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Hudgens JW, Gallagher ES, Karageorgos I, Anderson KW, Filliben JJ, Huang RYC, Chen GD, Bou-Assaf GM, Espada A, Chalmers MJ, Harguindey E, Zhang HM, Walters BT, Zhang J, Venable J, Steckler C, Park I, Brock A, Lu XJ, Pandey R, Chandramohan A, Anand GS, Nirudodhi SN, Sperry JB, Rouse JC, Carroll JA, Rand KD, Leurs U, Weis DD, Al-Naqshabandi MA, Hageman TS, Deredge D, Wintrode PL, Papanastasiou M, Lambris JD, Li S, Urata S
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Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb

ANALYTICAL CHEMISTRY 2019 JUN 4; 91(11):7336-7345
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A) from 15 laboratories. Laboratories reported similar to 89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (similar to 78 900 centroids), giving similar to 100% coverage, and similar to 10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of < s(Lab)> <= (0.15 +/- 0.01) Da (1 sigma((x) over bar)). All laboratories achieved < s(Lab)> <= 0.4 Da. For immersions of protein at T-HDx = (3.6 to 25) degrees C and for D2O exchange times of t(HDX) = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is sigma(Laboratories)(reproducibility15) (t(HDX)) = (9.0 +/- 0.9) % (1 sigma). A nine laboratory cohort that immersed samples at T-HDX = 25 degrees C exhibited reproducibility of sigma(25C cohort)(reproducibility) (t(HDX)) = (6.5 +/- 0.6) % for back-exchange corrected, deuterium uptake measurements.
Litke JL, Jaffrey SR
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Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

NATURE BIOTECHNOLOGY 2019 JUN; 37(6):667-675
RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
Etoc F, Brivanlou A
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A boost towards totipotency for stem cells

NATURE CELL BIOLOGY 2019 JUN; 21(6):671-673
Zhou J, Park CY, Theesfeld CL, Wong AK, Yuan Y, Scheckel C, Fak JJ, Funk J, Yao K, Tajima Y, Packer A, Darnell RB, Troyanskaya OG
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Whole-genome deep-learning analysis identifies contribution of noncoding mutations to autism risk

NATURE GENETICS 2019 JUN; 51(6):973-980
We address the challenge of detecting the contribution of noncoding mutations to disease with a deep-learning-based framework that predicts the specific regulatory effects and the deleterious impact of genetic variants. Applying this framework to 1,790 autism spectrum disorder (ASD) simplex families reveals a role in disease for noncoding mutations-ASD probands harbor both transcriptional- and post-transcriptional-regulation-disrupting de novo mutations of significantly higher functional impact than those in unaffected siblings. Further analysis suggests involvement of noncoding mutations in synaptic transmission and neuronal development and, taken together with previous studies, reveals a convergent genetic landscape of coding and noncoding mutations in ASD. We demonstrate that sequences carrying prioritized mutations identified in probands possess allele-specific regulatory activity, and we highlight a link between noncoding mutations and heterogeneity in the IQ of ASD probands. Our predictive genomics framework illuminates the role of noncoding mutations in ASD and prioritizes mutations with high impact for further study, and is broadly applicable to complex human diseases.
Stoeckle M
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The eDNA Revolution

SEA TECHNOLOGY 2019 JUN; 60(6):7-7
Hartweger H, McGuire AT, Horning M, Taylor JJ, Dosenovic P, Yost D, Gazumyan A, Seaman MS, Stamatatos L, Jankovic M, Nussenzweig MC
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HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells

JOURNAL OF EXPERIMENTAL MEDICINE 2019 JUN; 216(6):1301-1310
A small number of HIV-1-infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1-3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.
Xie W, Lama L, Adura C, Tomita D, Glickman JF, Tuschl T, Patel DJ
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Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2019 JUN 11; 116(24):11946-11955
The cyclic GMP-AMP synthase (cGAS)-cGAMP-STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of autoimmune diseases. Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGAS(CD)-DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGAS(CD)-DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS-DNA interface. We also report the crystal structure of the h-cGAS(CD)-DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGAS(CD), thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site-binding inhibitors.
Valverde DP, Yu SL, Boggavarapu V, Kumar N, Lees JA, Walz T, Reinisch KM, Melia TJ
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ATG2 transports lipids to promote autophagosome biogenesis

JOURNAL OF CELL BIOLOGY 2019 JUN; 218(6):1787-1798
During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER-autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.
Minuesa G, Albanese SK, Xie W, Kazansky Y, Worroll D, Chow A, Schurer A, Park SM, Rotsides CZ, Taggart J, Rizzi A, Naden LN, Chou T, Gourkanti S, Cappel D, Passarelli MC, Fairchild L, Adura C, Glickman JF, Schulman J, Famulare C, Patel M, Eibl JK, Ross GM, Bhattacharya S, Tan DS, Leslie CS, Beuming T, Patel DJ, Goldgur Y, Chodera JD, Kharas MG
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Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia

NATURE COMMUNICATIONS 2019 JUN 19; 10(?):? Article 2691
The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.
Xu M, Kolding J, Cohen JE
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Sequential analysis and design of fixed-precision sampling of Lake Kariba fishes using Taylor's power law

CANADIAN JOURNAL OF FISHERIES AND AQUATIC SCIENCES 2019 JUN; 76(6):904-917
Taylor's power law (TPL), which states that the variance of abundance is a power function of mean abundance, has been used to design sampling of agricultural pests and fish species. We show that TPL holds for means and variances of abundance of accumulated fish samples in the fished and unfished areas separately of Lake Kariba (between Zambia and Zimbabwe), measuring abundance indices by number and weight separately. We use TPL parameters estimated from sequentially accumulated samples to update a stopping line of fixed precision 0.1 after each new sample from a sampling day. In these Lake Kariba data, depending on the sampling area and abundance measure, our updated stopping-line method requires 21% to 41% of the number of sampling days and 19% to 40% of the number of samples that are planned a priori and performed under systematic sampling. Our novel method yields mean abundance estimates similar to those from systematic sampling and provides a conservative approach to reaching a fixed sampling precision level with reduced sampling labor and time. Using mixed-effect modeling for cumulative means and variances with either number or weight from both fished and unfished areas, we find that fishing increases the slope of TPL. This study provides the conceptual framework and an empirical case study for implementing a sequential sampling method for fish assemblages of an inland lake. The possible limitations and applications of our method for sampling in other environments are discussed.