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1.
Adv Sci (Weinh) ; : e2309702, 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38704672

RESUMO

This paper presents the first scanning electron microscopy (SEM)-based DNA imaging in biological samples. This novel approach incorporates a metal-free electro-stain reagent, formulated by combining DNA-binding proteins and synthetic polymers to enhance the visibility of 2-nm-thick DNA under SEM. Notably, DNA molecules stain with proteins and polymers appear as dark lines under SEM. The resulting DNA images exhibit a thickness of 15.0±4.0 nm. As SEM is the primary platform, it integrates seamlessly with various chemically functionalized large surfaces with the aid of microfluidic devices. The approach allows high-resolution imaging of various DNA structures including linear, circular, single-stranded DNA and RNA, originating from nuclear and mitochondrial genomes. Furthermore, quantum dots are successfully visualized as bright labels that are sequence-specifically incorporated into DNA molecules, which highlights the potential for SEM-based optical DNA mapping. In conclusion, DNA imaging using SEM with the novel electro-stain offers electron microscopic resolution with the ease of optical microscopy.

2.
Talanta ; 252: 123826, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35998444

RESUMO

Microscopic visualization of DNA molecules is a simple, intuitive, and powerful method. Nonetheless, DNA-molecule quantification methods that employ microscopic visualization have not been reported so far. In this study, a new quantitative approach is presented that enables the counting of individual DNA molecules that have been rendered visible by fluorescence microscopy. Toward this, a microfluidic device was employed that directed DNA molecules into microchannels and deposited the molecules onto a positively charged surface. This microfluidic device had a vertically tapered channel inlet structure that prevented the accumulation of excess DNA molecules in the channel inlet while creating a tapering flow, thereby ensuring the even distribution of the DNA molecules in the microchannels. The channel heights and the density of positive charges on the surface were optimized for analysis. The linearity of this method with respect to the determination of the concentration of DNA in solutions was subsequently determined. The limit of detection was 0.48 fg/µL, which corresponds to 64 molecules of 7.25 kbp DNA in 1 µL of sample. This quantitative approach was finally used to count two types of plasmids co-transformed in an E. coli cell; a measurement that is typically considered challenging with gel electrophoresis.


Assuntos
Técnicas Analíticas Microfluídicas , Escherichia coli/genética , DNA/genética , DNA/análise , Microscopia de Fluorescência , Plasmídeos
3.
Anal Chem ; 94(50): 17422-17430, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36454685

RESUMO

As observed in the COVID-19 pandemic, RNA viruses continue to rapidly evolve through mutations. In the absence of effective therapeutics, early detection of new severely pathogenic viruses and quarantine of infected people are critical for reducing the spread of the viral infections. However, conventional detection methods require a substantial amount of time to develop probes specific to new viruses, thereby impeding immediate response to the emergence of viral pathogens. In this study, we identified multiple types of viruses by obtaining the spectral fingerprint of their surface proteins with probe-free surface-enhanced Raman scattering (SERS). In addition, the SERS-based method can remarkably distinguish influenza virus variants with several surface protein point mutations from their parental strain. Principal component analysis (PCA) of the SERS spectra systematically captured the key Raman bands to distinguish the variants. Our results show that the combination of SERS and PCA can be a promising tool for rapid detection of newly emerging mutant viruses without a virus-specific probe.


Assuntos
COVID-19 , Orthomyxoviridae , Vírus , Humanos , Análise Espectral Raman/métodos , Mutação Puntual , Pandemias
4.
Adv Mater ; 33(43): e2102991, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34510585

