration dynamics in phase-separating IDPs. Here, by studying IDP polymers of similar composition but distinct liquid-like dynamics and stability upon separation, namely, thermal hysteresis, we probe at a nanoscopic level hydration/dehydration dynamics in IDPs as they reversibly switch between phase separation states. Using continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we observe distinct backbone and amino acid side-chain hydration dynamics in these IDPs. This nanoscopic view reveals that side-chain rehydration creates a dynamic water shield around the main-chain backbone that effectively and counterintuitively prevents water penetration and governs IDP solubility. We find that the strength of this superficial water shell is a sequence feature of IDPs that encodes for the stability of their phase-separated assemblies. Our findings expose and offer an initial understanding of how the complexity of nanoscopic water-IDP interactions dictate their rich phase separation behavior.
- Published
- 2021
5. Versatile biomanufacturing through stimulus-responsive cell–material feedback
- Author
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Anna J. Lee, Lingchong You, Zihe Liu, Xi Zhang, Shuqiang Huang, Stefan Roberts, Ashutosh Chilkoti, Tatyana A. Sysoeva, Zhuojun Dai, Michael Dzuricky, and Xiaoyu Yang
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0303 health sciences ,Bacterial lysis ,Lysis ,Cell material ,Stimuli responsive ,business.industry ,Functional protein ,Chemistry ,030302 biochemistry & molecular biology ,Bioengineering ,Nanotechnology ,Cell Biology ,Modular design ,Protein–protein interaction ,03 medical and health sciences ,Bioreactors ,Bacterial Proteins ,Gene Expression Regulation ,Escherichia coli ,Biomanufacturing ,Genetic Engineering ,business ,Molecular Biology ,Plasmids ,030304 developmental biology - Abstract
Small-scale production of biologics has great potential for enhancing the accessibility of biomanufacturing. By exploiting cell-material feedback, we have designed a concise platform to achieve versatile production, analysis and purification of diverse proteins and protein complexes. The core of our technology is a microbial swarmbot, which consists of a stimulus-sensitive polymeric microcapsule encapsulating engineered bacteria. By sensing the confinement, the bacteria undergo programmed partial lysis at a high local density. Conversely, the encapsulating material shrinks responding to the changing chemical environment caused by cell growth, squeezing out the protein products released by bacterial lysis. This platform is then integrated with downstream modules to enable quantification of enzymatic kinetics, purification of diverse proteins, quantitative control of protein interactions and assembly of functional protein complexes and multienzyme metabolic pathways. Our work demonstrates the use of the cell-material feedback to engineer a modular and flexible platform with sophisticated yet well-defined programmed functions.
- Published
- 2019
6. Advances in Understanding Stimulus-Responsive Phase Behavior of Intrinsically Disordered Protein Polymers
- Author
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Kiersten M. Ruff, Ashutosh Chilkoti, Stefan Roberts, and Rohit V. Pappu
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Models, Molecular ,Repetitive Sequences, Amino Acid ,0301 basic medicine ,Phase transition ,Stimuli responsive ,Polymers ,Protein Conformation ,Hydrostatic pressure ,Bioengineering ,Sequence (biology) ,Phase Transition ,03 medical and health sciences ,Structural Biology ,Phase (matter) ,Hydrostatic Pressure ,Molecular Biology ,Peptide sequence ,Organelles ,chemistry.chemical_classification ,Temperature ,Polymer ,Hydrogen-Ion Concentration ,Amino acid ,Intrinsically Disordered Proteins ,030104 developmental biology ,chemistry ,Biophysics - Abstract
Proteins and synthetic polymers can undergo phase transitions in response to changes to intensive solution parameters such as temperature, proton chemical potentials (pH), and hydrostatic pressure. For proteins and protein-based polymers, the information required for stimulus-responsive phase transitions is encoded in their amino acid sequence. Here, we review some of the key physical principles that govern the phase transitions of archetypal intrinsically disordered protein polymers (IDPPs). These are disordered proteins with repetitive amino acid sequences. Advances in recombinant technologies have enabled the design and synthesis of protein sequences of a variety of sequence complexities and lengths. We summarize insights that have been gleaned from the design and characterization of IDPPs that undergo thermo-responsive phase transitions and build on these insights to present a general framework for IDPPs with pH and pressure responsive phase behavior. In doing so, we connect the stimulus-responsive phase behavior of IDPPs with repetitive sequences to the coil-to-globule transitions that these sequences undergo at the single-chain level in response to changes in stimuli. The proposed framework and ongoing studies of stimulus-responsive phase behavior of designed IDPPs have direct implications in bioengineering, where designing sequences with bespoke material properties broadens the spectrum of applications, and in biology and medicine for understanding the sequence-specific driving forces for the formation of protein-based membraneless organelles as well as biological matrices that act as scaffolds for cells and mediators of cell-to-cell communication.
