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Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called 'focused interrogation of cells by nucleic acid detection and sequencing' (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV+ cells, as well as other cell subsets that were previously difficult to recover and sequence.
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Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.
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Astrócitos , Encefalomielite Autoimune Experimental , Memória Epigenética , Esclerose Múltipla , Animais , Feminino , Humanos , Masculino , Camundongos , Acetilcoenzima A/metabolismo , Astrócitos/enzimologia , Astrócitos/metabolismo , Astrócitos/patologia , ATP Citrato (pro-S)-Liase/metabolismo , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Sistemas CRISPR-Cas , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Esclerose Múltipla/enzimologia , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Análise da Expressão Gênica de Célula Única , Transposases/metabolismoRESUMO
Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE) 1-8 . However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY + p300 + memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY + p300 + astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro ; scRNA-seq and immunohistochemistry studies detected increased ACLY + p300 + astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.
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BACKGROUND: Clear cell Renal cell carcinoma (ccRCC) is an immunogenic tumor. B7 family members, such as CTLA-4, PD-1, and PD-L1, are the main components of immune checkpoints that regulate various immune responses. Specifically, B7-H3 regulates T cell-mediated immune responses against cancer. This study aimed to analyze the association between B7-H3 and CTLA-4 expression and the prognostic factors of ccRCC to provide a basis for their potential use as predictive factors and in immunotherapy. METHODS: Formalin-fixed paraffin-embedded specimens were obtained from 244 ccRCC patients, and B7-H3, CTLA-4, and PD-L1 expressions were evaluated using immunohistochemical staining. RESULTS: B7-H3 and CTLA-4 were positive in 73 (29.9%) and 57 (23.4%) of the 244 patients, respectively. B7-H3 expression was significantly associated with PD-L1 expression (P < 0.0001); however, CTLA-4 expression was not (P = 0.842). Kaplan-Meier analysis showed that positive B7-H3 expression was associated with poor progression-free survival (PFS) (P < 0.0001), whereas CTLA-4 expression was not (P = 0.457). Multivariate analysis revealed that B7-H3 was correlated with poor PFS (P = 0.031), whereas CTLA-4 was not (P = 0.173). CONCLUSIONS: To the best of our knowledge, this study is the first to investigate B7-H3 and PD-L1 expression and survival in ccRCC. B7-H3 expression is an independent prognostic factor for ccRCC. Furthermore, multiple immune cell inhibitory targets, such as B7-H3 and PD-L1, can be used for therapeutic tumor regression in a clinical setting.
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Carcinoma de Células Renais , Neoplasias Renais , Humanos , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/análise , Carcinoma de Células Renais/patologia , Estimativa de Kaplan-Meier , Neoplasias Renais/patologia , PrognósticoRESUMO
Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.
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Astrócitos , Encefalomielite Autoimune Experimental , Microfluídica , Esclerose Múltipla , Ácidos Nucleicos , Análise da Expressão Gênica de Célula Única , Animais , Humanos , Camundongos , Astrócitos/metabolismo , Astrócitos/patologia , Regulação da Expressão Gênica , Camundongos Knockout , Esclerose Múltipla/patologia , Microfluídica/métodos , Análise da Expressão Gênica de Célula Única/métodos , Ácidos Nucleicos/análise , Edição de GenesRESUMO
Genotyping of single nucleotide variants (SNVs) has enabled the assessment of disease-related risk factors and significantly improved the potency of diagnosis and prognosis. Meanwhile, genotyping of SNVs is challenging due to the high sequence similarity between wild-type (WT) and SNV. To increase the discrimination between WT and SNV, probes are modified with nucleic acid analogues such as locked nucleic acid (LNA), or deliberate mismatches are introduced to the probe sequence. However, nucleic acid analogues have limitation in high cost and complexity in their synthesis. And a generalized methodology has not been proposed for determining the position and type of deliberate mismatches at the designated experimental conditions to the best of our knowledge. Herein, we propose a reliable workflow for designing mismatch-introduced probes (MIPs) based on nucleic acid thermodynamic analysis and rejection sampling. The theoretical hybridization state of MIP was calculated using nucleic acid thermodynamics, and the detectability was estimated by rejection sampling that simulates the errors from experimental environments. We fabricated MIPs for SNVs in epidermal growth factor receptor, and experimentally demonstrated optimized detectability. The detectability increased up to 7.19-fold depending on the position and type of mismatch; moreover, the optimized MIP showed higher detectability than the LNA probe. This indicates that the workflow can be broadly applied to the optimization of probe sequence for the detection of various disease-related SNVs.