RESUMO

Cryogenic-electron microscopy (cryo-EM) is the preferred method to determine 3D structures of proteins and to study diverse material systems that intrinsically have radiation or air sensitivity. Current cryo-EM sample preparation methods provide limited control over the sample quality, which limits the efficiency and high throughput of 3D structure analysis. This is partly because it is difficult to control the thickness of the vitreous ice that embeds specimens, in the range of nanoscale, depending on the size and type of materials of interest. Thus, there is a need for fine regulation of the thickness of vitreous ice to deliver consistent high signal-to-noise ratios for low-contrast biological specimens. Herein, an advanced silicon-chip-based device is developed which has a regular array of micropatterned holes with a graphene oxide (GO) window on freestanding silicon nitride (Six Ny ). Accurately regulated depths of micropatterned holes enable precise control of vitreous ice thickness. Furthermore, GO window with affinity for biomolecules can facilitate concentration of the sample molecules at a higher level. Incorporation of micropatterned chips with a GO window enhances cryo-EM imaging for various nanoscale biological samples including human immunodeficiency viral particles, groEL tetradecamers, apoferritin octahedral, aldolase homotetramer complexes, and tau filaments, as well as inorganic materials.


Assuntos
Grafite
5.
Pharmaceutics ; 13(6)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071708

RESUMO

Mitochondria are intracellular energy generators involved in various cellular processes. Therefore, mitochondrial dysfunction often leads to multiple serious diseases, including neurodegenerative and cardiovascular diseases. A better understanding of the underlying mitochondrial dysfunctions of the molecular mechanism will provide important hints on how to mitigate the symptoms of mitochondrial diseases and eventually cure them. In this review, we first summarize the key parts of the genetic processes that control the physiology and functions of mitochondria and discuss how alterations of the processes cause mitochondrial diseases. We then list up the relevant core genetic components involved in these processes and explore the mutations of the components that link to the diseases. Lastly, we discuss recent attempts to apply multiple genetic methods to alleviate and further reverse the adverse effects of the core component mutations on the physiology and functions of mitochondria.

6.
Biotechnol Bioeng ; 117(12): 3924-3937, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32816306

RESUMO

Retroviral vectors show long-term gene expression in gene therapy through the integration of transgenes into the human cell genome. Murine leukemia virus (MLV), a well-studied gammaretrovirus, has been often used as a representative retroviral vector. However, frequent integrations of MLV-based vectors into transcriptional start sites (TSSs) could lead to the activation of oncogenes by enhancer effects of the genetic components within the vectors. Therefore, the MLV integration preference for TSSs limits its wider use in clinical applications. To reduce the integration preference of MLV-based vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted histones and leucine zippers, having DNA-binding property, into internal sites of MLV integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild-type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer MLV-based retroviral vectors for use in clinical applications.


Assuntos
Gammaretrovirus , Vetores Genéticos , Integrases , Proteínas Virais , Integração Viral , Gammaretrovirus/enzimologia , Gammaretrovirus/genética , Células HEK293 , Células HeLa , Humanos , Integrases/genética , Integrases/metabolismo , Zíper de Leucina , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
Yonsei Med J ; 61(6): 492-505, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32469173

RESUMO

PURPOSE: To elucidate the brain's intrinsic response to injury, we tracked the response of neural stem/progenitor cells (NSPCs) located in ventricular-subventricular zone (V-SVZ) to hypoxic-ischemic brain injury (HI). We also evaluated whether transduction of V-SVZ NSPCs with neurogenic factor NeuroD1 could enhance their neurogenesis in HI. MATERIALS AND METHODS: Unilateral HI was induced in ICR neonatal mice. To label proliferative V-SVZ NSPCs in response to HI, bromodeoxyuridine (BrdU) and retroviral particles encoding LacZ or NeuroD1/GFP were injected. The cellular responses of NSPCs were analyzed by immunohistochemistry. RESULTS: Unilateral HI increased the number of BrdU+ newly-born cells in the V-SVZ ipsilateral to the lesion while injury reduced the number of newly-born cells reaching the ipsilateral olfactory bulb, which is the programmed destination of migratory V-SVZ NSPCs in the intact brain. These newly-born cells were directed from this pathway towards the lesions. HI significantly increased the number of newly-born cells in the cortex and striatum by the altered migration of V-SVZ cells. Many of these newly-born cells differentiated into active neurons and glia. LacZ-expressing V-SVZ NSPCs also showed extensive migration towards the non-neurogenic regions ipsilateral to the lesion, and expressed the neuronal marker NeuN. NeuroD1+/GFP+ V-SVZ NSPCs almost differentiated into neurons in the peri-infarct regions. CONCLUSION: HI promotes the establishment of a substantial number of new neurons in non-neurogenic regions, suggesting intrinsic repair mechanisms of the brain, by controlling the behavior of endogenous NSPCs. The activation of NeuroD1 expression may improve the therapeutic potential of endogenous NSPCs by increasing their neuronal differentiation in HI.