- Published
- 2018
7. Intrinsically disordered proteins access a range of hysteretic phase separation behaviors
- Author
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Michael Dzuricky, Patrick Weber, Nan Li, Felipe Garcia Quiroz, Isaac Weitzhandler, Ashutosh Chilkoti, Yaroslava G. Yingling, and Stefan Roberts
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Phase transition ,Materials science ,Proline ,Non-equilibrium thermodynamics ,Molecular Dynamics Simulation ,010402 general chemistry ,Intrinsically disordered proteins ,01 natural sciences ,Lower critical solution temperature ,Phase Transition ,03 medical and health sciences ,Molecular dynamics ,Rare Diseases ,Upper critical solution temperature ,Phase (matter) ,Antifreeze Proteins ,Urea ,Amino Acids ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Circular Dichroism ,Temperature ,Hydrogen Bonding ,0104 chemical sciences ,Intrinsically Disordered Proteins ,Hysteresis ,Chemical physics ,Nanoparticles ,Hydrophobic and Hydrophilic Interactions - Abstract
The phase separation behavior of intrinsically disordered proteins (IDPs) is thought of as analogous to that of polymers that undergo equilibrium lower or upper critical solution temperature (LCST and UCST, respectively) phase transition. This view, however, ignores possible nonequilibrium properties of protein assemblies. Here, by studying IDP polymers (IDPPs) composed of repeat motifs that encode LCST or UCST phase behavior, we discovered that IDPs can access a wide spectrum of nonequilibrium, hysteretic phase behaviors. Experimentally and through simulations, we show that hysteresis in IDPPs is tunable and that it emerges through increasingly stable interchain interactions in the insoluble phase. To explore the utility of hysteretic IDPPs, we engineer self-assembling nanostructures with tunable stability. These findings shine light on the rich phase separation behavior of IDPs and illustrate hysteresis as a design parameter to program nonequilibrium phase behavior in self-assembling materials.
- Published
- 2019
8. Injectable tissue integrating networks from recombinant polypeptides with tunable order
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Ashutosh Chilkoti, Terrence G. Oas, Stefan Roberts, Andrew Hunt, Tyler S. Harmon, Vincent N. Miao, Yi Wen, Rohit V. Pappu, Jeffrey L. Schaal, Joel H. Collier, and Kan Jonathan Li
- Subjects
0301 basic medicine ,Materials science ,02 engineering and technology ,Viscoelasticity ,Article ,Injections ,law.invention ,03 medical and health sciences ,Tissue engineering ,law ,General Materials Science ,Amino Acid Sequence ,Nanoscopic scale ,Minimal inflammation ,Quantitative Biology::Biomolecules ,Viscosity ,Functional protein ,Mechanical Engineering ,Temperature ,General Chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Elasticity ,Recombinant Proteins ,Porous scaffold ,Elastin ,030104 developmental biology ,Mechanics of Materials ,Chemical physics ,Structural stability ,Recombinant DNA ,Peptides ,0210 nano-technology ,Porosity - Abstract
Emergent properties of natural biomaterials result from the collective effects of nanoscale interactions among ordered and disordered domains. Here, using recombinant sequence design, we have created a set of partially ordered polypeptides to study emergent hierarchical structures by precisely encoding nanoscale order-disorder interactions. These materials, which combine the stimuli-responsiveness of disordered elastin-like polypeptides and the structural stability of polyalanine helices, are thermally responsive with tunable thermal hysteresis and the ability to reversibly form porous, viscoelastic networks above threshold temperatures. Through coarse-grain simulations, we show that hysteresis arises from physical crosslinking due to mesoscale phase separation of ordered and disordered domains. On injection of partially ordered polypeptides designed to transition at body temperature, they form stable, porous scaffolds that rapidly integrate into surrounding tissue with minimal inflammation and a high degree of vascularization. Sequence-level modulation of structural order and disorder is an untapped principle for the design of functional protein-based biomaterials.
