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MIT Open Access Articles
Mostrando recursos 1 - 20 de 16,503
Target specificity of the CRISPR-Cas9 system - Wu, Xuebing; Kriz, Andrea J.; Sharp, Phillip A.
The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system.
CRISPR-mediated direct mutation of cancer genes in the mouse liver - Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Cai, Wenxin; Bronson, Roderick; Crowley, Denise G.; Zhang, Feng; Sharp, Phillip A.; Joshi, Nik; Yang, Gillian R.; Anderson, Daniel Griffith; Jacks, Tyler E.
The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten and p53 (also known as TP53 and Trp53), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid...
A Circuitous Route to Noncoding RNA - Wilusz, Jeremy E.; Sharp, Phillip A.
Most genetic information is expressed as, and transacted by, proteins. Yet, less than 2% of the human genome actually codes for proteins, prompting a search for functions for the other 98% of the genome, once considered to be mostly “junk DNA.” Transcription is pervasive, however, and high-throughput sequencing has identified tens of thousands of distinct RNAs generated from the non—protein—coding portion of the genome (1). These so-called noncoding RNAs vary in length, but like protein-coding RNAs, appear to be linear molecules with 5′ and 3′ termini, reflecting the defined start and end points of RNA polymerase on the DNA template....
Optimized E.coli expression strain LOBSTR eliminates common contaminants from His-tag purification - Andersen, Kasper R.; Leksa, Nina C.; Schwartz, Thomas
His-tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His-tag is the co-purification of contaminating histidine-rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (low background strain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging...
An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors - Jonas, Oliver H.; Landry, Heather M.; Fuller, Jason E.; Cima, Michael J.; Langer, Robert; Santini, John T., Jr.; Baselga, Jose; Tepper, Robert I.
Current anticancer chemotherapy relies on a limited set of in vitro or indirect prognostic markers of tumor response to available drugs. A more accurate analysis of drug sensitivity would involve studying tumor response in vivo. To this end, we have developed an implantable device that can perform drug sensitivity testing of several anticancer agents simultaneously inside the living tumor. The device contained reservoirs that released microdoses of single agents or drug combinations into spatially distinct regions of the tumor. The local drug concentrations were chosen to be representative of concentrations achieved during systemic treatment. Local efficacy and drug concentration profiles...
Experimental and Analytical Study of Exponential Power Excursion in Plate-Type Fuel - Sargentini, Lucia; Bucci, Matteo; Su, Guanyu; Buongiorno, Jacopo; McKrell, Thomas J.
This paper presents an investigation of transient heat transfer, which may occur in nuclear reactors with plate-type fuel during a reactivity initiated accident. Analytical solutions of the heat transfer equation were developed to describe the behavior of plate-type fuel and the MIT pool boiling facility during exponential power excursions governed by conduction heat transfer. Experimental results for non-boiling transient heat transfer tests are also presented and compared to the analytical solutions. Infrared thermometry was used to measure the temperature on the heater surface. The test matrix included exponential power escalations with periods τ as short as 5 milliseconds and water...
Thermal Expansion, Heat Capacity, and Thermal Conductivity of Nickel Ferrite (NiFe[subscript 2]O[subscript 4]) - Nelson, Andrew T.; White, Joshua T.; Andersson, David A.; Aguiar, Jeffery A.; McClellan, Kenneth J.; Byler, Darrin D.; Short, Michael P.; Stanek, Christopher R.
Nickel ferrite (NiFe[subscript 2]O[subscript 4]) is a major constituent of the corrosion deposits formed on the exterior of nuclear fuel cladding tubes during operation. NiFe[subscript 2]O[subscript 4] has attracted much recent interest, mainly due to the impact of these deposits, known as CRUD, on the operation of commercial nuclear reactors. Although advances have been made in modeling CRUD nucleation and growth under a wide range of conditions, the thermophysical properties of NiFe[subscript 2]O[subscript 4] at high temperatures have only been approximated, thereby limiting the accuracy of such models. In this study, samples of NiFe[subscript 2]O[subscript 4] were synthesized to provide...
mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase - Zoncu, Roberto; Bar-Peled, Liron; Efeyan, Alejo; Wang, Shuyu; Sancak, Yasemin; Sabatini, David M.; Efeyan, Alejo
The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulated by amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), which promote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. We found that the vacuolar H+–adenosine triphosphatase ATPase (v-ATPase) is necessary for amino acids to activate mTORC1. The v-ATPase engages in extensive amino acid–sensitive interactions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to the lysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acids to regulate the v-ATPase-Ragulator interaction and...