Assuntos
Hipóxia-Isquemia Encefálica/terapia , Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Neurogênese , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Hipóxia-Isquemia Encefálica/patologia , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/metabolismo , Nestina/metabolismo
8.
Int J Mol Sci ; 20(17)2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31450647

RESUMO

Various recent experimental observations indicate that growing cells on engineered materials can alter their physiology, function, and fate. This finding suggests that better molecular-level understanding of the interactions between cells and materials may guide the design and construction of sophisticated artificial substrates, potentially enabling control of cells for use in various biomedical applications. In this review, we introduce recent research results that shed light on molecular events and mechanisms involved in the interactions between cells and materials. We discuss the development of materials with distinct physical, chemical, and biological features, cellular sensing of the engineered materials, transfer of the sensing information to the cell nucleus, subsequent changes in physical and chemical states of genomic DNA, and finally the resulting cellular behavior changes. Ongoing efforts to advance materials engineering and the cell-material interface will eventually expand the cell-based applications in therapies and tissue regenerations.


Assuntos
Materiais Biocompatíveis , Sobrevivência Celular , Engenharia Tecidual , Alicerces Teciduais , Animais , Materiais Biocompatíveis/química , Fenômenos Biofísicos , Técnicas de Cultura de Células , Sobrevivência Celular/genética , Fenômenos Químicos , Expressão Gênica , Humanos , Mecanotransdução Celular , Engenharia Tecidual/métodos , Alicerces Teciduais/química
9.
Sci Rep ; 9(1): 10965, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358847

RESUMO

Despite advances in the construction of catalysts based on metal oxide nanoparticles (MO NPs) for various industrial, biomedical, and daily-life applications, the biosafety concerns about these NPs still remain. Recently, the need to analyze and improve the safety of MO NPs along with attempts to enhance their catalytic performance has been strongly perceived. Here, we prepared multiple variants of Fe-doped zirconium oxide (Fe@ZrO2) NPs under different pH conditions; then, we assessed their toxicity and finally screened the variant that exhibited the best catalytic performance. To assess the NP toxicity, the prepared NPs were introduced into three types of human cells originally obtained from different body parts likely to be most affected by NPs (skin, lung, and kidney). Experimental results from conventional cellular toxicity assays including recently available live-cell imaging indicated that none of the variants exerted severe negative effects on the viability of the human cells and most NPs were intracellular localized outside of nucleus, by which severe genotoxicity is unexpected. In contrast, Fe@ZrO2 NPs synthesized under a basic condition (pH = 13.0), exhibited the highest catalytic activities for three different reactions; each was biochemical (L-cysteine oxidation) or photochemical one (4-chlorophenol degradation and OH radical formation with benzoic acid). This study demonstrates that catalytic Fe@ZrO2 NPs with enhanced activities and modest or insignificant toxicity can be effectively developed and further suggests a potential for the use of these particles in conventional chemical reactions as well as in recently emerging biomedical and daily-life nanotechnology applications.