- Published
- 2018
9. Engineering the Architecture of Elastin‐Like Polypeptides: From Unimers to Hierarchical Self‐Assembly
- Author
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Nadia Kirmani, Stefan Roberts, Soumen Saha, Ashutosh Chilkoti, and Samagya Banskota
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Pharmacology ,chemistry.chemical_classification ,Materials science ,Biocompatibility ,Tropoelastin ,biology ,Biochemistry (medical) ,Supramolecular chemistry ,Pharmaceutical Science ,Medicine (miscellaneous) ,Sequence (biology) ,Nanotechnology ,Polymer ,Article ,chemistry ,Nanofiber ,biology.protein ,Pharmacology (medical) ,Self-assembly ,Elastin ,Genetics (clinical) - Abstract
Well-defined tunable nanostructures formed through the hierarchical self-assembly of peptide building blocks have drawn significant attention due to their potential applications in biomedical science. Artificial protein polymers derived from elastin-like polypeptides (ELPs), which are based on the repeating sequence of tropoelastin (the water-soluble precursor to elastin), provide a promising platform for creating nanostructures due to their biocompatibility, ease of synthesis, and customizable architecture. By designing the sequence and composition of ELPs at the gene level, their physicochemical properties can be controlled to a degree that is unmatched by synthetic polymers. A variety of ELP-based nanostructures are designed, inspired by the self-assembly of elastin and other proteins in biological systems. The choice of building blocks determines not only the physical properties of the nanostructures, but also their self-assembly into architectures ranging from spherical micelles to elongated nanofibers. This review focuses on the molecular determinants of ELP and ELP-hybrid self-assembly and formation of spherical, rod-like, worm-like, fibrillar, and vesicle architectures. A brief discussion of the potential biomedical applications of these supramolecular assemblies is also included.
- Published
- 2020
10. Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides
- Author
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Ashutosh Chilkoti, Nan Li, Stefan Roberts, Yaroslava G. Yingling, and Felipe Garcia Quiroz
- Subjects
Circular dichroism ,Phase transition ,Polymers and Plastics ,Amino Acid Motifs ,Bioengineering ,02 engineering and technology ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Lower critical solution temperature ,Phase Transition ,Biomaterials ,Molecular dynamics ,Protein Domains ,Phase (matter) ,Materials Chemistry ,Protein secondary structure ,Hydrogen bond ,Chemistry ,Transition temperature ,Temperature ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Elastin ,Crystallography ,0210 nano-technology - Abstract
Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature ( Tt). However, poly(VPGVG) resolubilizes upon cooling below its Tt, whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the Tt. The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, especially valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above Tt, where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the observed thermal hysteresis for poly(VGPVG).