Testing the Substrate-Envelope Hypothesis with Designed Pairs of Compounds - Shen, Yang; Altman, Michael D.; Ali, Akbar; Nalam, Madhavi N. L.; Cao, Hong; Rana, Tariq M.; Schiffer, Celia A.; Tidor, Bruce
Acquired resistance to therapeutic agents is a significant barrier to the development of clinically effective treatments for diseases in which evolution occurs on clinical time scales, frequently arising from target mutations. We previously reported a general strategy to design effective inhibitors for rapidly mutating enzyme targets, which we demonstrated for HIV-1 protease inhibition [Altman et al. J. Am. Chem. Soc. 2008, 130, 6099–6113]. Specifically, we developed a computational inverse design procedure with the added constraint that designed inhibitors bind entirely inside the substrate envelope, a consensus volume occupied by natural substrates. The rationale for the substrate-envelope constraint is that it...
Tyrosine/Cysteine Cluster Sensitizing Human γD-Crystallin to Ultraviolet Radiation-Induced Photoaggregation in Vitro - Schafheimer, Steven Nathaniel; Schey, Kevin; Wang, Zhen; King, Jonathan Alan
Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. HγD-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs....
Single-photon sensitive light-in-fight imaging - Gariepy, Genevieve; Henderson, Robert; Li, Chunyong; Thomson, Robert R.; Buller, Gerald S.; Raskar, Ramesh; Leach, Jonathan; Faccio, Daniele; Heshmat Dehkordi, Barmak; Krstajic, Nikola
The ability to record images with extreme temporal resolution enables a diverse range of applications, such as fluorescence lifetime imaging, time-of-flight depth imaging and characterization of ultrafast processes. Recently, ultrafast imaging schemes have emerged, which require either long acquisition times or raster scanning and have a requirement for sufficient signal that can only be achieved when light is reflected off an object or diffused by a strongly scattering medium. Here we present a demonstration of the potential of single-photon detector arrays for visualization and rapid characterization of events evolving on picosecond time scales. The single-photon sensitivity, temporal resolution and full-field...
Postmortem examination of patient H.M.’s brain based on histological sectioning and digital 3D reconstruction - Annese, Jacopo; Schenker-Ahmed, Natalie M.; Bartsch, Hauke; Maechler, Paul; Sheh, Colleen; Thomas, Natasha; Kayano, Junya; Ghatan, Alexander; Bresler, Noah; Frosch, Matthew P.; Klaming, Ruth; Corkin, Suzanne Hammond
Modern scientific knowledge of how memory functions are organized in the human brain originated from the case of Henry G. Molaison (H.M.), an epileptic patient whose amnesia ensued unexpectedly following a bilateral surgical ablation of medial temporal lobe structures, including the hippocampus. The neuroanatomical extent of the 1953 operation could not be assessed definitively during H.M.’s life. Here we describe the results of a procedure designed to reconstruct a microscopic anatomical model of the whole brain and conduct detailed 3D measurements in the medial temporal lobe region. This approach, combined with cellular-level imaging of stained histological slices, demonstrates a significant...
The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera - Waterhouse, Robert M.
Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and...
Bioinspired micrograting arrays mimicking the reverse color diffraction elements evolved by the butterfly Pierella luna - England, Grant; Kolle, Mathias; Kim, Philseok; Khan, Mughees; Munoz, Philip; Mazur, Eric; Aizenberg, Joanna
Recently, diffraction elements that reverse the color sequence normally observed in planar diffraction gratings have been found in the wing scales of the butterfly Pierella luna. Here, we describe the creation of an artificial photonic material mimicking this reverse color-order diffraction effect. The bioinspired system consists of ordered arrays of vertically oriented microdiffraction gratings. We present a detailed analysis and modeling of the coupling of diffraction resulting from individual structural components and demonstrate its strong dependence on the orientation of the individual miniature gratings. This photonic material could provide a basis for novel developments in biosensing, anticounterfeiting, and efficient light...
Search for anomalous production of prompt same-sign lepton pairs and pair-produced doubly charged Higgs bosons with √s = 8 TeV pp collisions using the ATLAS detector - Taylor, Frank E.