Assuntos
Ferro/química , Nanopartículas Metálicas/química , Zircônio/química , Células A549 , Catálise , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio
10.
Nanoscale ; 11(12): 5693-5704, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30865198

RESUMO

Controlling the interactions between cells and viruses is critical for treating infected patients, preventing viral infections, and improving virus-based therapeutics. Chemical methods using small molecules and biological methods using proteins and nucleic acids are employed for achieving this control, albeit with limitations. We found, for the first time, that retroviral DNA integration patterns in the human genome, the result of complicated interactions between cells and viruses, can be engineered by adapting cells to the defined nanotopography of silica bead monolayers. Compared with cells on a flat glass surface, cells on beads with the highest curvature harbored retroviral DNAs at genomic sites near transcriptional start sites and CpG islands during infections at more than 50% higher frequencies. Furthermore, cells on the same type of bead layers contained retroviral DNAs in the genomic regions near cis-regulatory elements at frequencies that were 2.6-fold higher than that of cells on flat glass surfaces. Systems-level genetic network analysis showed that for cells on nanobeads with the highest curvature, the genes that would be affected by cis-regulatory elements near the retroviral integration sites perform biological functions related to chromatin structure and antiviral activities. Our unexpected observations suggest that novel engineering approaches based on materials with specific nanotopography can improve control over viral events.


Assuntos
DNA Viral/metabolismo , Vírus da Leucemia Murina/genética , Nanotecnologia/métodos , Ilhas de CpG , Redes Reguladoras de Genes , Genoma Humano , Células HEK293 , Humanos , Nanoestruturas/química , Dióxido de Silício/química
11.
Nanotechnology ; 30(34): 345502, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-30865941

RESUMO

The direct method of detecting a virus with extremely low concentration is recommended for the diagnosis of viral disease. In this study, coplanar-gate graphene field-effect transistors (GFETs) were built on flexible polyethylene terephthalate substrates for the attomolar detection of a virus. The GFETs exhibited a very low detection limit of 47.8 aM with relatively low source/drain voltage due to aqueous dielectric media which stabilizes viruses and antibodies for specific bonding. The antibody as a probe molecule was decorated on a graphene surface using 1-pyrenebutanoic acid succinimidyl ester that had previously been immobilized on a graphene surface. The Dirac point voltage shifted downward after dropping the virus solution, due to the electrostatic gating effect of graphene in the antigen (namely, virus)-antibody complex. The virus detection platform used in this study is expected to be beneficial for direct diagnosis in saline environments, since the performances of GFETs were not significantly affected by the presence of Na+ and Cl-. Furthermore, since our flexible and transparent virus sensors can be used in a wearable device, they provide a simple and fast method for diagnosing viruses.


Assuntos
Grafite/química , Imunoensaio/métodos , Plásticos/química , Transistores Eletrônicos , Vírus/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , HIV-1/imunologia , HIV-1/isolamento & purificação , Imunoensaio/instrumentação , Limite de Detecção , Polietilenotereftalatos/química , Eletricidade Estática , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/isolamento & purificação , Vírus/imunologia
12.
Anal Chem ; 91(9): 5677-5684, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30829035

RESUMO

Rapid diagnosis and quarantine of influenza virus mutant-infected people is critical to contain the fatal viral infection spread because effective antiviral drugs are normally not available. Conventional methods, however, cannot be used for the diagnosis because these methods need predefined labels, likely also unavailable for just emerging viruses. Here, we propose label-free identification of cells infected with different influenza viruses based on surface-enhanced Raman spectroscopy (SERS) and principal component analysis (PCA). Viral envelope proteins that are displayed on the surface of cells after infection of influenza viruses were targeted for this identification. Cells that expressed the envelope proteins of A/WSN/33 H1N1 or A/California/04/2009 H1N1 influenza viruses produced distinct SERS signals. Cells that displayed combinations of the envelope proteins from these two viral variants, an indication of emergence of a new virus, also generated characteristic SERS patterns. However, the cell's own surface proteins often hindered the identification of virally infected cells by producing SERS peaks similar to viral ones. PCA of the obtained SERS patterns could effectively capture the virus-specific signal components from the jumbled SERS peaks. Our study demonstrates a potential of combination of SERS and PCA to identify newly emerging influenza viruses through sensing the cells infected with the viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/classificação , Influenza Humana/diagnóstico , Análise de Componente Principal/métodos , Análise Espectral Raman/métodos , Proteínas do Envelope Viral/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/metabolismo , Influenza Humana/virologia
13.
Analyst ; 144(3): 921-927, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30310901