- Published
- 2018
11. Design of Sequence-Specific Polymers by Genetic Engineering
- Author
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Kelli M. Luginbuhl, Davoud Mozhdehi, Ashutosh Chilkoti, and Stefan Roberts
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chemistry.chemical_classification ,chemistry ,Posttranslational modification ,Polymer ,Computational biology ,Sequence (medicine) - Published
- 2017
12. Fusion of fibroblast growth factor 21 to a thermally responsive biopolymer forms an injectable depot with sustained anti-diabetic action
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Ashutosh Chilkoti, Caslin A. Gilroy, and Stefan Roberts
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0301 basic medicine ,Male ,Materials science ,FGF21 ,Lysis ,Hot Temperature ,Pharmaceutical Science ,Mice, Obese ,Peptide ,02 engineering and technology ,Pharmacology ,Article ,Body Temperature ,Diabetes Mellitus, Experimental ,03 medical and health sciences ,Mice ,Random Allocation ,Biopolymers ,Drug Delivery Systems ,In vivo ,Animals ,Humans ,Hypoglycemic Agents ,chemistry.chemical_classification ,Coacervate ,Dose-Response Relationship, Drug ,3T3 Cells ,021001 nanoscience & nanotechnology ,Controlled release ,Fusion protein ,Elastin ,Fibroblast Growth Factors ,030104 developmental biology ,HEK293 Cells ,chemistry ,Delayed-Action Preparations ,Drug delivery ,Biophysics ,0210 nano-technology - Abstract
Fibroblast growth factor 21 (FGF21) is under investigation as a type 2 diabetes protein drug, but its efficacy is impeded by rapid in vivo clearance and by costly production methods. To improve the protein's therapeutic utility, we recombinantly expressed FGF21 as a fusion with an elastin-like polypeptide (ELP), a peptide polymer that exhibits reversible thermal phase behavior. Below a critical temperature, ELPs exist as miscible unimers, while above, they associate into a coacervate. The thermal responsiveness of ELPs is retained upon fusion to proteins, which has notable consequences for the production and in vivo delivery of FGF21. First, the ELP acts as a solubility enhancer during E. coli expression, yielding active fusion protein from the soluble cell lysate fraction and eliminating the protein refolding steps that are required for purification of FGF21 from inclusion bodies. Second, the ELP's phase transition behavior is exploited for facile chromatography-free purification of the ELP-FGF21 fusion. Third, the composition and molecular weight of the ELP are designed such that the ELP-FGF21 fusion undergoes a phase transition triggered solely by body heat, resulting in an immiscible viscous phase upon subcutaneous (s.c.) injection and thereby creating an injectable depot. Indeed, a single s.c. injection of ELP-FGF21 affords up to five days of sustained glycemic control in ob/ob mice. The ELP fusion partner massively streamlines production and purification of FGF21, while providing a controlled release method for delivery that reduces the frequency of injection, thereby enhancing the pharmacological properties of FGF21 as a protein drug to treat metabolic disease.
- Published
- 2017
13. Elastin-like polypeptides as models of intrinsically disordered proteins
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Michael Dzuricky, Stefan Roberts, and Ashutosh Chilkoti
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Models, Molecular ,Biopolymer ,Protein Conformation ,Biophysics ,engineering.material ,Intrinsically disordered proteins ,Biochemistry ,Pentapeptide repeat ,Article ,Protein structure ,Structural Biology ,Protein purification ,Genetics ,Humans ,Transition Temperature ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Phase transition ,Tropoelastin ,biology ,Chemistry ,Cell Biology ,Protein engineering ,Elastin ,Intrinsically Disordered Proteins ,Models, Chemical ,Tandem repeat ,Intrinsically disordered protein ,engineering ,biology.protein ,Elastin-like polypeptide ,Peptides ,Hydrophobic and Hydrophilic Interactions - Abstract
Elastin-like polypeptides (ELPs) are a class of stimuli-responsive biopolymers inspired by the intrinsically disordered domains of tropoelastin that are composed of repeats of the VPGXG pentapeptide motif, where X is a “guest residue”. They undergo a reversible, thermally triggered lower critical solution temperature (LCST) phase transition, which has been utilized for a variety of applications including protein purification, affinity capture, immunoassays, and drug delivery. ELPs have been extensively studied as protein polymers and as biomaterials, but their relationship to other disordered proteins has heretofore not been established. The biophysical properties of ELPs that lend them their unique material behavior are similar to the properties of many intrinsically disordered proteins (IDP). Their low sequence complexity, phase behavior, and elastic properties make them an interesting “minimal” artificial IDP, and the study of ELPs can hence provide insights into the behavior of other more complex IDPs. Motivated by this emerging realization of the similarities between ELPs and IDPs, this review discusses the biophysical properties of ELPs, their biomedical utility, and their relationship to other disordered polypeptide sequences.