A low-background inclusive search for new physics in events with same-sign dileptons is presented. The search uses proton-proton collisions corresponding to 20.3 fb[superscript −1] of integrated luminosity taken in 2012 at a centre-of-mass energy of 8 TeV with the ATLAS detector at the LHC. Pairs of isolated leptons with the same electric charge and large transverse momenta of the type e [superscript ±] e [superscript ±], e [superscript ±] μ [superscript ±], and μ [superscript ±] μ [superscript ±] are selected and their invariant mass distribution is examined. No excess of events above the expected level of Standard Model background...
Search for disappearing tracks in proton-proton collisions at √s = 8 TeV - Apyan, Aram; Barbieri, Richard Alexander; Busza, Wit; Cali, Ivan Amos; Chan, Matthew Hans; Di Matteo, Leonardo; Gomez-Ceballos, Guillelmo; Goncharov, Maxim; Gulhan, Doga Can; Klute, Markus; Lai, Yue Shi; Lee, Yen-Jie; Levin, Andrew Michael; Luckey, P. David, Jr.; Paus, Christoph M. E.; Ralph, Duncan Kelley; Roland, Christof E.; Roland, Gunther M.; Stephans, George S. F.; Sumorok, Konstanty C.; Velicanu, Dragos Alexandru; Veverka, Jan; Wyslouch, Boleslaw; Yang, Mingming; Zanetti, Marco; Zhukova, Victoria
A search is presented for long-lived charged particles that decay within the CMS detector and produce the signature of a disappearing track. Disappearing tracks are identified as those with little or no associated calorimeter energy deposits and with missing hits in the outer layers of the tracker. The search uses proton-proton collision data recorded at √s = 8 TeV that corresponds to an integrated luminosity of 19.5 fb[superscript −1]. The results of the search are interpreted in the context of the anomaly-mediated supersymmetry breaking (AMSB) model. The number of observed events is in agreement with the background expectation, and limits...
Search for invisible particles produced in association with single-top-quarks in proton–proton collisions at √s = 8 TeV with the ATLAS detector - Taylor, Frank E.
A search for the production of single-top-quarks in association with missing energy is performed in proton–proton collisions at a centre-of-mass energy of √s = 8 TeV with the ATLAS experiment at the large hadron collider using data collected in 2012, corresponding to an integrated luminosity of 20.3 fb[superscript −1]. In this search, the W boson from the top quark is required to decay into an electron or a muon and a neutrino. No deviation from the standard model prediction is observed, and upper limits are set on the production cross-section for resonant and non-resonant production of an invisible exotic state...
Search for dark matter in events with heavy quarks and missing transverse momentum in pp collisions with the ATLAS detector - Taylor, Frank E.
This article reports on a search for dark matter pair production in association with bottom or top quarks in 20.3 fb[superscript −1] of pp collisions collected at √s = 8 TeV by the ATLAS detector at the LHC. Events with large missing transverse momentum are selected when produced in association with high-momentum jets of which one or more are identified as jets containing b -quarks. Final states with top quarks are selected by requiring a high jet multiplicity and in some cases a single lepton. The data are found to be consistent with the Standard Model expectations and limits are...
Measurements of the W production cross sections in association with jets with the ATLAS detector - Taylor, Frank E.
This paper presents cross sections for the production of a W boson in association with jets, measured in proton–proton collisions at √s = 7TeV with the ATLAS experiment at the large hadron collider. With an integrated luminosity of 4.6 fb[superscript −1], this data set allows for an exploration of a large kinematic range, including jet production up to a transverse momentum of 1 TeV and multiplicities up to seven associated jets. The production cross sections for W bosons are measured in both the electron and muon decay channels. Differential cross sections for many observables are also presented including measurements of...
Searches for heavy long-lived charged particles with the ATLAS detector in proton-proton collisions at √s = 8 TeV - Taylor, Frank E.
Searches for heavy long-lived charged particles are performed using a data sample of 19.1 fb[superscript −1] from proton-proton collisions at a centre-of-mass energy of √s = 8 TeV collected by the ATLAS detector at the Large Hadron Collider. No excess is observed above the estimated background and limits are placed on the mass of long-lived particles in various supersymmetric models. Long-lived tau sleptons in models with gauge-mediated symmetry breaking are excluded up to masses between 440 and 385 GeV for tan β between 10 and 50, with a 290 GeV limit in the case where only direct tau slepton production...