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1.
Viruses ; 14(4)2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-35458458

RESUMO

Low pathogenic H9N2 avian influenza viruses have spread in wild birds and poultry worldwide. Recently, the number of human cases of H9N2 virus infection has increased in China and other countries, heightening pandemic concerns. In Japan, H9N2 viruses are not yet enzootic; however, avian influenza viruses, including H5N1, H7N9, H5N6, and H9N2, have been repeatedly detected in raw poultry meat carried by international flight passengers from Asian countries to Japan. Although H9N2 virus-contaminated poultry products intercepted by the animal quarantine service at the Japan border have been characterized in chickens and ducks, the biological properties of those H9N2 viruses in mammals remain unclear. Here, we characterized the biological features of two H9N2 virus isolates [A/chicken/Japan/AQ-HE28-50/2016 (Ck/HE28-50) and A/chicken/Japan/AQ-HE28-57/2016 (Ck/HE28-57)] in a mouse model. We found that these H9N2 viruses replicate well in the respiratory tract of infected mice without adaptation, and that Ck/HE28-57 caused body weight loss in the infected mice. Our results indicate that H9N2 avian influenza viruses isolated from raw chicken meat products illegally brought to Japan can potentially infect and cause disease in mammals.


Assuntos
Virus da Influenza A Subtipo H5N1 , Subtipo H7N9 do Vírus da Influenza A , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Doenças das Aves Domésticas , Animais , Galinhas , China , Modelos Animais de Doenças , Vírus da Influenza A Subtipo H9N2/genética , Mamíferos , Camundongos , Filogenia , Aves Domésticas , Produtos Avícolas
2.
Nat Commun ; 12(1): 751, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531495

RESUMO

Optogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Potenciação de Longa Duração/fisiologia , Optogenética/métodos , Sinapses/metabolismo , Animais , Eletrofisiologia , Feminino , Células HeLa , Hipocampo/metabolismo , Hipocampo/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sinapses/fisiologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-33257455

RESUMO

H7N9 highly pathogenic avian influenza virus (HPAIV) infection in a human was first reported in 2017. A/duck/Japan/AQ-HE29-22/2017 (H7N9) (Dk/HE29-22), found in imported duck meat at an airport in Japan, possesses a hemagglutinin with a multibasic cleavage site, indicating high pathogenicity in chickens, as in the case of other H7 HPAIVs. In the present study, we examined the pathogenicity of Dk/HE29-22 and the effectiveness of a cap-dependent endonuclease inhibitor (baloxavir) and neuraminidase inhibitors (oseltamivir and zanamivir) against infection with this strain in a macaque model (n = 3 for each group). All of the macaques infected with Dk/HE29-22 showed severe signs of disease and pneumonia even after the virus had disappeared from lung samples. Virus titers in macaques treated with baloxavir were significantly lower than those in the other treated groups. After infection, levels of interferon alpha and beta (IFN-α and IFN-ß) in the blood of macaques in the baloxavir group were the highest among the groups, whereas levels of tumor necrosis factor alpha (TNF-α) and interleukin 13 (IL-13) were slightly increased in the untreated group. In addition, immune checkpoint proteins, including programmed death 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT), were expressed at high levels in the untreated group, especially in one macaque that showed severe signs of disease, indicating that negative feedback responses against vigorous inflammation may contribute to disease progression. In the group treated with baloxavir, the percentages of PD-1-, CTLA-4-, and TIGIT-positive T lymphocytes were lower than those in the untreated group, indicating that reduction in virus titers may prevent expression of immune checkpoint molecules from downregulation of T cell responses.


