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1.
BMC Genomics ; 25(1): 227, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429743

RESUMO

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.


Assuntos
Artefatos , Genoma Humano , Humanos , Biblioteca Gênica , Análise de Sequência de DNA/métodos , DNA de Neoplasias , Sequenciamento de Nucleotídeos em Larga Escala/métodos
2.
J Vis Exp ; (204)2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38465925

RESUMO

Transcriptomics allows to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Avaliação Pré-Clínica de Medicamentos , Biblioteca Gênica , Custos e Análise de Custo
3.
Methods Mol Biol ; 2774: 1-13, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38441754

RESUMO

Directed evolution is an efficient strategy for obtaining desired biomolecules. Since the 1990s, the emergence of display techniques has enabled high-throughput screening of functional proteins. However, classical methods require library construction by plasmid cloning and are limited by transformation efficiencies, typically limiting library sizes to ~106-107 variants. More recently, in vitro techniques have emerged that avoid cloning, allowing library sizes of >1012 members. One of these, CIS display, is a DNA-based display technique which allows high-throughput selection of biomolecules in vitro. CIS display creates the genotype-phenotype link required for selection by a DNA replication initiator protein, RepA, that binds exclusively to the template from which it has been expressed. This method has been successfully used to evolve new protein-protein interactions but has not been used before to select DNA-binding proteins, which are major components in mammalian synthetic biology. In this chapter, we describe a directed evolution method using CIS display to efficiently select functional DNA-binding proteins from pools of nonbinding proteins. The method is illustrated by enriching the minimal transcription factor Cro from a low starting frequency (1 in 109). This protocol is also applicable to engineering other DNA-binding proteins or transcription factors from combinatorial libraries.


Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , Animais , Fatores de Transcrição/genética , Biblioteca Gênica , Proteínas de Ligação a DNA/genética , Clonagem Molecular , DNA/genética , Mamíferos
4.
Methods Mol Biol ; 2774: 135-152, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38441763

RESUMO

Sequencing-based, massively parallel genetic assays have enabled simultaneous characterization of the genotype-phenotype relationships for libraries encoding thousands of unique protein variants. Since plasmid transfection and lentiviral transduction have characteristics that limit multiplexing with pooled libraries, we developed a mammalian synthetic biology platform that harnesses the Bxb1 bacteriophage DNA recombinase to insert single promoterless plasmids encoding a transgene of interest into a pre-engineered "landing pad" site within the cell genome. The transgene is expressed behind a genomically integrated promoter, ensuring only one transgene is expressed per cell, preserving a strict genotype-phenotype link. Upon selecting cells based on a desired phenotype, the transgene can be sequenced to ascribe each variant a phenotypic score. We describe how to create and utilize landing pad cells for large-scale, library-based genetic experiments. Using the provided examples, the experimental template can be adapted to explore protein variants in diverse biological problems within mammalian cells.


Assuntos
Bacteriófagos , Genômica , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Biblioteca Gênica , Bioensaio , Proteínas Mutantes , Mamíferos
5.
Eur Rev Med Pharmacol Sci ; 28(5): 1976-1986, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38497880

RESUMO

OBJECTIVE: Leaving Against Medical Advice (LAMA) is a prevalent issue in healthcare settings that may lead to negative patient outcomes. We conducted a systematic review and meta-analysis to assess the impact of LAMA on patient outcomes. MATERIALS AND METHODS: A comprehensive literature search was performed across PubMed, MEDLINE, Embase, Cochrane Library, CINAHL, PsycINFO, Web of Science, and Scopus. Studies reporting adverse outcomes, including mortality and hospital readmission rates, in patients who underwent LAMA were included. The odds ratios (ORs) with 95% confidence intervals (CIs) were pooled using a random-effects model. RESULTS: Eight studies were included in the review, with four contributing to the meta-analysis on 1-year mortality and five to the meta-analysis on hospital readmission rates. LAMA was not significantly associated with higher 1-year mortality [OR = 0.66, 95% CI (0.38, 1.16), p = 0.15] or hospital readmission rates [OR = 0.61, 95% CI (0.30, 1.23), p = 0.16] across the studies. However, there was substantial heterogeneity in the results (I2 = 91% for mortality; I2 = 99% for readmissions). CONCLUSIONS: While individual studies reported varying outcomes, the pooled results did not show a significant association between LAMA and increased 1-year mortality or hospital readmission rates. However, the high degree of heterogeneity suggests the influence of diverse patient populations, healthcare settings, and study methodologies on these outcomes. Further research is needed to better understand the factors contributing to the adverse outcomes associated with LAMA and to develop targeted interventions to mitigate them.


