RESUMO
Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for understanding viral spread and evolution as well as for vaccine development. Existing RNA sequencing methods are demanding on user technique and time and, thus, not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We developed a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. We demonstrate the utility of our approach on pharyngeal, sputum, and stool samples collected from coronavirus disease 2019 (COVID-19) patients, successfully obtaining whole metatranscriptomes and complete high-depth, high-coverage SARS-CoV-2 genomes with high yield and robustness. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate molecular epidemiology studies during current and future outbreaks.
Assuntos
COVID-19/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , RNA Viral/genética , SARS-CoV-2/genética , Sequenciamento Completo do Genoma , Animais , Humanos , Camundongos , Células NIH 3T3 , RNA Viral/metabolismo , SARS-CoV-2/metabolismoRESUMO
Somatic mutations that accumulate in normal tissues are associated with ageing and disease1,2. Here we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies of 9 organs from 5 donors. We found that somatic mutation accumulations and clonal expansions were widespread, although to variable extents, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for in tissues from the oesophagus and cardia. Endogenous mutational processes with the SBS1 and SBS5 mutational signatures are ubiquitous among normal tissues, although they exhibit different relative activities. Exogenous mutational processes operate in multiple tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimetre resolution. In the oesophagus and cardia, macroscopic somatic clones that expanded to hundreds of micrometres were frequently seen, whereas in tissues such as the colon, rectum and duodenum, somatic clones were microscopic in size and evolved independently, possibly restricted by local tissue microstructures. Our study depicts a body map of somatic mutations and clonal expansions from the same individual.
Assuntos
Células Clonais/metabolismo , Saúde , Mutagênese , Mutação , Especificidade de Órgãos , Idoso de 80 Anos ou mais , Biópsia , Cadáver , Cárdia/metabolismo , Proliferação de Células , Células Clonais/citologia , Esôfago/metabolismo , Feminino , Genômica , Humanos , MasculinoRESUMO
Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Analíticas Microfluídicas , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , Automação Laboratorial , Sequência de Bases , Linhagem Celular , Biologia Computacional , Análise Custo-Benefício , Código de Barras de DNA Taxonômico , Perfilação da Expressão Gênica/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Técnicas Analíticas Microfluídicas/economia , Reprodutibilidade dos Testes , Análise de Sequência de RNA/economia , Análise de Célula Única/economia , Fluxo de TrabalhoRESUMO
The interactome networks at the DNA, RNA, and protein levels are crucial for cellular functions, and the diverse variations of these networks are heavily involved in the establishment of different cell states. We have developed a diffusion-based method, Hi-C to geometry (CTG), to obtain reliable geometric information on the chromatin from Hi-C data. CTG produces a consistent and reproducible framework for the 3D genomic structure and provides a reliable and quantitative understanding of the alterations of genomic structures under different cellular conditions. The genomic structure yielded by CTG serves as an architectural blueprint of the dynamic gene regulatory network, based on which cell-specific correspondence between gene-gene and corresponding protein-protein physical interactions, as well as transcription correlation, is revealed. We also find that gene fusion events are significantly enriched between genes of short CTG distances and are thus close in 3D space. These findings indicate that 3D chromatin structure is at least partially correlated with downstream processes such as transcription, gene regulation, and even regulatory networking through affecting protein-protein interactions.