RESUMO

The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/análise , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos , DNA/química , DNA/metabolismo , Humanos
14.
Mol Ther Methods Clin Dev ; 12: 58-70, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30534579

RESUMO

The unique ability of retroviruses to integrate genes into host genomes is of great value for long-term expression in gene therapy, but only when integrations occur at safe genomic sites. To reap the benefit of using retroviruses without severe detrimental effects, we developed several murine leukemia virus (MLV)-based gammaretroviral vectors with safer integration patterns by perturbing the structure of the integrase via insertion of DNA-binding zinc-finger domains (ZFDs) into an internal position of the enzyme. ZFD insertion significantly reduced the inherent, strong MLV integration preference for genomic regions near transcriptional start sites (TSSs), which are the most dangerous spots. The altered retroviral integration pattern was related to increased formation of residual primer-binding site sequences at the 3' end of proviruses. Several ZFD insertion mutants showed lower frequencies of integrations into the TSS genome regions when having the residual primer-binding site sequences in the proviruses. Our findings not only can extend the use of retroviruses in biomedical applications, but also provide a glimpse into the mechanisms underlying retroviral integration.

15.
Molecules ; 23(9)2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30208599

RESUMO

Mitochondria are the energy-producing organelles of cells. Mitochondrial dysfunctions link to various syndromes and diseases including myoclonic epilepsy and ragged-red fiber disease (MERRF), Leigh syndrome (LS), and Leber hereditary optic neuropathy (LHON). Primary mitochondrial diseases often result from mutations of mitochondrial genomes and nuclear genes that encode the mitochondrial components. However, complete intracellular correction of the mutated genetic parts relevant to mitochondrial structures and functions is technically challenging. Instead, there have been diverse attempts to provide corrected genetic materials with cells. In this review, we discuss recent novel physical, chemical and biological strategies, and methods to introduce genetic cargos into mitochondria of eukaryotic cells. Effective mitochondria-targeting gene delivery systems can reverse multiple mitochondrial disorders by enabling cells to produce functional mitochondrial components.


Assuntos
Terapia Genética/métodos , Doenças Mitocondriais/terapia , Animais , Técnicas de Transferência de Genes , Humanos , Doenças Mitocondriais/genética , Terapia de Alvo Molecular , Mutação
16.
Mol Cells ; 40(5): 339-345, 2017 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-28535668

RESUMO

Retroviral and lentiviral vectors are mostly pseudotyped and often purified and concentrated via ultracentrifugation. In this study, we quantified and compared the stabilities of retroviral [murine leukemia virus (MLV)-based] and lentiviral [human immunodeficiency virus (HIV)-1-based] vectors pseudotyped with relatively mechanically stable envelope proteins, vesicular stomatitis virus glycoproteins (VSVGs), and the influenza virus WSN strain envelope proteins against ultracentrifugation. Lentiviral genomic and functional particles were more stable than the corresponding retroviral particles against ultracentrifugation when pseudotyped with VSVGs. However, both retroviral and lentiviral particles were unstable when pseudotyped with the influenza virus WSN strain envelope proteins. Therefore, the stabilities of pseudotyped retroviral and lentiviral vectors against ultracentrifugation process are a function of not only the type of envelope proteins, but also the type of viral internal core (MLV or HIV-1 core). In addition, the fraction of functional viral particles among genomic viral particles greatly varied at times during packaging, depending on the type of envelope proteins used for pseudotyping and the viral internal core.