Assuntos
Readmissão do Paciente , Pacientes , Humanos , Biblioteca Gênica , Razão de Chances
6.
Proc Natl Acad Sci U S A ; 121(11): e2311726121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38451939

RESUMO

Proteins are a diverse class of biomolecules responsible for wide-ranging cellular functions, from catalyzing reactions to recognizing pathogens. The ability to evolve proteins rapidly and inexpensively toward improved properties is a common objective for protein engineers. Powerful high-throughput methods like fluorescent activated cell sorting and next-generation sequencing have dramatically improved directed evolution experiments. However, it is unclear how to best leverage these data to characterize protein fitness landscapes more completely and identify lead candidates. In this work, we develop a simple yet powerful framework to improve protein optimization by predicting continuous protein properties from simple directed evolution experiments using interpretable, linear machine learning models. Importantly, we find that these models, which use data from simple but imprecise experimental estimates of protein fitness, have predictive capabilities that approach more precise but expensive data. Evaluated across five diverse protein engineering tasks, continuous properties are consistently predicted from readily available deep sequencing data, demonstrating that protein fitness space can be reasonably well modeled by linear relationships among sequence mutations. To prospectively test the utility of this approach, we generated a library of stapled peptides and applied the framework to predict affinity and specificity from simple cell sorting data. We then coupled integer linear programming, a method to optimize protein fitness from linear weights, with mutation scores from machine learning to identify variants in unseen sequence space that have improved and co-optimal properties. This approach represents a versatile tool for improved analysis and identification of protein variants across many domains of protein engineering.


Assuntos
Aprendizado de Máquina , Proteínas , Proteínas/metabolismo , Engenharia de Proteínas/métodos , Mutação , Biblioteca Gênica
7.
STAR Protoc ; 5(1): 102908, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38461411

RESUMO

Processing dissociated cells for transcriptomics is challenging when targeting small brain structures, like brainstem nuclei, where cell yield may be low. Here, we present a protocol for dissecting, dissociating, and cryopreserving mouse brainstem that allows asynchronous sample collection and downstream processing of cells obtained from brainstem tissue in neonatal mice. Although we demonstrate this protocol with the isolated preBötzinger complex and downstream SmartSeq3 cDNA library preparation, it could be readily adapted for other brainstem areas and library preparation approaches.


Assuntos
Tronco Encefálico , Análise da Expressão Gênica de Célula Única , Camundongos , Animais , Núcleo Celular , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica
8.
PLoS One ; 19(3): e0300865, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38517905

RESUMO

Entomological sampling and storage conditions often prioritise efficiency, practicality and conservation of morphological characteristics, and may therefore be suboptimal for DNA preservation. This practice can impact downstream molecular applications, such as the generation of high-throughput genomic libraries, which often requires substantial DNA input amounts. Here, we use a practical Tn5 transposase tagmentation-based library preparation method optimised for 96-well plates and low yield DNA extracts from insect legs that were stored under sub-optimal conditions for DNA preservation. The samples were kept in field vehicles for extended periods of time, before long-term storage in ethanol in the freezer, or dry at room temperature. By reducing DNA input to 6ng, more samples with sub-optimal DNA yields could be processed. We matched this low DNA input with a 6-fold dilution of a commercially available tagmentation enzyme, significantly reducing library preparation costs. Costs and workload were further suppressed by direct post-amplification pooling of individual libraries. We generated medium coverage (>3-fold) genomes for 88 out of 90 specimens, with an average of approximately 10-fold coverage. While samples stored in ethanol yielded significantly less DNA compared to those which were stored dry, these samples had superior sequencing statistics, with longer sequencing reads and higher rates of endogenous DNA. Furthermore, we find that the efficiency of tagmentation-based library preparation can be improved by a thorough post-amplification bead clean-up which selects against both short and large DNA fragments. By opening opportunities for the use of sub-optimally preserved, low yield DNA extracts, we broaden the scope of whole genome studies of insect specimens. We therefore expect these results and this protocol to be valuable for a range of applications in the field of entomology.