Assuntos
Subtipo H7N9 do Vírus da Influenza A , Influenza Aviária , Influenza Humana , Infecções por Orthomyxoviridae , Pneumonia Viral , Animais , Galinhas , Endonucleases , Humanos , Macaca fascicularis , Neuraminidase
4.
Jpn J Infect Dis ; 2021 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-34980710

RESUMO

Circulation of avian influenza A viruses in poultry is a public health concern because these viruses may cause severe disease in humans and have the potential to become more transmissible among humans. Monitoring the susceptibility of these viruses to antivirals is important for influenza pandemic preparedness. However, information about their antiviral susceptibility is limited. Here, we determined the susceptibilities of avian influenza A(H5N1), A(H5N2), A(H5N8), A(H7N7), A(H7N9), A(H9N1), and A(H9N2) viruses isolated in Japan to the antivirals approved for use there: the M2 inhibitor amantadine; the neuraminidase inhibitors oseltamivir, peramivir, zanamivir, and laninamivir; and the RNA polymerase inhibitors baloxavir and favipiravir. Genotypic methods that detect amino acid substitutions associated with antiviral resistance and phenotypic methods that assess viral susceptibility to drugs revealed that these avian influenza A viruses are susceptible to neuraminidase inhibitors and RNA polymerase inhibitors. These results suggest that the neuraminidase inhibitors and the RNA polymerase inhibitors currently approved in Japan could be a treatment option against influenza A virus infections in humans.

5.
Opt Lett ; 45(11): 3171-3174, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32479487

RESUMO

The internal modification of glass using ultrashort pulse lasers has been attracting attention in a wide range of applications. However, the remarkably low processing speed has impeded its use in the industry. In this study, we achieved ultrafast internal modification of glass by coaxially focusing a single-pulse femtosecond laser and continuous-wave (CW) laser with the wavelength that is transparent to the glass. Compared with the conventional method, the processing speed increased by a factor of 500. The observation of high-speed phenomena revealed that the CW laser was absorbed by the seed electrons that were generated by the femtosecond laser pulse. This technique may help expand the applications of femtosecond lasers in the industry.

6.
Sci Rep ; 10(1): 8441, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439885

RESUMO

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.


Assuntos
Simulação por Computador , Variação Genética , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Aviária/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Virais/genética , Animais , Aves , Técnicas In Vitro , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/genética , Influenza Aviária/virologia , RNA Viral/genética , Curva ROC
7.
Transbound Emerg Dis ; 67(2): 792-798, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31650680

RESUMO

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi-basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ-HE29-22/2017 (HE29-22) and A/duck/Japan/AQ-HE29-52/2017 (HE29-52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ-HE28-3/2016 (HE28-3)] in mice and ferrets. In mice, HE29-52 was more pathogenic than HE29-22 and HE28-3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Influenza Humana/virologia , Doenças das Aves Domésticas/virologia , Produtos Avícolas/virologia , Animais , Embrião de Galinha , Cães , Patos , Feminino , Furões , Humanos , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Japão/epidemiologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Doenças das Aves Domésticas/epidemiologia
8.
Transbound Emerg Dis ; 66(6): 2342-2352, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31293102

RESUMO

The first human case of zoonotic H7N9 avian influenza virus (AIV) infection was reported in March 2013 in China. This virus continues to circulate in poultry in China while mutating to highly pathogenic AIVs (HPAIVs). Through monitoring at airports in Japan, a novel H7N3 reassortant of the zoonotic H7N9 HPAIVs, A/duck/Japan/AQ-HE30-1/2018 (HE30-1), was detected in a poultry meat product illegally brought by a passenger from China into Japan. We analysed the genetic, pathogenic and antigenic characteristics of HE30-1 by comparing it with previous zoonotic H7N9 AIVs and their reassortants. Phylogenetic analysis of the entire HE30-1 genomic sequence revealed that it comprised at least three different sources; the HA (H7), PB1, PA, NP, M and NS segments of HE30-1 were directly derived from H7N9 AIVs, whereas the NA (N3) and PB2 segments of HE30-1 were unrelated to zoonotic H7N9. Experimental infection revealed that HE30-1 was lethal in chickens but not in domestic or mallard ducks. HE30-1 was shed from and replicated in domestic and mallard ducks and chickens, whereas previous zoonotic H7N9 AIVs have not adapted well to ducks. This finding suggests the possibility that HE30-1 may disseminate to remote area by wild bird migration once it establishes in wild bird population. A haemagglutination-inhibition assay indicated that antigenic drift has occurred among the reassortants of zoonotic H7N9 AIVs; HE30-1 showed similar antigenicity to some of those H7N9 AIVs, suggesting it might be prevented by the H5/H7 inactivated vaccine that was introduced in China in 2017. Our study reports the emergence of a new reassortant of zoonotic H7N9 AIVs with novel viral characteristics and warns of the challenge we still face to control the zoonotic H7N9 AIVs and their reassortants.