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Pareamento Incorreto de Bases , Ácidos Nucleicos , Sondas de DNA , Hibridização de Ácido Nucleico/métodos , TermodinâmicaRESUMO
The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity. However, it remains challenging to develop vaccine nanoparticles that can preserve and present conformational epitopes of protein antigens for induction of neutralizing antibody responses. Here, we have designed a new lipid-based nanoparticle vaccine platform (NVP) that presents viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses. We show that NVP was readily taken up by dendritic cells (DCs) and promoted DC maturation and antigen presentation. NVP loaded with BG505.SOSIP.664 (SOSIP) or SARS-CoV-2 receptor-binding domain (RBD) was readily recognized by neutralizing antibodies, indicating the conformational display of antigens on the surfaces of NVP. Rabbits immunized with SOSIP-NVP elicited strong neutralizing antibody responses against HIV-1. Furthermore, mice immunized with RBD-NVP induced robust and long-lasting antibody responses against RBD from SARS-CoV-2. These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens.
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Vacinas contra a AIDS/química , Vacinas contra COVID-19/química , Imunidade Humoral/efeitos dos fármacos , Lipídeos/química , Nanopartículas , Vacinas Virais/química , Vacinas contra a AIDS/administração & dosagem , Adjuvantes Imunológicos , Animais , Apresentação de Antígeno , Reações Antígeno-Anticorpo , Vacinas contra COVID-19/administração & dosagem , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , HIV-1 , Humanos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , SARS-CoV-2 , Vacinas Virais/administração & dosagemRESUMO
Owing to its precise manipulation in nanoscale, DNA as a genetic code becomes a promising and generic material in lots of nanotechnological outstanding exploitations. The nanoscale assembly of nucleic acids in aqueous solution has showed very remarkable capability that is not achievable from any other material resources. In the meantime, their striking role played by effective intracellular interactions have been identified, making these more attractive for a variety of biological applications. Lately, a number of interesting attempts have been made to augment their marvelous diagnostic and therapeutic capabilities, as being integrated with inorganic compounds involving gold, iron oxide, quantum dot, upconversion, etc. It was profoundly studied how structural DNA-inorganic hybrid materials have complemented with each other in a synergistic way for better-graded biological performances. Such hybrid materials consisting of both structural DNAs and inorganics are gradually receiving much attention as a practical and future-oriented material substitute. However, any special review articles highlighting the significant and innovative materials have yet to be published. At the first time, we here demonstrate novel hybrid complexes made of structural DNAs and inorganics for some practical applications.
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Effective intermolecular interaction is required between probe and target molecules for successful detection of biomarkers. Here, we demonstrate that localization of probes on DNA nanostructures improves detection sensitivity and reaction rate. The structural flexibility of DNA nanostructures enabled frequent intramolecular interactions among the localized probes. The Smoluchowski coagulation method and the coarse-grained molecular dynamic software oxDNA were used for theoretical estimation of inter- and intramolecular behaviors of the DNA nanostructures as well as adequate experiments verifying the improvements in sensitivity with probe localization. Remarkably, the probe-localized DNA nanostructure had an increased sensitivity up to 274 times higher than that of the same probes without localization. We believe this achievement represents a wide applicability as a potential design strategy for robust, reliable, and sensitive biosensors.