Assuntos
DNA , Sequenciamento de Nucleotídeos em Larga Escala , Transposases , DNA/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Etanol , Análise de Sequência de DNA/métodos
9.
Methods Mol Biol ; 2754: 131-146, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38512665

RESUMO

Tau protein was extensively studied using nuclear magnetic resonance spectroscopy, providing a powerful way to determine interaction sites between Tau and partner proteins. Here we used this analytical tool to describe the epitopes of Tau-specific VHHs (variable domain of the heavy chain of the heavy chain-only antibodies, aka nanobodies) selected from a synthetic library. An in vitro Tau aggregation assay was subsequently used as a functional screen to check VHH efficacy as aggregation inhibitors. We have observed a correlation between the targeted epitope and the aggregation-inhibition capacity of a series of Tau-specific VHHs.


Assuntos
Anticorpos de Domínio Único , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/química , Proteínas tau/genética , Epitopos , Cadeias Pesadas de Imunoglobulinas/química , Biblioteca Gênica
10.
Sci Rep ; 14(1): 6756, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514891

RESUMO

Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different Escherichia coli strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience.


Assuntos
Elementos de DNA Transponíveis , Genes Bacterianos , Mutagênese Insercional , Elementos de DNA Transponíveis/genética , Análise Custo-Benefício , Sequência de Bases , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biblioteca Gênica
11.
Genome Biol ; 25(1): 72, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38504331

RESUMO

DANCE is the first standard, generic, and extensible benchmark platform for accessing and evaluating computational methods across the spectrum of benchmark datasets for numerous single-cell analysis tasks. Currently, DANCE supports 3 modules and 8 popular tasks with 32 state-of-art methods on 21 benchmark datasets. People can easily reproduce the results of supported algorithms across major benchmark datasets via minimal efforts, such as using only one command line. In addition, DANCE provides an ecosystem of deep learning architectures and tools for researchers to facilitate their own model development. DANCE is an open-source Python package that welcomes all kinds of contributions.


Assuntos
Benchmarking , Aprendizado Profundo , Humanos , Algoritmos , Biblioteca Gênica , Análise de Célula Única
12.
Methods Mol Biol ; 2776: 243-257, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38502509

RESUMO

Global understanding of plastid gene expression has always been impaired by its complexity. RNA splicing, editing, and intercistronic processing create multiple transcripts isoforms that can hardly be resolved using traditional molecular biology techniques. During the last decade, the wide adoption of RNA-seq-based techniques has, however, allowed an unprecedented understanding of all the different steps of chloroplast gene expression, from transcription to translation. Current strategies are nonetheless unable to identify and quantify full length transcripts isoforms, a limitation that can now be overcome using Nanopore Sequencing. We here provide a complete protocol to produce, from total leaf RNA, cDNA libraries ready for Nanopore sequencing. While most Nanopore protocols take advantage of the mRNA polyA tail we here first ligate an RNA adapter to the 3' ends of the RNAs and use it to initiate the template switching reverse transcription. The cDNA is then prepared and indexed for use with the regular Oxford Nanopore v14 chemistry. This protocol is of particular interest to researchers willing to simultaneously study the multiple post-transcriptional processes prevalent in the chloroplast.


Assuntos
Sequenciamento por Nanoporos , Transcriptoma , Sequenciamento por Nanoporos/métodos , Biblioteca Gênica , RNA/genética , Isoformas de Proteínas/genética , Cloroplastos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos
13.
Sci Rep ; 14(1): 3130, 2024 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-38326509

RESUMO

The Tambaqui is one of the most representative Amazon fish species, being highly exploited in fisheries, aquaculture and as a research model. Nonetheless, data about functional genome are still required to evaluate reproductive and nutrition parameters as well as resistance to pathogens. The of next-generation sequencing has allows assessing the transcriptional processes in non-model species by providing comprehensive gene collections to be used as a database in further genomic applications and increased performance of captive populations. In this study, we relied on RNAseq approach to generate the first transcriptome of the telencephalon from adult males and females of Colossoma macropomum, resulting in a reference dataset for future functional studies. We retrieved 896,238 transcripts, including the identification of 267,785 contigs and 203,790 genes. From this total, 91 transcripts were differentially expressed, being 63 and 28 of them positively regulated for females and males, respectively. The functional annotation resulted in a library of 40 candidate genes for females and 20 for males. The functional enrichment classes comprised reproductive processes (GO:0,048,609; GO:0,003,006; GO:0,044,703; GO:0,032,504; GO:0,019,953) being related to sex differentiation (e.g., SAFB) and immune response (e.g., SLC2A6, AHNAK, NLRC3, NLRP3 and IgC MHC I alpha3), thus indicating that the genes in the neurotranscriptome of Tambaqui participate in sex differentiation and homeostasis of captive specimens. These data are useful to design the selection of genes related to sex determination and animal welfare in raising systems of Tambaqui.


