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As part of the ongoing bacterial-phage arms race, CRISPR-Cas systems in bacteria clear invading phages whereas anti-CRISPR proteins (Acrs) in phages inhibit CRISPR defenses. Known Acrs have proven extremely diverse, complicating their identification. Here, we report a deep learning algorithm for Acr identification that revealed an Acr against type VI-B CRISPR-Cas systems. The algorithm predicted numerous putative Acrs spanning almost all CRISPR-Cas types and subtypes, including over 7,000 putative type IV and VI Acrs not predicted by other algorithms. By performing a cell-free screen for Acr hits against type VI-B systems, we identified a potent inhibitor of Cas13b nucleases we named AcrVIB1. AcrVIB1 blocks Cas13b-mediated defense against a targeted plasmid and lytic phage, and its inhibitory function principally occurs upstream of ribonucleoprotein complex formation. Overall, our work helps expand the known Acr universe, aiding our understanding of the bacteria-phage arms race and the use of Acrs to control CRISPR technologies.
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Bacteriófagos , Aprendizado Profundo , Bactérias/genética , Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/genética , Endonucleases/metabolismoRESUMO
MOTIVATION: The CRISPR-Cas9 system is a Type II CRISPR system that has rapidly become the most versatile and widespread tool for genome engineering. It consists of two components, the Cas9 effector protein, and a single guide RNA that combines the spacer (for identifying the target) with the tracrRNA, a trans-activating small RNA required for both crRNA maturation and interference. While there are well-established methods for screening Cas effector proteins and CRISPR arrays, the detection of tracrRNA remains the bottleneck in detecting Class 2 CRISPR systems. RESULTS: We introduce a new pipeline CRISPRtracrRNA for screening and evaluation of tracrRNA candidates in genomes. This pipeline combines evidence from different components of the Cas9-sgRNA complex. The core is a newly developed structural model via covariance models from a sequence-structure alignment of experimentally validated tracrRNAs. As additional evidence, we determine the terminator signal (required for the tracrRNA transcription) and the RNA-RNA interaction between the CRISPR array repeat and the 5'-part of the tracrRNA. Repeats are detected via an ML-based approach (CRISPRidenify). Providing further evidence, we detect the cassette containing the Cas9 (Type II CRISPR systems) and Cas12 (Type V CRISPR systems) effector protein. Our tool is the first for detecting tracrRNA for Type V systems. AVAILABILITY AND IMPLEMENTATION: The implementation of the CRISPRtracrRNA is available on GitHub upon requesting the access permission, (https://github.com/BackofenLab/CRISPRtracrRNA). Data generated in this study can be obtained upon request to the corresponding person: Rolf Backofen (backofen@informatik.uni-freiburg.de). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Sistemas CRISPR-Cas , RNA , Humanos , Genoma , RNA/genética , Alinhamento de Sequência , Pequeno RNA não Traduzido/genéticaRESUMO
In this work, we have developed selective methods for the synthesis of quinoline-2-carboxylates and quinoline-3-carboxylates as well as (indolin-2-ylidene)acetates through copper-, silver-, or phosphine-catalyzed reaction of propiolates with 2'-amino-2,2,2-trifluoroacetophenones. The approaches proposed ensure synthesis of substituted quinoline carboxylates and (indolin-2-ylidene)acetates in good yields. Introduction of alkynones into the reaction with 2'-amino-2,2,2-trifluoroacetophenones gives acyl substituted derivatives in good yields.
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In this study, we developed a selective method for synthesis of multi-substituted quinoline-2-ylphosphonates and quinoline-3-ylphosphonates by copper- or gold-catalyzed reactions of phosphoryl-substituted conjugated ynones with 2'-amino-2,2,2-trifluoroacetophenones. The approach proposed makes it possible to obtain various substituted quinolines in good yields. It is also shown that (4,4,4-trifluoro-3-oxobut-1-yn-1-yl)phosphonate reacts with 2-aminoaryl ketones under non-catalytic conditions with formation of 4-substituted quinoline-2-ylphosphonates in high yields.