Assuntos
Patos/virologia , Vírus da Influenza A Subtipo H7N3/genética , Vírus da Influenza A Subtipo H7N3/patogenicidade , Vírus Reordenados , Animais , China , Genoma Viral , Influenza Aviária/virologia , Japão , Filogenia , Sequenciamento Completo do Genoma
9.
J Vet Med Sci ; 81(3): 444-448, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30674734

RESUMO

A new reassortant H7N3 avian influenza virus (AIV) was isolated from a duck meat product that was illegally taken on board a passenger flight from China to Japan in March 2018. Sequencing analysis revealed that the H7N3 isolate, A/duck/Japan/AQ-HE30-1/2018 (Dk/HE30-1) (H7N3), was a reassortant highly pathogenic avian influenza virus (HPAIV) that contained the haemagglutinin (HA) gene of Chinese H7N9 HPAIV. Dk/HE30-1 (H7N3) possessed a novel polybasic sequence motif PEVPKRRRTAR/GLF at the HA cleavage site that has never previously been reported in H7 HPAIVs. The HA antigenicity of Dk/HE30-1 (H7N3) slightly differed from that of H7N9 HPAIVs previously reported. These findings will help further our knowledge of the circulation and genetic evolution of emerging AIVs in endemic areas.


Assuntos
Vírus da Influenza A Subtipo H7N3/isolamento & purificação , Produtos da Carne/virologia , Viagem , Aeronaves , Animais , Patos , Contaminação de Alimentos , Vírus da Influenza A Subtipo H7N3/genética , Vírus da Influenza A Subtipo H7N3/patogenicidade , Japão , Filogenia , Vírus Reordenados , Virulência
10.
Elife ; 72018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30063210

RESUMO

During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas do Tecido Nervoso/química , Receptores de Superfície Celular/química , Proteínas rap de Ligação ao GTP/química , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Guanosina Difosfato/química , Guanosina Trifosfato/química , Mutação , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Neurônios/química , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Sinapses/química , Sinapses/genética , Sinapses/patologia , Proteínas rap de Ligação ao GTP/genética
11.
Virology ; 524: 10-17, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30138834

RESUMO

H7N9 highly and low pathogenic avian influenza viruses (HPAIV and LPAIV, respectively) have been isolated from duck meat products that were brought illegally into Japan by flight passengers in their hand luggage. These H7N9 virus isolates were phylogenetically closely related to those prevailing in China. Antigenic analysis revealed that the hemagglutinin of the H7N9 HPAIV isolate was slightly different from those of the H7N9 LPAIV and older H7 strains. These meat products contaminated with AIVs repeatedly brought into Japan lead to increased risks of poultry and public health. Continuous border disease control based on the detection and culling of infected poultry and meat products is, thus, essential for the prevention of introduction and spread of AIVs.


Assuntos
Antígenos Virais/imunologia , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Doenças das Aves Domésticas/virologia , Aves Domésticas/virologia , Animais , China , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Japão/epidemiologia , Filogenia , Doenças das Aves Domésticas/epidemiologia , Saúde Pública , Risco , Viagem , Zoonoses
12.
PLoS One ; 12(11): e0188778, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29190677

RESUMO

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named "actin-pl-clusters". In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent "actin podosome-like clusters".


Assuntos
Actinas/metabolismo , Podossomos/metabolismo , Imagem Individual de Molécula/métodos , Animais , Células Cultivadas
13.
Sci Rep ; 7(1): 6791, 2017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28754922

RESUMO

Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities such as protein-protein interactions and conformational changes. Here, using saturation mutagenesis, we developed a dark yellow fluorescent protein named ShadowY that can serve as an acceptor for FLIM-FRET. ShadowY is spectrally similar to the previously reported dark YFP but has a much smaller quantum yield, greater extinction coefficient, and superior folding property. When ShadowY was paired with mEGFP or a Clover mutant (CloverT153M/F223R) and applied to a single-molecule FRET sensor to monitor a light-dependent conformational change of the light-oxygen-voltage domain 2 (LOV2) in HeLa cells, we observed a large FRET signal change with low cell-to-cell variability, allowing for precise measurement of individual cell responses. In addition, an application of ShadowY to a separate-type Ras FRET sensor revealed an EGF-dependent large FRET signal increase. Thus, ShadowY in combination with mEGFP or CloverT153M/F223R is a promising FLIM-FRET acceptor.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Mutação , Domínios Proteicos
14.
Sci Rep ; 7: 46840, 2017 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-28589959

RESUMO

This corrects the article DOI: 10.1038/srep15334.