Assuntos
Vetores Genéticos/química , Lentivirus/genética , Retroviridae/genética , Ultracentrifugação , Proteínas do Envelope Viral/genética , Vetores Genéticos/genética , Células HEK293 , HIV-1/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A/genética , Vírus da Leucemia Murina/genética , Glicoproteínas de Membrana/genética , Neuraminidase/genética , Transdução Genética , Proteínas Virais/genética , Vírion/química
17.
Biotechnol Lett ; 38(8): 1285-91, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27146205

RESUMO

OBJECTIVES: To develop a method for reliable quantification of viral vectors, which is necessary for determining the optimal dose of vector particles in clinical trials to obtain the desired effects without severe unwanted immune responses. RESULTS: A significant level of vector plasmid remained in retroviral and lentiviral vector samples, which led to overestimation of viral titers when using the conventional RT-qPCR-based genomic titration method. To address this problem, we developed a new method in which the residual plasmid was quantified by an additional RT-qPCR step, and standard molecules and primer sets were optimized. The obtained counts were then used to correct the conventionally measured genomic titers of viral samples. While the conventional method produced significantly higher genomic titers for mutant retroviral vectors than for wild-type vectors, our method produced slightly higher or equivalent titers, corresponding with the general idea that mutation of viral components mostly results in reduced or, at best, retained titers. CONCLUSION: Subtraction of the number of residual vector plasmid molecules from the conventionally measured genomic titer can yield reliable quantification of retroviral and lentiviral vector samples, a prerequisite to advancing the safety of gene therapy applications.


Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linhagem Celular , Humanos , Plasmídeos/genética , Transdução Genética
18.
Anal Chem ; 87(23): 11652-9, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26528878

RESUMO

In this work, we demonstrate in situ virus identification based on surface-enhanced Raman scattering (SERS). We hypothesized that newly emerging influenza viruses possess surface proteins and lipids that can generate distinctive Raman signals. To test this hypothesis, SERS signals were measured from the surface of a noninfluenza virus, two different influenza viruses, and a genetically shuffled influenza virus. To ensure the safety for experimenters we constructed nonreplicating pseudotyped viruses that display main influenza virus surface components. Pseudotype with influenza virus components produced enhanced Raman peaks, on gold nanoparticles, that are easily distinguishable from those of pseudotype with a noninfluenza virus component, vesicular stomatitis virus G protein (VSVG). Furthermore, virus with the surface components of a newly emerging influenza strain, A/California/04/2009 (H1N1), generated Raman peaks different from those of viruses with components of the conventional laboratory-adapted influenza strain, A/WSN/33 (H1N1). Interestingly, the virus simultaneously displaying surface components of both influenza strains, a model mutant with genome reassortment, also produced a Raman signal pattern that is clearly distinguishable from those of each strain. This work highlights that SERS can provide a powerful label-free strategy to quickly identify newly emerging and potentially fatal influenza viruses.


Assuntos
Orthomyxoviridae/química , Orthomyxoviridae/isolamento & purificação , Ouro/química , Lipídeos/análise , Nanopartículas Metálicas/química , Análise Espectral Raman , Propriedades de Superfície , Proteínas Virais/análise
19.
BMB Rep ; 48(1): 6-12, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25104401

RESUMO

Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future.


Assuntos
Marcação de Genes , Genoma , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/metabolismo , Desoxirribonucleases/metabolismo , Integrases/metabolismo , Recombinases/metabolismo
20.
Nat Neurosci ; 15(10): 1399-406, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22983209

RESUMO

Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs, progenitors, and, ultimately, neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, although a few endogenous factors are known to regulate neuronal differentiation. Astrocytes have been implicated, but their role in juxtacrine (that is, cell-cell contact dependent) signaling in NSC niches has not been investigated. We found that ephrin-B2 presented from rodent hippocampal astrocytes regulated neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies revealed a previously unknown role for ephrin-B2 in instructing neuronal differentiation. In addition, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activated ß-catenin in vitro and in vivo independently of Wnt signaling and upregulated proneural transcription factors. Ephrin-B2(+) astrocytes therefore promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.


Assuntos
Astrócitos/fisiologia , Efrina-B2/fisiologia , Hipocampo/fisiologia , Neurogênese/fisiologia , Animais , Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Efrina-B2/biossíntese , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Receptor EphB4/biossíntese , Receptor EphB4/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Regulação para Cima , beta Catenina/metabolismo
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