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CRISPR-Cas are adaptive immune systems that degrade foreign genetic elements in archaea and bacteria. In carrying out their immune functions, CRISPR-Cas systems heavily rely on RNA components. These CRISPR (cr) RNAs are repeat-spacer units that are produced by processing of pre-crRNA, the transcript of CRISPR arrays, and guide Cas protein(s) to the cognate invading nucleic acids, enabling their destruction. Several bioinformatics tools have been developed to detect CRISPR arrays based solely on DNA sequences, but all these tools employ the same strategy of looking for repetitive patterns, which might correspond to CRISPR array repeats. The identified patterns are evaluated using a fixed, built-in scoring function, and arrays exceeding a cut-off value are reported. Here, we instead introduce a data-driven approach that uses machine learning to detect and differentiate true CRISPR arrays from false ones based on several features. Our CRISPR detection tool, CRISPRidentify, performs three steps: detection, feature extraction and classification based on manually curated sets of positive and negative examples of CRISPR arrays. The identified CRISPR arrays are then reported to the user accompanied by detailed annotation. We demonstrate that our approach identifies not only previously detected CRISPR arrays, but also CRISPR array candidates not detected by other tools. Compared to other methods, our tool has a drastically reduced false positive rate. In contrast to the existing tools, our approach not only provides the user with the basic statistics on the identified CRISPR arrays but also produces a certainty score as a practical measure of the likelihood that a given genomic region is a CRISPR array.
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Aprendizado de Máquina , Software , Genoma Arqueal , Genoma BacterianoRESUMO
CRISPR-Cas systems are adaptive immune systems in prokaryotes, providing resistance against invading viruses and plasmids. The identification of CRISPR loci is currently a non-standardized, ambiguous process, requiring the manual combination of multiple tools, where existing tools detect only parts of the CRISPR-systems, and lack quality control, annotation and assessment capabilities of the detected CRISPR loci. Our CRISPRloci server provides the first resource for the prediction and assessment of all possible CRISPR loci. The server integrates a series of advanced Machine Learning tools within a seamless web interface featuring: (i) prediction of all CRISPR arrays in the correct orientation; (ii) definition of CRISPR leaders for each locus; and (iii) annotation of cas genes and their unambiguous classification. As a result, CRISPRloci is able to accurately determine the CRISPR array and associated information, such as: the Cas subtypes; cassette boundaries; accuracy of the repeat structure, orientation and leader sequence; virus-host interactions; self-targeting; as well as the annotation of cas genes, all of which have been missing from existing tools. This annotation is presented in an interactive interface, making it easy for scientists to gain an overview of the CRISPR system in their organism of interest. Predictions are also rendered in GFF format, enabling in-depth genome browser inspection. In summary, CRISPRloci constitutes a full suite for CRISPR-Cas system characterization that offers annotation quality previously available only after manual inspection.
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Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Anotação de Sequência Molecular , Software , Proteínas Associadas a CRISPR/classificação , Proteínas Associadas a CRISPR/genética , Aprendizado de MáquinaRESUMO
We simulate scattering of electrons by a chain of antiferromagnetically coupled quantum Heisenberg spins, to analyze spin-transfer effects not described by the classical models of magnetism. Our simulations demonstrate efficient excitation of dynamical states that would be forbidden by the semiclassical symmetries, such as generation of multiple magnetic excitation quanta by a single electron. Furthermore, quantum interference of spin wave functions enables generation of magnetization dynamics with amplitudes exceeding the transferred magnetic moment. The efficiency of excitation is almost independent of the electron spin polarization, and is governed mainly by the transfer of energy. Nonclassical spin transfer may thus enable efficient electronic control of antiferromagnets not limited by the classical constraints.
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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are essential genetic elements in many archaeal and bacterial genomes, playing a key role in a prokaryote adaptive immune system against invasive foreign elements. In recent years, the CRISPR-Cas system has also been engineered to facilitate target gene editing in eukaryotic genomes. Bioinformatics played an essential role in the detection and analysis of CRISPR systems and here we review the bioinformatics-based efforts that pushed the field of CRISPR-Cas research further. We discuss the bioinformatics tools that have been published over the last few years and, finally, present the most popular tools for the design of CRISPR-Cas9 guides.
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Sistemas CRISPR-Cas/genética , Biologia Computacional/métodos , Edição de Genes , Algoritmos , Biologia Computacional/tendências , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Metal-catalyzed (Cu, Ag, Au) reactions of alkynylphosphonates with 1-(2-aminophenyl)-2,2,2-trifluoroethan-1-ones were developed. Terminal alkyne diethyl ethynylphosphonate reacted with ketones to give different products depending on the catalyst used. With a CuI/PPh3 catalytic system, the formation of CF3-containing indoline derivatives was observed with good yields. The use of AgSbF6 as a catalyst led to quinoline derivatives in high yields. The less reactive 2-substituted ethynylphosphonates required gold complexes as catalysts to provide the corresponding 2-aryl(alkyl) substituted 4-(trifluoromethyl)quinolin-3-ylphosphonates with good yields.