Assuntos
Cromatina , Redes Reguladoras de Genes , Cromatina/genética , Regulação da Expressão Gênica , Cromossomos , DNARESUMO
Spatial transcriptomics technology has revolutionized our understanding of cell types and tissue organization, opening possibilities for researchers to explore transcript distributions at subcellular levels. However, existing methods have limitations in resolution, sensitivity, or speed. To overcome these challenges, we introduce SPRINTseq (Spatially Resolved and signal-diluted Next-generation Targeted sequencing), an innovative in situ sequencing strategy that combines hybrid block coding and molecular dilution strategies. Our method enables fast and sensitive high-resolution data acquisition, as demonstrated by recovering over 142 million transcripts using a 108-gene panel from 453,843 cells from four mouse brain coronal slices in less than 2 d. Using this advanced technology, we uncover the cellular and subcellular molecular architecture of Alzheimer's disease, providing additional information into abnormal cellular behaviors and their subcellular mRNA distribution. This improved spatial transcriptomics technology holds great promise for exploring complex biological processes and disease mechanisms.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Camundongos , RNA Mensageiro/genética , TranscriptomaRESUMO
Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations' stochastic natures. Using a Tn5-transposase-assisted single-cell whole-genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, whereas monosomy X occurred most frequently in over-30-yr-old females. In the monosomy X single cells from individuals with phased genomes and identified X-inactivation ratios in bulk, the inactive X Chromosomes were lost more often than the active ones.
Assuntos
Variações do Número de Cópias de DNA , Genômica , Aneuploidia , Feminino , Humanos , Linfócitos , Sequenciamento Completo do GenomaRESUMO
Both the composition of cell types and their spatial distribution in a tissue play a critical role in cellular function, organ development, and disease progression. For example, intratumor heterogeneity and the distribution of transcriptional and genetic events in single cells drive the genesis and development of cancer. However, it can be challenging to fully characterize the molecular profile of cells in a tissue with high spatial resolution because microscopy has limited ability to extract comprehensive genomic information, and the spatial resolution of genomic techniques tends to be limited by dissection. There is a growing need for tools that can be used to explore the relationship between histological features, gene expression patterns, and spatially correlated genomic alterations in healthy and diseased tissue samples. Here, we present a technique that combines label-free histology with spatially resolved multiomics in unfixed and unstained tissue sections. This approach leverages stimulated Raman scattering microscopy to provide chemical contrast that reveals histological tissue architecture, allowing for high-resolution in situ laser microdissection of regions of interests. These microtissue samples are then processed for DNA and RNA sequencing to identify unique genetic profiles that correspond to distinct anatomical regions. We demonstrate the capabilities of this technique by mapping gene expression and copy number alterations to histologically defined regions in human oral squamous cell carcinoma (OSCC). Our approach provides complementary insights in tumorigenesis and offers an integrative tool for macroscale cancer tissues with spatial multiomics assessments.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Bucais , Carcinoma de Células Escamosas/genética , Variações do Número de Cópias de DNA/genética , Perfilação da Expressão Gênica/métodos , Genômica , Humanos , Análise de Sequência de RNARESUMO
Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life.
Assuntos
DNA Mitocondrial , Células-Tronco Pluripotentes , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Haplótipos , Heteroplasmia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oócitos/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single cells well below the conventional NGS detection limit and provided accurate quantitation of heteroplasmy level. iMiGseq resolved the complete haplotype of individual mtDNA in single oocytes and revealed genetic linkage of de novo mutations. iMiGseq detected sequential acquisition of detrimental mutations, including large deletions, in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells. iMiGseq identified unintended heteroplasmy shifts in mitoTALEN editing, while showing no appreciable level of unintended mutations in DdCBE-mediated mtDNA base editing. Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also evaluate the safety of various mtDNA editing strategies.
Assuntos
DNA Mitocondrial , Genoma Mitocondrial , DNA Mitocondrial/genética , Heteroplasmia/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , MutaçãoRESUMO
BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , Edição de Genes , Humanos , Edição de Genes/métodos , Quebras de DNA , Reparo de DNA por Recombinação , Deleção de Sequência , DNA Polimerase teta , Proteína de Replicação A/metabolismo , Proteína de Replicação A/genéticaRESUMO
BACKGROUND: p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. METHODS: p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation. In addition, p66Shc affects mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation were observed by transmission electron microscopy (TEM). Mitochondrial function was evaluated by detected cytoplastic mitochondrial DNA (mtDNA) and mitochondrial membrane potential (MMP). RESULTS: High glucose induces the expression and mitochondrial translocation of p66Shc, which promotes MAMs formation and stimulates PINK1-PRKN-mediated mitophagy. Moreover, mitochondrial localized p66Shc reduces MMP and triggers cytosolic mtDNA release, thus activates cGAS/STING signaling and ultimately leads to enhanced autophagy and cellular senescence. Specially, we found p66Shc is required for the interaction between STING and LC3II, as well as between STING and ATG5, thereby regulates cGAS/STING-mediated autophagy. We also identified hundreds of genes associated several biological processes including aging are co-regulated by p66Shc and ATG5, deletion either of which results in diminished cellular senescence. CONCLUSION: p66Shc is not only implicated in the initiation of autophagy by promoting MAMs formation, but also helps stabilizing active autophagic flux by activating cGAS/STING pathway in trophoblast.
