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The selective hydrogenation of acetylene catalyzed by Pd nanoparticles is industrially used to increase the purity of ethylene. Despite the implementation of Pd based catalysts on an industrial scale, little is known about metal-support interactions on a fundamental level due to the complexity of these systems. In this study, the influence of metal-support interactions between Pd nanoparticles and two electronically modified a-SiO2 thin films on acetylene hydrogenation is investigated under ultra-high vacuum (UHV) conditions. The hydrogenation is performed under isothermal reaction conditions using a pulsed molecular beam reactive scattering (pMBRS) technique. Besides the activity and selectivity of clean Pd particles also the impact of dehydrogenated species intentionally introduced a priori is elucidated, whereas the active phase of the catalyst is additionally characterized by CO infrared reflection-absorption spectroscopy (IRRAS) and post-mortem temperature-programmed reaction (TPR). Metal-support interactions are found to influence the catalytic properties of Pd particles by charge-transfer, where positive charging leads to increased activity for acetylene hydrogenation. However, the increased activity is accompanied by formation of undesired byproducts. The active sites for acetylene and ethylene hydrogenation are shown to be different as previously proposed by the A and E model. The availability of the two different active sites on the Pd nanoparticles is determined by dehydrogenated species, whose nature and stability can be tuned by metal-support interactions. Based on these findings an electronic model is proposed how selectivity for acetylene hydrogenation can be steered solely by metal-support interactions leading to blocking of unselective sites in situ.
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Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3'-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr's performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.
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Nanoporos , Poli A/metabolismo , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/métodos , Poli T/metabolismo , PoliadenilaciónRESUMEN
The CRISPR-Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR-Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR-Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.no.
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Sistemas CRISPR-Cas/genética , Bases de Datos Genéticas , Marcación de Gen , Genoma/genética , ARN Guía de Kinetoplastida/genética , Programas Informáticos , Animales , Edición Génica/métodos , HumanosRESUMEN
The hydrogenation of ethylene and acetylene was studied on a Pdn/MgO/Mo(100) model system containing palladium particles with a narrow size distribution around Pd26 (Pd20 to Pd35). Reactivity measurements were carried out in an ultrahigh vacuum chamber under isothermal conditions in the presence of deuterium. The catalyst system can readily hydrogenate both of these small molecules, and for acetylene, an alternative reaction network exists, in which it is trimerized to benzene. Distinct deactivation behavior was found for the two molecules and ascribed to different adsorption sites formed and influenced by the carbonaceous overlayer formed during the course of the reaction. These findings extend the A-E-model by Borodzinski and Golȩbiowski to extremely small particles and low partial pressures and show that it is possible to study realistic catalytic sites under highly defined conditions.
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In the concept of anode-free lithium-ion batteries, cells are manufactured with a bare anode current collector where the lithium metal anode is electrochemically formed from the lithium-containing cathode during the first charge cycle. While this concept has many attractive aspects from a manufacturing and energy density standpoint, stable plating and stripping remain challenging. We have investigated gold, platinum, and amorphous carbon as seed layers placed between the copper current collector and the lithium phosphorus oxynitride thin-film solid electrolyte. These layers guide lithium nucleation and improve the plating and stripping dynamics. All seed layers facilitate reversible lithium plating and stripping even at high current densities up to 8 mA cm-2. Of particular note is the amorphous carbon seed layer, which allowed a significant reduction in plating potential from 300 mV to as low as 50 mV. These results underscore the critical role of seed layers in improving the efficiency of anode-free solid-state batteries and open the door to simplified manufacturing of anode-free battery designs.
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Fabry disease (FD) is a rare genetic lysosomal storage disorder, resulting from partial or complete lack of alpha-galactosidase A (α-GAL) enzyme, leading to systemic accumulation of substrate glycosphingolipids with a broad range of tissue damage. Current in vivo models are laborious, expensive, and fail to adequately mirror the complex FD physiopathology. To address these issues, we developed an innovative FD model in zebrafish. Zebrafish GLA gene encoding α-GAL enzyme presents a high (>70%) homology with its human counterpart, and the corresponding protein has a similar tissue distribution, as evaluated by immunohistochemistry. Moreover, a similar enzymatic activity in different life stages could be demonstrated. By using CRISPR/Cas9 technology, we generated a mutant zebrafish with decreased GLA gene expression, and decreased expression of the specific gene product in the kidney. Mutant animals showed higher plasma creatinine levels and proteinuria. Transmission electron microscopy (TEM) studies documented an increased podocyte foot process width (FPW) in mutant, as compared to wild type zebrafish. This zebrafish model reliably mirrors distinct features of human FD and could be advantageously used for the identification of novel biomarkers and for an effective screening of innovative therapeutic approaches.