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Técnicas Biossensoriais , Sondas de DNA/química , DNA/análise , Nanoestruturas/química , Biomarcadores/análise , DNA/química , Humanos , Simulação de Dinâmica Molecular , Sensibilidade e Especificidade , SoftwareRESUMO
Nanoparticles (NPs) of protein-based materials have become one of the most promising candidates for drug carriers in drug-delivery systems because of their in vivo nontoxicity, biodegradability, compatibility with hydrophilic drugs, and adaptability to the human body. Many studies have investigated the fabrication of protein NPs from human serum albumin (HSA) as a new drug carrier. It is important for these NPs to remain in the blood until they reach their therapeutic target to achieve the desired effect; the quicker the clearance of drugs from the body, the shorter is the residence time of drugs in the body, which eventually reduces drug efficacy. Macrophage uptake is a major mechanism for clearance of NPs from the body, so, reducing the degree of macrophage uptake is a major challenge in drug-delivery systems. Original studies of HSA NP uptake by macrophages showed that denatured HSA and HSA NPs synthesized with 80% (v/v) ethanol showed a high degree of macrophage uptake. We found that HSA NPs synthesized with lower ethanol content at pH 7 showed lower macrophage uptake in in vitro macrophage cellular uptake experiments. The effects of the preparation parameters of ethanol concentration, pH, and glutaraldehyde on the macrophage uptake of NPs were thoroughly studied. This newly developed protein NP with lower macrophage uptake has potential application as a drug carrier for many delivery systems.
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Sistema Fagocitário Mononuclear , Nanopartículas/química , Albumina Sérica Humana/química , Animais , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Sistemas de Liberação de Medicamentos , Etanol/química , Feminino , Glutaral/química , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Macrófagos/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/química , Neoplasias Experimentais/metabolismo , Células PC-3 , Tamanho da Partícula , Albumina Sérica Humana/síntese química , Albumina Sérica Humana/metabolismo , Propriedades de Superfície , Distribuição TecidualRESUMO
Immunostimulatory oligodeoxynucleotides (DNAs) have been widely studied in pharmaceutical and biomedical research fields for applications in cancer immunotherapy and vaccination. Toll-like receptors (TLRs) are critical for the instruction and orchestration of the host immune system composed of innate and adaptive immunity. In particular, TLR9 responds to DNAs with unmethylated deoxycytosine-deoxyguanosine (CpG) motifs, thereby inducing the activation of innate immune cells, such as dendritic cells, and consequently, adaptive immune cells. In this study, we developed two kinds of Y-shaped double-stranded DNA nanostructures (Y-DNAs), including a single unit composed of three DNA strands (YS-DNA) and a ligated multiunit complex formed by crosslinking each YS-DNA (YL-DNA), and investigated whether they have immunostimulatory activity in innate immune cells. YS-DNA and YL-DNA induced the production of immune cytokines such as IL-12 and TNF-α and the expression of costimulatory molecules such as CD80 and CD86 in primary mouse dendritic cells and macrophage cells (RAW264.7 cells). A Coprecipitation study demonstrated that YL-DNA was directly associated with TLR9. The induction of immune cytokines by YS-DNA and YL-DNA was abolished in TLR9-deficient primary mouse dendritic cells. The results demonstrated that Y-DNAs induced the activation of dendritic cells and macrophages mediated by the activation of TLR9, as shown by the expression of immune cytokines and costimulatory molecules. The results suggest that Y-DNA nanostructures provide a beneficial strategy for immunotherapy by modulating the immune system.
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DNA/química , Imunidade Celular/efeitos dos fármacos , Nanoestruturas/química , Oligodesoxirribonucleotídeos/química , Receptor Toll-Like 9/metabolismo , Animais , DNA/administração & dosagem , DNA/imunologia , Células HEK293 , Humanos , Imunidade Celular/fisiologia , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanoestruturas/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Receptor Toll-Like 9/imunologiaRESUMO
In biological systems, a few sequence differences diversify the hybridization profile of nucleotides and enable the quantitative control of cellular metabolism in a cooperative manner. In this respect, the information required for a better understanding may not be in each nucleotide sequence, but representative information contained among them. Existing methodologies for nucleotide sequence design have been optimized to track the function of the genetic molecule and predict interaction with others. However, there has been no attempt to extract new sequence information to represent their inheritance function. Here, we tried to conceptually reveal the presence of a representative sequence from groups of nucleotides. The combined application of the K-means clustering algorithm and the social network analysis theorem enabled the effective calculation of the representative sequence. First, a "common sequence" is made that has the highest hybridization property to analog sequences. Next, the sequence complementary to the common sequence is designated as a 'representative sequence'. Based on this, we obtained a representative sequence from multiple analog sequences that are 8â»10-bases long. Their hybridization was empirically tested, which confirmed that the common sequence had the highest hybridization tendency, and the representative sequence better alignment with the analogs compared to a mere complementary.