Assuntos
Caraciformes , Animais , Masculino , Feminino , Caraciformes/genética , Aquicultura , Pesqueiros , Genômica , Biblioteca Gênica
14.
Exp Mol Med ; 56(2): 453-460, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38413820

RESUMO

The major drawbacks of RNA sequencing (RNA-seq), a remarkably accurate transcriptome profiling method, is its high cost and poor scalability. Here, we report a highly scalable and cost-effective method for transcriptomics profiling called Bulk transcriptOme profiling of cell Lysate in a single poT (BOLT-seq), which is performed using unpurified bulk 3'-end mRNA in crude cell lysates. During BOLT-seq, RNA/DNA hybrids are directly subjected to tagmentation, and second-strand cDNA synthesis and RNA purification are omitted, allowing libraries to be constructed in 2 h of hands-on time. BOLT-seq was successfully used to cluster small molecule drugs based on their mechanisms of action and intended targets. BOLT-seq competes effectively with alternative library construction and transcriptome profiling methods.


Assuntos
Perfilação da Expressão Gênica , RNA , RNA/genética , RNA Mensageiro/genética , Biblioteca Gênica , DNA Complementar/genética , Perfilação da Expressão Gênica/métodos
15.
Mol Biol Rep ; 51(1): 367, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38411701

RESUMO

BACKGROUND: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility. METHODS: Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)6-tagged uvsY. RESULTS: Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY. CONCLUSIONS: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.


Assuntos
Aminoácidos , Recombinases , Recombinases/genética , Solubilidade , Biblioteca Gênica , Mutagênese
16.
Brief Bioinform ; 25(2)2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38366803

RESUMO

The evolution in single-cell RNA sequencing (scRNA-seq) technology has opened a new avenue for researchers to inspect cellular heterogeneity with single-cell precision. One crucial aspect of this technology is cell-type annotation, which is fundamental for any subsequent analysis in single-cell data mining. Recently, the scientific community has seen a surge in the development of automatic annotation methods aimed at this task. However, these methods generally operate at a steady-state total cell-type capacity, significantly restricting the cell annotation systems'capacity for continuous knowledge acquisition. Furthermore, creating a unified scRNA-seq annotation system remains challenged by the need to progressively expand its understanding of ever-increasing cell-type concepts derived from a continuous data stream. In response to these challenges, this paper presents a novel and challenging setting for annotation, namely cell-type incremental annotation. This concept is designed to perpetually enhance cell-type knowledge, gleaned from continuously incoming data. This task encounters difficulty with data stream samples that can only be observed once, leading to catastrophic forgetting. To address this problem, we introduce our breakthrough methodology termed scEVOLVE, an incremental annotation method. This innovative approach is built upon the methodology of contrastive sample replay combined with the fundamental principle of partition confidence maximization. Specifically, we initially retain and replay sections of the old data in each subsequent training phase, then establish a unique prototypical learning objective to mitigate the cell-type imbalance problem, as an alternative to using cross-entropy. To effectively emulate a model that trains concurrently with complete data, we introduce a cell-type decorrelation strategy that efficiently scatters feature representations of each cell type uniformly. We constructed the scEVOLVE framework with simplicity and ease of integration into most deep softmax-based single-cell annotation methods. Thorough experiments conducted on a range of meticulously constructed benchmarks consistently prove that our methodology can incrementally learn numerous cell types over an extended period, outperforming other strategies that fail quickly. As far as our knowledge extends, this is the first attempt to propose and formulate an end-to-end algorithm framework to address this new, practical task. Additionally, scEVOLVE, coded in Python using the Pytorch machine-learning library, is freely accessible at https://github.com/aimeeyaoyao/scEVOLVE.