Assuntos
Autofagossomos , Trofoblastos Extravilosos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Autofagossomos/metabolismo , Autofagia , DNA Mitocondrial/metabolismo , Trofoblastos/metabolismo , Glucose/metabolismo , Nucleotidiltransferases/metabolismoRESUMO
BACKGROUND: Rhodococcus equi (R. equi) is a Gram-positive zoonotic pathogen that frequently leads to illness and death in young horses (foals). This study presents the complete genome sequence of R. equi strain BJ13, which was isolated from a thoroughbred racehorse breeding farm in Beijing, China. RESULTS: The BJ13 genome has a length of 5.30 Mb and consists of a complete chromosome and a plasmid measuring 5.22 Mb and 0.08 Mb, respectively. We predicted 4,929 coding gene open reading frames, along with 52 tRNAs and 12 rRNAs. Through analysis of mobile genetic elements, we identified 6 gene islands and 1 prophage gene. Pathogenic system analysis predicted the presence of 418 virulence factors and 225 drug resistance genes. Secretion system analysis revealed the prediction of 297 secreted proteins and 1,106 transmembrane proteins. BJ13 exhibits genomic features, virulence-associated genes, potential drug resistance, and a virulence plasmid structure that may contribute to the evolution of its pathogenicity. Lastly, the pathogenicity of the isolated strain was assessed through animal experiments, which resulted in inflammatory reactions or damage in the lungs, liver, and spleen of mice. Moreover, by the 7th day post-infection, the mortality rate of the mice reached 50.0%, indicating complex immune regulatory mechanisms, including overexpression of IL-10 and increased production of pro-inflammatory cytokines like TNF-α. These findings validate the strong pathogenicity of the isolated strain and provide insights for studying the pathogenic mechanisms of Rhodococcus equi infection. CONCLUSIONS: The complete genome sequence of R. equi strain BJ13 provides valuable insights into its genomic characteristics, virulence potential, drug resistance, and secretion systems. The strong pathogenicity observed in animal experiments underscores the need for further investigation into the pathogenic mechanisms of R. equi infection.
Assuntos
Infecções por Actinomycetales , Genoma Bacteriano , Doenças dos Cavalos , Rhodococcus equi , Sequenciamento Completo do Genoma , Rhodococcus equi/patogenicidade , Rhodococcus equi/genética , Animais , Cavalos , Doenças dos Cavalos/microbiologia , Infecções por Actinomycetales/veterinária , Infecções por Actinomycetales/microbiologia , Virulência/genética , Camundongos , Fatores de Virulência/genética , FemininoRESUMO
H11N9 viruses in wild birds might have provided the NA gene of human H7N9 virus in early 2013 in China, which evolved with highly pathogenic strains in 2017 and caused severe fatalities. To investigate the prevalence and evolution of the H11N9 influenza viruses, 16,781 samples were collected and analyzed during 2016-2020. As a result, a novel strain of influenza A (H11N9) virus with several characteristics that increase virulence was isolated. This strain had reduced pathogenicity in chicken and mice and was able to replicate in mice without prior adaptation. Phylogenetic analyses showed that it was a sextuple-reassortant virus of H11N9, H3N8, H3N6, H7N9, H9N2, and H6N8 viruses present in China, similar to the H11N9 strains in Japan and Korea during the same period. This was the H11N9 strain isolated from China most recently, which add a record to viruses in wild birds. This study identified a new H11N9 reassortant in a wild bird with key mutation contributing to virulence. Therefore, comprehensive surveillance and enhanced biosecurity precautions are particularly important for the prediction and prevention of potential pandemics resulting from reassortant viruses with continuous evolution and expanding geographic distributions.