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PURPOSE: Our study compared the rates of union achieved with the Ilizarov method in congenital pseudarthrosis of the tibia (CPT) associated with neurofibromatosis type 1 (NF1) or CPT of idiopathic origin in paediatric patients. METHODS: We studied the outcomes of 28 children that were treated for CPT between 2005 and 2013. Group 1 included children (n = 14, mean age = 9.7 years) with CPT associated with NF1 while group 2 were CPT cases that had radiographic confirmation of dysplastic lesions in the tibia but lacked clinical NF1 manifestations (n = 14, mean age = 8.6 years). There was no statistical difference between the groups regarding their age or number of previous operations per patient. Individual technical solutions were planned for each patient but coaptation of bone fragments and autologous local tissue grafting to achieve a greater bone thickness and contact area at the pseudarthrosis level were mainly used. Refracture-free rate after the first operation, number of re-operations per patient, and union rates in the groups were compared. RESULTS: Bone union and weight bearing were obtained in all the cases after the first operation. Refracture-free rate was 42.86 % in group 1 and 35.71% in group 2 (no statistical difference, p > 0.05). Mean number of re-operations per patient was 1.07 and 0.78 respectively (p > 0.05). Subsequent treatment for refractures with the Ilizarov techniques gained 92.86% of union in both groups at the follow-ups by completion of the study (range, 2-9 years). CONCLUSIONS: The Ilizarov method yields comparable results in the management of CPT associated with NF1 or tibial dysplasia of idiopathic origin in paediatric cases. Further research should focus on the ways to support the Ilizarov method in order to reduce the number of repetitive surgeries or eliminate them.
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Técnica de Ilizarov , Neurofibromatose 1/complicações , Pseudoartrose/congênito , Tíbia/cirurgia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Neurofibromatose 1/cirurgia , Complicações Pós-Operatórias/etiologia , Pseudoartrose/complicações , Pseudoartrose/cirurgia , Reoperação , Estudos Retrospectivos , Suporte de CargaRESUMO
LiF crystal and film detectors were used to measure the far-field fluence profile of a self-amplified spontaneous-emission free-electron laser beam and diffraction imaging with high spatial resolution. In these measurements the photoluminescence (PL) response of LiF crystal and film was compared over a wide range of soft x-ray fluences. It was found that the soft x-ray fluence dependences of LiF crystal and film differ. At low fluence, the LiF crystal shows higher PL response compared to LiF film, while this comparison is the opposite at higher fluence. Accurate measurement of LiF crystal and film PL response is important for precise characterization of the spatial, spectral, and coherence features of x-ray beams across the full profile and in localized areas. For such measurements, crucial LiF detector attributes are high spatial resolution and high dynamic range.
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Optical features of point defects photoluminescence in LiF crystals, irradiated by soft X-ray pulses of the Free Electron Laser with wavelengths of 17.2 - 61.5 nm, were measured. We found that peak of photoluminescence spectra lies near of 530 nm and are associated with emission of F3+ centers. Our results suggest that redistribution of photoluminescence peak intensity from the red to the green part of the spectra is associated with a shortening of the applied laser pulses down to pico - or femtosecond durations. Dependence of peak intensity of photoluminescence spectra from the soft X-ray irradiation fluence was measured and the absence of quenching phenomena, even at relatively high fluencies was found, which is very important for wide applications of LiF crystal X-ray imaging detectors.
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Understanding the mechanism of crack propagation during bone cutting is necessary for the development of realistic bone cutting models. This article studies the on-line fractural behaviour of cortical bone caused by penetration with a sharp metallic wedge mounted on an on-line loading stage within an X-ray microfocus computed tomography system. The experimental results demonstrated anisotropy in crack propagation depending on the penetration direction with regard to the longitudinal bone axis and relate the crack growth to the extent of penetration. Scanning electron microscopy is performed to analyse the mechanism of cracking in the two phase microstructure of compact bone.