15.
Cell Biochem Biophys ; 75(3-4): 399-412, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28646414

RESUMO

The central mechanism for the transmission of the prion protein misfolding is the structural conversion of the normal cellular prion protein to the pathogenic misfolded prion protein, by the interaction with misfolded prion protein. This process might be enhanced due to the homo-dimerization/oligomerization of normal prion protein. However, the behaviors of normal prion protein in the plasma membrane have remained largely unknown. Here, using single fluorescent-molecule imaging, we found that both prion protein and Thy1, a control glycosylphosphatidylinositol-anchored protein, exhibited very similar intermittent transient immobilizations lasting for a few seconds within an area of 24.2 and 3.5 nm in diameter in CHO-K1 and hippocampal neurons cultured for 1- and 2-weeks, respectively. Prion protein molecules were immobile during 72% of the time, approximately 1.4× more than Thy1, due to prion protein's higher immobilization frequency. When mobile, prion protein diffused 1.7× slower than Thy1. Prion protein's slower diffusion might be caused by its transient interaction with other prion protein molecules, whereas its brief immobilization might be due to temporary association with prion protein clusters. Prion protein molecules might be newly recruited to prion protein clusters all the time, and simultaneously, prion protein molecules in the cluster might be departing continuously. Such dynamic interactions of normal prion protein molecules would strongly enhance the spreading of misfolded prion protein.


Assuntos
Membrana Celular/metabolismo , Glicosilfosfatidilinositóis/química , Proteínas Priônicas/metabolismo , Antígenos Thy-1/metabolismo , Animais , Células CHO , Membrana Celular/química , Células Cultivadas , Cricetinae , Cricetulus , Difusão , Corantes Fluorescentes/química , Glicosilfosfatidilinositóis/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Microscopia de Fluorescência , Proteínas Priônicas/química , Ratos , Ratos Wistar , Antígenos Thy-1/química
17.
Neuron ; 94(1): 37-47.e5, 2017 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-28318784

RESUMO

Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1-2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.


Assuntos
Aprendizagem da Esquiva/fisiologia , Região CA1 Hipocampal/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espinhas Dendríticas/metabolismo , Plasticidade Neuronal/fisiologia , Células Piramidais/metabolismo , Animais , Animais Recém-Nascidos , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Espinhas Dendríticas/fisiologia , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Células HeLa , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Humanos , Immunoblotting , Imuno-Histoquímica , Cinética , Potenciação de Longa Duração/fisiologia , Camundongos , Microscopia de Fluorescência , Neurônios/metabolismo , Neurônios/fisiologia , Optogenética , Células Piramidais/fisiologia , Proteínas de Ligação a RNA , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras , Proteínas Supressoras de Tumor/genética
18.
Sci Rep ; 5: 15334, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26469148

RESUMO

Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Mutagênese , Plasmídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Teoria Quântica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismo
19.
PLoS One ; 10(3): e0121109, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25799407

RESUMO

Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors.


Assuntos
Técnicas Biossensoriais/métodos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Mutação , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Phys Rev Lett ; 111(16): 162001, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24182255

RESUMO

On the basis of lattice simulations using highly improved staggered quarks for twelve-flavor QCD with several bare fermion masses, we observe a flavor-singlet scalar state lighter than the pion in the correlators of fermionic interpolating operators. The same state is also investigated using correlators of gluonic interpolating operators. Combined with our previous study that showed twelve-flavor QCD to be consistent with being in the conformal window, we infer that the lightness of the scalar state is due to infrared conformality. This result shed some light on the possibility of a light composite Higgs boson ("technidilaton") in walking technicolor theories.

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