Assuntos
Vírus da Influenza A Subtipo H3N8 , Subtipo H7N9 do Vírus da Influenza A , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Animais , Camundongos , Humanos , Patos , Subtipo H7N9 do Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H9N2/genética , Filogenia , Animais Selvagens , Galinhas , Vírus Reordenados/genéticaRESUMO
The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.
Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Reação em Cadeia da Polimerase/métodos , DNA/sangue , Variações do Número de Cópias de DNA/genética , Emulsões/química , Desenho de Equipamento , Feminino , Humanos , Gravidez , Diagnóstico Pré-Natal/métodos , Esclerose Tuberosa/genéticaRESUMO
Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.
Assuntos
DNA/genética , RNA/genética , Análise de Sequência de RNA/métodos , Quimera/genética , DNA Complementar/genética , Biblioteca Gênica , Células HEK293 , Células HeLa , Humanos , Análise de Célula Única , Transposases/metabolismoRESUMO
BACKGROUND: Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. RESULTS: Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. CONCLUSIONS: SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.
Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , DNA , DNA Complementar/genética , DNA Complementar/metabolismo , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Fatores de Transcrição/genéticaRESUMO
Doublet emission from open-shell molecules has demonstrated its research and application value in recent years. However, understandings of the photoluminescence mechanism of open-shell molecules are far less than that of closed-shell molecules, leading to challenges in molecular design of efficient doublet emission systems. Here we report a cerium(III) 4-(9H-carbozol-9-yl)phenyl-tris(pyrazolyl)borate complex Ce(CzPhTp)3 with a new luminescence mechanism of delayed doublet emission, which also represents the first example with metal-centered delayed photoluminescence. The energy gap between the doublet and triplet excited states of Ce(CzPhTp)3 is reduced by the management of the inner and outer coordination spheres, thereby promoting efficient energy transfer between the two excited states and activating the delayed emission. The photoluminescence mechanism discovered may provide a new way for the design of efficient doublet emission and bring insights into rational molecular design and energy level regulation in open-shell molecules.
RESUMO
In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems.
Assuntos
Proteínas , Prótons , Elétrons , Íons/química , Espectrometria de Massas/métodos , Proteínas/químicaRESUMO
Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex's architecture and subunits' identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for top-down MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody-antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.
Assuntos
Medição da Troca de Deutério , Microfluídica , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Conformação Proteica , Proteínas/químicaRESUMO
Exploring photocatalysts to foster CO2 photoreduction into high value-added chemicals is of great significance. Lead halide perovskites (LHPs) have recently been extensively investigated as photocatalysts, owing to their facile fabrication and prominent optoelectronic properties. However, the toxicity of lead and instability will hinder their future large-scale applications. To address these challenges, a series of lead-free Sb-based all-inorganic mixed halide perovskite Cs3 Sb2 (Brx I1- x )9 (0 ≤ x ≤ 1) nanoplatelets (NPLs) is synthesized. The perovskite NPLs are prepared using a ligand-assisted re-precipitation approach at 50 °C. The authors observe the tunability of their optical band gaps from 2.1 to 2.5 eV, and they can maintain the excellent stability over 120 h under heating at 100 °C or UV light irradiation. The resultant materials are employed as efficient photocatalysts for visible-light driven CO2 reduction at the gas-solid interface. The Cs3 Sb2 (Br0.7 I0.3 )9 perovskite NPLs afford an impressive overall yield of 27.7 µmol g-1 for the selective photocatalytic conversion of CO2 into CO. This study represents a significant demonstration for practical artificial photosynthesis by using LHP materials as photocatalysts.