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Fêmur/cirurgia , Fêmur/ultraestrutura , Modelos Biológicos , Animais , Bovinos , Fêmur/diagnóstico por imagem , Técnicas In Vitro , RadiografiaRESUMO
Ru(II) complexes with polypyridyl ligands play a central role in the development of photocatalytic organic reactions. This work is aimed at the structural modification of such complexes to increase their photocatalytic efficiency and adapt them for the preparation of reusable photocatalytic systems. Nine [Ru(phen)(bpy)2]2+-type complexes (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline) (Ru-Pcat) bearing the P(O)(OEt)2 substituent attached to the phen core directly or through a 1,4-phenylene linker were synthesized and characterized by spectroscopic and electrochemical techniques. The coordination mode of phen ligands was confirmed by single crystal X-ray analysis. The (spectro)electrochemical data show that the first electron transfer in Ru-Pcat takes place on the phen ligand. The emission maxima and quantum yields are strongly affected by the substitution pattern, reaching the far-red region (697 nm) for Ru-3,8P2. The singlet oxygen quantum yields of Ru-Pcat were evaluated using the chemical trapping method. Finally, the photocatalytic performance of Ru-Pcat in the oxidation of sulfides with molecular oxygen was investigated. Both dialkyl and alkyl aryl sulfides were quantitatively transformed into sulfoxides under irradiation with a blue LED in the acetonitrile-water mixture (10 : 1) using a low loading of 0.005-0.05 mol% Ru(II) photocatalysts. To rationalize the effect of phosphonate substituents on the photocatalytic efficiency, comparative kinetic studies of (1) 4-nitrothioanisole oxidation proceeding predominantly via the electron transfer pathway and (2) oxidation of dibutyl sulfide wherein singlet oxygen serves as an oxidant have been performed. It was demonstrated that complexes with the P(O)(OEt)2 substituent at positions 4 and 7 outperform the benchmark photocatalyst Ru-(bpy)3 and the parent complex Ru-phen in the reactions proceeding through electron transfer (reductive quenching photocatalytic cycle). The TON in the oxidation of 4-methoxythioanisole was found to be as high as 1 000 000 that is, to our knowledge, the highest among previously reported photocatalysts. In contrast, upon separating the P(O)(OEt)2 group and the phen core with the 1,4-phenylene linker, singlet oxygen quantum yields significantly increase that favors reactions proceeding through energy transfer (the oxidation of dibutyl sulfide in our case). Thus, both series of Ru(II) complexes prepared in this work are promising for the improvement of known photocatalytic reactions and the development of new transformations.
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We observe an optical signature induced by the modulation of electron density inside a bulk transparent solid that is quasiperiodically ionized on an attosecond time scale by electric field peaks of a focused few-cycle laser pulse. The emitted optical signal resulting from the attosecond ionization dynamics is spatially, temporally and spectrally isolated from concomitant optical responses through the use of a noncollinear pump-probe technique. The method holds promise for developing an attosecond metrology for bulk solids, in which, unlike in the established attosecond metrology of gases and surfaces, direct detection of charged particles is unfeasible.
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We have experimentally detected optical harmonics that are generated due to a tunneling-ionization-induced modulation of the electron density. The optical signature of electron tunneling can be isolated from concomitant optical responses by using a noncollinear pump-probe setup. Whereas previously demonstrated tools for attosecond metrology of gases, plasmas, and surfaces rely on direct detection of charged particles, detection of the background-free time-resolved optical signal, which uniquely originates from electron tunneling, offers an interesting alternative that is especially suited for systems in which free electrons cannot be directly measured.
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In the title compound, [Cu2(OH)2{C12H7N2(PO3C2H5)}2(H2O)2]·7H2O, two Cu2+ cations are bridged by two hydroxide groups, forming a centrosymmetric binuclear complex. Each Cu2+cation is further coordinated by the N atoms of a bidentate ethyl (1,10-phenanthrolin-3-yl)phospho-nate anion and a water mol-ecule in a square-pyramidal geometry. In the crystal, a network of O-Hâ¯O hydrogen bonds involving the P(O)(O-)(OEt) groups, bridging hydroxyl groups, coordinated and uncoordinated water mol-ecules generates a three-dimensional supra-molecular structure. The ethyl group exhibits disorder and was modelled over three sites with occupancies of 0.455, 0.384 and 0.161.
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A series of ditopic macrocyclic receptors with variable cavity sizes, containing nitrogen or mixed (nitrogen-oxygen) donor sites and an externally directed 1,10-phenanthroline fragment, were prepared by means of a palladium-catalyzed amination reaction. A ditopic mixed NO-ligand (4 a) was coordinated to [Ru(bpy)2 ]2+ (bpy=2,2'-bipyridine) to prepare a luminescent and chromogenic complex, [Ru(bpy)2 (4 a)][PF]2 , which provided the selective dual-channel detection of CuII ions.
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We introduce a bandwidth-unlimited, dispersion- and shear-self-calibrated, timing-jitter-free pulse measurement technique based on a quasi-linear temporal phase modulation in a gas weakly ionized by a long pump pulse. Results of a 5 fs pulse characterization are reported.