Assuntos
Algoritmos , Análise da Expressão Gênica de Célula Única , Benchmarking , Entropia , Biblioteca Gênica , Análise de Sequência de RNA , Perfilação da Expressão Gênica , Análise por Conglomerados
17.
J Clin Microbiol ; 62(3): e0010322, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38315007

RESUMO

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. In addition, the enormous demand for WGS on supply chains and the resulting shortages of laboratory supplies necessitated the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method (the BCCDC cutdown method) that can be used in a high-throughput scenario, where one technologist can perform 576 library preparations (6 plates of 96 samples) over the course of one 8-hour shift. The same protocol can also be used in a rapid turnaround time scenario, from primary samples (up to 96 samples) to loading on a sequencer in an 8-hour shift. This new method uses Freed et al.'s 1,200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and condensed Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original protocol, the application of this new method using hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% of the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results demonstrate that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 WGS protocol. IMPORTANCE: Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Análise Custo-Benefício , Pandemias , Biblioteca Gênica , DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos
18.
Gene ; 908: 148278, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38360121

RESUMO

Eucommia ulmoides (E. ulmoides) is widely cultivated and exhibits remarkable adaptability in China. It is the most promising rubber source plant in the temperate zone. E. ulmoides gum (EUG) is a trans-polyisoprene with a unique "rubber-plastic duality", and is widely used in advanced materials and biomedical fields. The transcription of Farnesyl pyrophosphate synthase (FPS), the rate-limiting enzyme of EUG biosynthesis, is controlled by regulatory mechanisms that remain poorly elucidated. In this research, 12 TGA transcription factors (TFs) in E. ulmoides were identified. Promoter prediction results revealed that the EuFPS1 promoter had binding sites for EuTGAs. Subsequently, the EuTGA1 was obtained by screening the E. ulmoides cDNA library using the EuFPS1 promoter as a bait. The individual yeast one­hybrid and dual-luciferase assays confirmed that in the tobacco plant, EuTGA1 interacted with the EuFPS1 promoter, resulting in a more than threefold increase in the activity of the EuFPS1. Subcellular localization study further revealed that EuTGA1 is localized in the nucleus and acts as a TF to regulate EuFPS1 expression. In addition, qRT-PCR analysis demonstrated that the expression trend of EuFPS1 and EuTGA1 was the same at different time of the year. Notably, low temperature and MeJA treatments down-regulated EuTGA1 expression. Additionally, the transient transformation of EuTGA1 enhanced NtFPS1 expression in tobacco plants. Overall, this study identified a TF that interacted with EuFPS1 promoter to positively regulate EuFPS1 expression. The findings of this study provide a theoretical basis for further research on the expression regulation of EuFPS1.


Assuntos
Eucommiaceae , Borracha , Borracha/metabolismo , Eucommiaceae/genética , Eucommiaceae/química , Eucommiaceae/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Biblioteca Gênica , Geraniltranstransferase/genética
19.
Antiviral Res ; 224: 105838, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38373533

RESUMO

Rabies, a viral zoonosis, is responsible for almost 59,000 deaths each year, despite the existence of an effective post-exposure prophylaxis. Indeed, rabies causes acute encephalomyelitis, with a case-fatality rate of 100 % after the onset of neurological clinical signs. Therefore, the development of therapies to inhibit the rabies virus (RABV) is crucial. Here, we identified, from a 30,000 compound library screening, phthalazinone derivative compounds as potent inhibitors of RABV infection and more broadly of Lyssavirus and even Mononegavirales infections. Combining in vitro experiments, structural modelling, in silico docking and in vivo assays, we demonstrated that phthalazinone derivatives display a strong inhibition of lyssaviruses infection by acting directly on the replication complex of the virus, and with noticeable effects in delaying the onset of the clinical signs in our mouse model.


Assuntos
Lyssavirus , Vírus da Raiva , Raiva , Animais , Camundongos , Raiva/prevenção & controle , Biblioteca Gênica , Modelos Animais de Doenças
20.
Org Lett ; 26(7): 1353-1357, 2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38335275

RESUMO

DNA-encoded chemical library (DECL) technology is a commonly employed screening platform in both the pharmaceutical industry and academia. To expand the chemical space of DECLs, new and robust DNA-compatible reactions are sought after. In particular, DNA-compatible cyclization reactions are highly valued, as these reactions tend to be atom economical and thus may provide lead- and drug-like molecules. Herein, we report two new methodologies employing DNA-conjugated thiosemicarbazides as a common precursor, yielding highly substituted 1,3,4-oxadiazoles and 1,2,4-triazoles. These two novel DNA-compatible reactions feature a high conversion efficiency and broad substrate scope under mild conditions that do not observably degrade DNA.


Assuntos
Oxidiazóis , Bibliotecas de Moléculas Pequenas , Ciclização , Biblioteca Gênica , DNA/metabolismo
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