- Published
- 2015
14. 2.5 Elastin-Like Polypeptides ☆
- Author
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Stefan Roberts, Felipe Garcia Quiroz, Ashutosh Chilkoti, Michael Dzuricky, Jeffrey L. Schaal, Simone A. Costa, and Joseph R. Simon
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Materials science ,integumentary system ,Tropoelastin ,biology ,Biocompatibility ,Nanotechnology ,02 engineering and technology ,engineering.material ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Smart polymer ,0104 chemical sciences ,Tissue engineering ,Drug delivery ,Self-healing hydrogels ,biology.protein ,engineering ,Biopolymer ,0210 nano-technology ,Elastin ,Biomedical engineering - Abstract
Elastin-like polypeptides are a family of stimuli-responsive biopolymers that have been extensively studied for biomedical applications. Derived from tropoelastin, they mimic many of elastin’s remarkable material properties, yet can be designed with a molecular precision surpassing synthetic polymers. Their biocompatibility and stimuli-responsive phase behavior have allowed them to be used in a variety of biomedical applications ranging from self-assembling, drug-loaded nanoparticles to injectable cell scaffolds and drug depots.
- Published
- 2017
15. Author Correction: Injectable tissue integrating networks from recombinant polypeptides with tunable order
- Author
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Andrew Hunt, Terrence G. Oas, Tyler S. Harmon, Jeffrey L. Schaal, Joel H. Collier, Rohit V. Pappu, Kan Jonathan Li, Vincent N. Miao, Ashutosh Chilkoti, Stefan Roberts, and Yi Wen
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0301 basic medicine ,Information retrieval ,Computer science ,Mechanical Engineering ,Published Erratum ,02 engineering and technology ,General Chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,03 medical and health sciences ,030104 developmental biology ,Mechanics of Materials ,Order (business) ,General Materials Science ,0210 nano-technology - Abstract
In the version of this Article originally published, one of the authors' names was incorrectly given as Jeffery Schaal; it should have been Jeffrey L. Schaal. This has been corrected in all versions of the Article.
- Published
- 2018
16. Diagnostic value of routine chest radiography in febrile, neutropenic children for early detection of pneumonia and mould infections
- Author
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Gabriela Maron, Randall T. Hayden, Jerry L. Shenep, Nowell R. York, Bassem I. Razzouk, Stefan Roberts, Gregory M. Wells, Nilay M. Gandhi, and Sue C. Kaste
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Adult ,Male ,medicine.medical_specialty ,Pediatrics ,Neutropenia ,Adolescent ,Fever ,Radiography ,Early detection ,Young Adult ,medicine ,Humans ,Young adult ,Child ,Intensive care medicine ,Lung ,Medical Audit ,medicine.diagnostic_test ,business.industry ,Fungi ,Infant ,Pneumonia ,medicine.disease ,respiratory tract diseases ,Early Diagnosis ,medicine.anatomical_structure ,Mycoses ,Oncology ,Child, Preschool ,Female ,Occult pneumonia ,business ,Chest radiograph - Abstract
Despite recent studies failing to demonstrate the value of routine chest radiography (CXR) in the initial evaluation of the febrile neutropenic patient with cancer, this screening test is advocated by some experts. We evaluated the benefits of CXR for early diagnosis of pulmonary infection at St. Jude Children’s Research Hospital (SJCRH) with emphasis on early recognition of mould infections. We reviewed the courses of 200 consecutive febrile neutropenic pediatric patients to determine if routine CXR at initial evaluation was useful in the identification of clinically occult pneumonia. We also reviewed all cases of proven or probable mould infections from the opening of SJCRH in 1962 until 1998 when routine CXR was no longer practiced in our institution to identify cases that were first recognized by routine CXR. Of 200 febrile neutropenic patients, pulmonary abnormalities consistent with pneumonia were detected by routine CXR in only five patients without pulmonary signs or symptoms. In only one case was a change in management considered. Of the 70 patients with pulmonary mould infection identified from 1962 to 1998, routine CXR was performed in 45 patients at the onset of a febrile, neutropenic episode in which a mould infection was diagnosed. Routine CXR was pivotal in the recognition of the mould infection in only two cases over this 36-year period. CXR is warranted in the evaluation of the newly febrile neutropenic pediatric oncology patient only when respiratory signs or symptoms are present.