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Biologia Computacional , Nucleotídeos , Oligonucleotídeos , Algoritmos , Sequência de Bases , Biologia Computacional/métodos , Nucleotídeos/química , Nucleotídeos/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Alinhamento de Sequência , SoftwareRESUMO
We screened, for the first time, plasmid-mediated colistin resistance mcr-3 genes among 636 Escherichia coli isolates collected from swine in South Korea. Whole-genome sequencing showed that the E. coli strain harbored the mcr-3 gene in a p17S-208 plasmid with an IncHI2-ST3 plasmid type and a size of 260,399 base pairs. The deduced amino acid sequences revealed that persistent evolution in the bacterial genome has resulted in mcr gene variants. There is a need for extensive surveillance to prevent the dissemination of colistin resistance mcr genes from animal to human.
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Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Plasmídeos/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Animais , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Genoma Bacteriano/genética , Testes de Sensibilidade Microbiana/métodos , República da Coreia , SuínosRESUMO
RNA biomarkers have been recently reported to be associated tightly with the diagnosis and prognosis of many diseases. Particularly, cancers considered to be a serious threat to primates are known to be vastly dominated by genetic networks where RNA plays a key role. RNAs are thus recognized as a major target group that can be used for numerous cancer treatments and it is still required to identify and enumerate them in an effective manner. Here, a new topological transformation-based nanobarcoding technique (TNT) is first reported using fluorescence-DNA barcodes engaged with graphene oxide (GOx ) for effectively discriminating short RNAs such as miRNAs and their single nucleotide polyporphisms in tissue and plasma. Through topological transformation into 3D DNA-RNA polygonal structures, various kinds of microRNAs have been read at the same time and analyzed quantitatively. Also, it positively discerned epidermal growth factor receptor (EGFR) mutations known as single base variations of typical lung cancer specific RNAs. A single variant of 0.785% in target EGFR mutations is explicitly detected. It is speculated that the TNT may be a versatile method for polymerase chain reaction (PCR)-free practical diagnosis of several clinical genetic deviations such as significant biotic RNA and genic fragments and would be a promising alternative to conventional PCR terrains.
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Código de Barras de DNA Taxonômico , Grafite , MicroRNAs/genética , Mutação , Polimorfismo de Nucleotídeo Único , Receptores ErbB/genética , HumanosRESUMO
BACKGROUND: Carriage of antibiotic-resistant foodborne pathogens by food production animals is one of many contributors to treatment failure in health care settings, and it necessitates an integrated approach to investigate the carriage of resistant pathogens harboring integrons in food-producing animals. METHODS: Escherichia coli isolates with reduced susceptibility to tetracycline antibiotics (n = 92) were tested for associations between carriage of class1 integrons, phylogenetic group affiliation and tetracycline resistance determinants using the MIC method, PFGE analysis, PCR and sequencing. RESULTS: Phylogroups B1 and A were the most common (58.7 and 19.6%, respectively), followed by groups D (20.7%) and B2 (1.1%). All isolates carried at least one of the tet genes examined. In addition, 88 (95.7%) of all tetracycline-resistant isolates carried tet(A) or tet(B), while 47 (51.1%) and 41 (44.6%) harbored only tet(A) or tet(B), respectively. Likewise, isolates harboring these genes had a higher chance (P < 0.05) of carrying class 1 integrons. Of the tested isolates, 38 (41.3%) carried the intI1 gene. Classical integrons with complete genes (sul1 and qacE∆1) at the 3'-CS were recognized in 27 isolates. PCR screening and subsequent sequencing demonstrated that 84.2% (32/38) of the intI1-positive isolates harbored resistance gene cassettes. Overall, seven gene cassettes were identified, either solely or combined with another gene cassette. The most common gene was aadA1 (10 isolates), followed by a combination of aadA1-dfrA1 (seven isolates), aadA1-dfrA12 (six isolates) and aadA1-aadA2-dfrA12 (three isolates). Genetic typing using PFGE showed minimum clonal relatedness with 28 different clusters and 12-25 discernible DNA fragments. CONCLUSIONS: This study brings new insight into the relationships between the presence of integrons, phylogenetic group association and characteristics of tetracycline antibiotic resistance determinants in commensal E. coli strains.