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The poly(A) tail is a homopolymeric stretch of adenosine at the 3'-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. Existing techniques for genome-wide estimation of poly(A) tail length are based on short-read sequencing. These methods are limited because they sequence a synthetic DNA copy of mRNA instead of the native transcripts. Furthermore, they can identify only a short segment of the transcript proximal to the poly(A) tail which makes it difficult to assign the measured poly(A) length uniquely to a single transcript isoform. With the introduction of native RNA sequencing by Oxford Nanopore Technologies, it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making transcriptome-wide isoform-specific poly(A) tail length assessment feasible. We developed tailfindr-an R-based package for estimating poly(A) tail length from Oxford Nanopore sequencing data. In this chapter, we describe in detail the pipeline for transcript isoform-specific poly(A) tail profiling based on native RNA Nanopore sequencing-from library preparation to downstream data analysis with tailfindr.
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Secuenciación de Nanoporos/métodos , Poli A/análisis , ARN/análisis , Análisis de Secuencia de ARN/métodos , Animales , Estudios de Factibilidad , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Poli A/genética , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , ARN/química , ARN/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/análisis , ARN Mensajero/genética , Transcriptoma , Pez Cebra/genéticaRESUMEN
The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on-target editing) with minimal editing at unintended locations (off-target editing). Although laboratory validation should ideally be used to detect off-target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA-DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user-friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock-out, sequence knock-in, and RNA knock-down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of gRNAs for gene knock-out Alternate Protocol 1: Design of gRNAs for dCas9 fusion/effector targeting Support Protocol: Design of gRNAs for targeting transgenic or plasmid sequences Basic Protocol 2: Design of gRNAs for RNA targeting Basic Protocol 3: Design of gRNAs for sequence knock-in Alternate Protocol 2: Design of gRNAs for knock-in using non-homologous end joining Basic Protocol 4: Design of gRNAs for knock-in using Cas9 nickases.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Sistemas CRISPR-Cas , Genoma , ARN Guía de Kinetoplastida/genéticaRESUMEN
Thin-film solar cells based on polycrystalline absorbers have reached very high conversion efficiencies of up to 23-25%. In order to elucidate the limiting factors that need to be overcome for even higher efficiency levels, it is essential to investigate microscopic origins of loss mechanisms in these devices. In the present work, a high efficiency (21% without anti-reflection coating) copper indium gallium diselenide (CIGSe) solar cell is characterized by means of a correlative microscopy approach and corroborated by means of photoluminescence spectroscopy. The values obtained by the experimental characterization are used as input parameters for two-dimensional device simulations, for which a real microstructure was used. It can be shown that electrostatic potential and lifetime fluctuations exhibit no substantial impact on the device performance. In contrast, nonradiative recombination at random grain boundaries can be identified as a significant loss mechanism for CIGSe solar cells, even for devices at a very high performance level.
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Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5' UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step.
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Iniciación de la Cadena Peptídica Traduccional/genética , Humanos , Subunidades Ribosómicas Pequeñas/metabolismoRESUMEN
Nature employs self-assembly to fabricate the most complex molecularly precise machinery known to man. Heteromolecular, two-dimensional self-assembled networks provide a route to spatially organize different building blocks relative to each other, enabling synthetic molecularly precise fabrication. Here we demonstrate optoelectronic function in a near-to-monolayer molecular architecture approaching atomically defined spatial disposition of all components. The active layer consists of a self-assembled terrylene-based dye, forming a bicomponent supramolecular network with melamine. The assembly at the graphene-diamond interface shows an absorption maximum at 740 nm whereby the photoresponse can be measured with a gallium counter electrode. We find photocurrents of 0.5 nA and open-circuit voltages of 270 mV employing 19 mW cm(-2) irradiation intensities at 710 nm. With an ex situ calculated contact area of 9.9 × 10(2) µm(2), an incident photon to current efficiency of 0.6% at 710 nm is estimated, opening up intriguing possibilities in bottom-up optoelectronic device fabrication with molecular resolution.