- Published
- 2012
17. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia
- Author
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Stephen P. Hunger, Elisa Laurenti, Pankaj Gupta, Linda Holmfeldt, Shann Ching Chen, David Zhao, Cheng Cheng, William E. Evans, Michael Rusch, Daniel Alford, Sheila A. Shurtleff, Faiyaz Notta, Elaine Coustan-Smith, David J. Dooling, Debbie Payne-Turner, John C. Obenauer, Xiang Chen, Jinghui Zhang, Michelle L. Hermiston, Lei Wei, Daniel J. McGoldrick, Mignon L. Loh, Deqing Pei, Charles Lu, Michael I. Barbato, Kathryn G. Roberts, Jing Ma, Kimberley P. Dunsmore, Kolja Eppert, Meenakshi Devidas, Elaine R. Mardis, Kiran Chand Bobba, Gang Wu, Chris Harris, Susan L. Heatley, James R. Downing, Guangchun Song, Sergei Doulatov, Jared Becksfort, Susana C. Raimondi, Richard K. Wilson, Jianmin Wang, Lucinda Fulton, Kerri Ochoa, Brent L. Wood, Xin Hong, Stanley Pounds, Stephen Espy, Matthew Parker, Robert Huether, Giuseppe Basso, Stuart S. Winter, Maria Kleppe, Stefan Roberts, Richard W. Kriwacki, Li Ding, Ching-Hon Pui, Anatoly Ulyanov, Timothy J. Ley, Jan Cools, J. Racquel Collins-Underwood, John E. Dick, Kristin A. Shimano, Dario Campana, Kimberly J. Johnson, Charles G. Mullighan, Robert S. Fulton, Clayton W. Naeve, and John Easton
- Subjects
Neuroblastoma RAS viral oncogene homolog ,Myeloid ,DNA Copy Number Variations ,T-Lymphocytes ,Molecular Sequence Data ,Biology ,Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ,medicine.disease_cause ,Article ,Translocation, Genetic ,Histones ,hemic and lymphatic diseases ,medicine ,Humans ,Genetic Predisposition to Disease ,Age of Onset ,Child ,EP300 ,Interleukin-7 receptor ,Janus Kinases ,Receptors, Interleukin-7 ,Multidisciplinary ,Genome, Human ,Stem Cells ,Genomics ,Sequence Analysis, DNA ,medicine.disease ,Hematopoiesis ,Leukemia, Myeloid, Acute ,Reelin Protein ,DNM2 ,Haematopoiesis ,Leukemia ,Genes, ras ,medicine.anatomical_structure ,Mutation ,Immunology ,Cancer research ,KRAS ,Signal Transduction - Abstract
Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.