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Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/genética , Integrons/genética , Resistência a Tetraciclina/genética , Animais , Antibacterianos/uso terapêutico , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Testes de Sensibilidade Microbiana/veterinária , Filogenia , República da Coreia , Tetraciclina/uso terapêuticoRESUMO
The magnetic properties of nanoparticles make them ideal for using in various applications, especially in biomedical applications. However, the magnetic force generated by a single nanoparticle is low. Herein, we describe the development of nanocomplexes (size of 100 nm) of many iron oxide nanoparticles (IONPs) encapsulated in poly(lactic- co-glycolic acid) (PLGA) using the simple method of emulsion solvent evaporation. The response of the IONP-encapsulated PLGA nanocomplexes (IPNs) to an external magnetic field could be controlled by modifying the amount of IONPs loaded into each nanocomplex. In a constant size of IPNs, larger loading numbers of IONPs resulted in more rapid responses to a magnetic field. In addition, nanocomplexes were coated with a silica layer to facilitate the addition of fluorescent dyes. This allowed visualization of the responses of the IPNs to an applied magnetic field corresponding to the IONP loading amount. We envision that these versatile, easy-to-fabricate IPNs with controllable magnetism will have important potential applications in diverse fields.
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Nanoparticles are used extensively to detect nucleic acid biomarkers due to their analytical applicability and sensitivity. Systems employing the surface plasmon resonance of gold nanomaterials are overwhelmingly considered to be candidates. The aggregation of gold nanomaterials mediated by the hybridization of target DNA at the interface causes a change in the surface plasmon resonance inherent in gold nanomaterials. Such changes can be measured by spectroscopy or even visualized by the naked eye, enabling effective and positive detection. The optical properties of gold nanoparticles are affected by their shape. The geometric appearance of the nanoparticles also affects their colloidal stability and aggregation behavior. In this study, we examined the effect of the geometric appearance of gold nanomaterials on DNA-mediated aggregation behavior through comparative experiments. Experimental and theoretical methods were used concurrently to derive accurate results and to support the hypotheses. Coarse-grained molecular dynamics simulations were performed with a large-scale atomic/molecular massively parallel simulator to understand the aggregation of nanoparticles with the same surface area and various aspect ratios. As a result, we confirmed that the aggregation sensitivity of nanoparticles was affected by the shape of the contact point with the gold nanomaterials. This study demonstrates that the design of a detection system should be accompanied by an in-depth review of the morphology of the nanoparticle.
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Sondas de DNA/química , DNA de Cadeia Simples/química , Ouro/química , Nanopartículas Metálicas/química , Nanotubos/química , Sequência de Bases , Sondas de DNA/genética , DNA de Cadeia Simples/genética , Humanos , Simulação de Dinâmica Molecular , Hibridização de Ácido Nucleico , Tamanho da Partícula , Ressonância de Plasmônio de Superfície/métodos , Telomerase/químicaRESUMO
We hereby report the first characterization of mcr-3 gene from healthy animals in South Korea. Out of 636 E. coli isolates, collected between 2014- 2017, nine colistin resistant isolates were screened for the presence of mcr-1 and mcr-3 genes. Nine (1.4%) isolates had shown resistance for colistin and among them three and two isolates were mcr-1 harboring and mcr-3 harboring strains, respectively. All the colistin-resistant isolates were multidrug-resistant. mcr-1 and mcr-3 genes were confirmed to be transferred to a recipient E. coli J53 AZR.