- Published
- 2012
18. Nanocrystal‐Based Time–Temperature Indicators
- Author
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Jie Zeng, Stefan Roberts, and Younan Xia
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Silver ,medicine.diagnostic_test ,Chemistry ,Organic Chemistry ,Temperature ,Nanoparticle ,Nanotechnology ,General Chemistry ,medicine.disease_cause ,Catalysis ,Nanocrystal ,Spectrophotometry ,medicine ,Nanoparticles ,Thermodynamics ,Indicators and Reagents ,Spectrophotometry, Ultraviolet ,Algorithms ,Ultraviolet - Published
- 2010
19. A Model for Hysteresis Observed in Phase Transitions of Thermally Responsive Intrinsically Disordered Protein Polymers
- Author
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Ashutosh Chilkoti, Tyler S. Harmon, Stefan Roberts, and Rohit V. Pappu
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chemistry.chemical_classification ,Phase transition ,Materials science ,010304 chemical physics ,Band gap ,Biophysics ,02 engineering and technology ,Polymer ,021001 nanoscience & nanotechnology ,01 natural sciences ,Lower critical solution temperature ,Crystallography ,Hysteresis ,chemistry ,Chemical physics ,Phase (matter) ,0103 physical sciences ,Phenomenological model ,0210 nano-technology ,Dispersion (chemistry) - Abstract
A new class of thermally responsive protein based block co-polymers shows tunable hysteresis in their thermal transitions. The sequences encompass intrinsically disordered regions comprising of repeats of Elastin-Like Polypeptides (ELPs). These are interspersed with alpha-helical polyalanine domains. The lower critical solution temperatures (LCSTs) measured along the heating and cooling arms are tunable and can be non-overlapping. The number and type of alanine-rich blocks determine the extent of hysteresis. We have developed a phenomenological model that reproduces the experimentally observed tunable hysteresis for block copolymeric sequences. This requires an imbalance between the strengths of homotypic interactions between alanine-rich regions and ELP repeats. Additionally, the ELPs and alanine-rich regions have to be immiscible with one another. These features engender micro-phase separation whereby the block copolymers form spherical clusters comprising of alanine-rich cores and ELP coronas. Upon raising the temperature above the LCST, the clusters are drawn to one another by favorable interactions among ELPs and these clusters further network via domain swapping of alanine-rich regions. Lowering the temperature below the LCST leads to dispersion of the clusters by weakening of homotypic interactions between ELPs. However, the domain swapped states persist and this maintains the physical networking of clusters thus giving rise to hysteresis. Domain swapping and the persistence of this state below the LCST are governed by the energy gap between the homotypic interactions of ELPs vis-a-vis alanine-rich regions. Our findings have direct bearing on the de novo design of responsive materials based on IDPs, where tunable hysteresis can be used to encode memory effects, and for understanding the complexities of sequence-encoded phase behavior of archetypal low complexity disordered proteins.
- Published
- 2017
20. CREST maps somatic structural variation in cancer genomes with base-pair resolution
- Author
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Clayton W. Naeve, Jing Ma, Susan L. Heatley, Chris Harris, Michael Rusch, John C. Obenauer, John Easton, Lei Wei, Ken Chen, Linda Holmfeldt, Charles G. Mullighan, Li Ding, Elaine R. Mardis, Debbie Payne-Turner, David Zhao, James R. Downing, Jinghui Zhang, Jianmin Wang, Stefan Roberts, Xian Fan, and Richard K. Wilson
- Subjects
Cancer genome sequencing ,Base Pair Mismatch ,Genomics ,Computational biology ,Biology ,Polymorphism, Single Nucleotide ,Biochemistry ,Genome ,Article ,Deep sequencing ,Structural variation ,Neoplasms ,Animals ,Humans ,Molecular Biology ,Exome sequencing ,Genetics ,DNA, Neoplasm ,Sequence Analysis, DNA ,Cell Biology ,Crest ,Sequence Alignment ,Algorithms ,Software ,Biotechnology ,Reference genome - Abstract
We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.
- Published
- 2011
21. Two molecular subgroups of Wilms' tumors with or without WT1 mutations
- Author
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Valérie, Schumacher, Stefanie, Schuhen, Sandra, Sonner, Angela, Weirich, Ivo, Leuschner, Dieter, Harms, Jonathan, Licht, Stefan, Roberts, and Brigitte, Royer-Pokora
- Subjects
Collagen Type IV ,Mice ,COS Cells ,Mutation ,NIH 3T3 Cells ,Animals ,Humans ,Laminin ,WT1 Proteins ,Wilms Tumor ,Kidney Neoplasms - Abstract
Wilms' tumors (WTs) exhibit more than one pattern of differentiation, each of which is associated with distinctive clinical features and treatment responses. Mutations in the WT1 gene are found predominantly in WTs with stromal histology. To better understand the biological and clinical features in different WTs, we have analyzed WTs with and without WT1 mutations for a set of parameters.Twenty-two new WTs were analyzed for WT1 mutations by PCR single-strand conformational polymorphism. Five tumors with WT1 mutations and six tumors without WT1 mutations were studied for the presence of WT1 transcripts and protein as well as for the expression of differentiation markers.Two new WT1 mutations were identified in stromal-predominant tumors, and none were identified in the other histological subtypes. Tumors with WT1 mutations expressed mutant messages, cytoplasmic truncated WT1 proteins, and muscle markers. In contrast, blastemal-predominant tumors without mutations showed nuclear WT1 protein staining. Both tumor types were positive for markers of early-induced mesenchyme and one marker of uninduced mesenchyme, but blastemal-predominant tumors also expressed cytokeratin, suggesting that these are further along the epithelial differentiation pathway.Our data show that the two-hit inactivation of WT1 is operative in stromal-predominant WTs. Cells without functional nuclear WT1 protein start a faulty differentiation program. In contrast, blastemal-predominant tumors express wild-type WT1 and show early signs of epithelialization. The extensive rhabdomyomatous differentiation and the presence of WT1 mutations may be used as a diagnostic tool to identify a tumor subtype that seems to respond poorly to chemotherapy. These studies provide a foundation for improvement in tumor classification and ultimately for the development of more individualized tumor treatments.
- Published
- 2003
22. Cover Picture: Nanocrystal‐Based Time–Temperature Indicators (Chem. Eur. J. 42/2010)
- Author
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Jie Zeng, Younan Xia, and Stefan Roberts
- Subjects
Nanocrystal ,Chemistry ,Organic Chemistry ,Nanotechnology ,Cover (algebra) ,General Chemistry ,Catalysis - Published
- 2010
23. Discovery of Novel Recurrent Mutations in Childhood Early T-Cell Precursor Acute Lymphoblastic Leukemia by Whole Genome Sequencing - a Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project
- Author
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Cheng Cheng, Brent L. Wood, Li Ding, Sergei Doulatov, Kiran Chand Bobba, Mignon L. Loh, Stephen P. Hunger, Jing Ma, James R. Downing, Michelle L. Hermiston, Meenakshi Devidas, Elaine R. Mardis, Guangchun Song, Lei Wei, Susan L. Heatley, David Zhao, Shann-Ching Chen, Jianmin Wang, Stefan Roberts, Lucinda Fulton, Stanley Pounds, Gang Wu, Elisa Laurenti, Chris Harris, Timothy J. Ley, Daniel Alford, Jan Cools, Susana C. Raimondi, Daniel J. McGoldrick, Maria Kleppe, Debbie Payne-Turner, Dario Campana, Richard K. Wilson, Kimberly P. Dunsmore, Ching-Hon Pui, Kathryn G. Roberts, Xiang Chen, John E. Dick, Clayton W. Naeve, Xin Hong, Kolja Eppert, Michael I. Barbato, Kristin A. Shimano, John Easton, William E. Evans, Michael Rusch, Richard W. Kriwacki, Kimberly J. Johnson, Charles Lu, Charles G. Mullighan, Racquel Collins-Underwood, Stephen Espy, Jared Becksfort, Robert S. Fulton, Robert Huether, David J. Dooling, Anatoly Ulyanov, Linda Holmfeldt, Pankaj Gupta, Sheila A. Shurtleff, Stuart S. Winter, Kerri Ochoa, Deqing Pei, Jinghui Zhang, John C. Obenauer, Faiyaz Notta, Elaine Coustan-Smith, and Giuseppe Basso
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Genetics ,Mutation ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,medicine.disease_cause ,Biochemistry ,Pediatric cancer ,Fusion gene ,Leukemia ,ETV6 ,Acute lymphocytic leukemia ,medicine ,Cancer research ,EP300 ,Exome sequencing - Abstract
Abstract 68 Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown. Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort. We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1). We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations. Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR. Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL. Disclosures: Evans: St. Jude Children's research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity's Board of Directors or advisory committees.
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