RESUMO
We analyzed the effects of olfactory receptors (ORs) on transient receptor potential vanilloid 1 (TRPV1) activation using HEK293T cells co-expressing TRPV1 and OR51E1. We demonstrate here that the effect of OR51E1 on TRPV1 activation varies depending on the two TRPV1 ligands: capsaicin and eugenol. Notably, both of these ligands are vanilloid analogs. OR51E1 enhanced the response of TRPV1 to capsaicin but diminished that to eugenol. OR51E2 also showed similar effects. Based on the susceptibility to the OR's modulatory effects, various TRPV1 ligands could be classified into capsaicin and eugenol types. Activation of OR51E1 enhanced cAMP production. In addition, forskolin exhibited almost identical effects as ORs on TRPV1 responses to both types of ligands. These results suggest that OR51E1-induced cAMP elevation leads to a modification of TRPV1, presumably phosphorylation of TRPV1, which amplifies the susceptibility of TRPV1 to the two types of ligands differently.
Assuntos
Capsaicina , AMP Cíclico , Eugenol , Receptores Odorantes , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Humanos , Eugenol/farmacologia , Células HEK293 , Capsaicina/farmacologia , AMP Cíclico/metabolismo , Receptores Odorantes/metabolismo , Receptores Odorantes/genética , Ligantes , Fosforilação/efeitos dos fármacos , Colforsina/farmacologiaRESUMO
Quartz-crystal-microbalance (QCM) biosensor is a typical label-free biosensor, and its sensitivity can be greatly improved by removing electrodes and wires that would be otherwise attached to the surfaces of the quartz resonator. The wireless-electrodeless QCM biosensor was then developed using a microelectro-mechanical systems (MEMS) process, although challenges remain in the sensitivity, the coupling efficiency, and the miniaturization (or mass production). In this study, we establish a MEMS process to obtain a large number of identical ultrasensitive and highly efficient sensor chips with dimensions of 6 mm square. The fundamental shear resonance frequency of the thinned AT-cut quartz resonator packaged in the microchannel exceeds 160 MHz, which is excited by antennas deposited on inner walls of the microchannel, significantly improving the electro-mechanical coupling efficiency in the wireless operation. The high sensitivity of the developed MEMS QCM biosensors is confirmed by the immunoglobulin G (IgG) detection using protein A and ZZ-tag displaying a bionanocapsule (ZZ-BNC), where we find that the ZZ-BNC can provide more effective binding sites and higher affinity to the target molecules, indicating a further enhancement in the sensitivity of the MEMS QCM biosensor. We then perform the label-free C-reactive protein (CRP) detection using the ZZ-BNC-functionalized MEMS QCM biosensor, which achieves a detection limit of 1 ng mL-1 or less even with direct detection.
Assuntos
Técnicas Biossensoriais , Sistemas Microeletromecânicos , Quartzo/química , Proteína C-Reativa , Miniaturização , Técnicas Biossensoriais/métodos , Técnicas de Microbalança de Cristal de Quartzo/métodosRESUMO
The detection sensitivity of immunostick colorimetric assay has been increased by using a bio-nanocapsule as a scaffold for oriented immobilization of immunoglobulin Gs. This immunostick produced â¼82-folds stronger coloration in the detection of food allergens and reduced detection time by a factor of 5.
Assuntos
Hipersensibilidade Alimentar , Nanocápsulas , Humanos , Colorimetria , Imunoglobulina G , Hipersensibilidade Alimentar/diagnóstico , AlérgenosRESUMO
Among the five human senses, light, sound, and force perceived by the eye, ear, and skin, respectively are physical phenomena, and therefore can be easily measured and expressed as objective, univocal, and simple digital data with physical quantity. However, as taste and odor molecules perceived by the tongue and nose are chemical phenomena, it has been difficult to express them as objective and univocal digital data, since no reference chemicals can be defined. Therefore, while the recording, saving, transmitting to remote locations, and replaying of human visual, auditory, and tactile information as digital data in digital devices have been realized (this series of data flow is defined as DX (digital transformation) in this review), the DX of human taste and odor information is not yet in the realization stage. Particularly, since there are at least 400,000 types of odor molecules and an infinite number of complex odors that are mixtures of these molecules, it has been considered extremely difficult to realize "human olfactory DX" by converting all odors perceived by human olfaction into digital data. In this review, we discuss the current status and future prospects of the development of "human olfactory DX", which we believe can be realized by utilizing odor sensors that employ the olfactory receptors (ORs) that support human olfaction as sensing molecules (i.e., human OR sensor).
Assuntos
Odorantes , Receptores Odorantes , Humanos , Olfato , Nariz , LínguaRESUMO
Cytoplasmic delivery of functional proteins into target cells remains challenging for many biological agents to exert their therapeutic effects. Extracellular vesicles (EVs) are expected to be a promising platform for protein delivery; however, efficient loading of proteins of interest (POIs) into EVs remains elusive. In this study, we utilized small compound-induced heterodimerization between FK506 binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain to sort bioactive proteins into EVs using the FRB-FKBP system. When CD81, a typical EV marker protein, and POI were fused with FKBP and FRB, respectively, rapamycin induced the binding of these proteins through the FKBP-FRB interaction and recruited the POIs into EVs. The released EVs, displaying the virus-derived membrane fusion protein, delivered the POI cargo into recipient cells and their functionality in the recipient cells was confirmed. Furthermore, we demonstrated that CD81 could be replaced with other EV-enriched proteins, such as CD63 or HIV Gag. Thus, the FRB-FKBP system enables the delivery of functional proteins and paves the way for EV-based protein delivery platforms.
Assuntos
Vesículas Extracelulares , Comunicação Celular , Vesículas Extracelulares/metabolismo , Sirolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo , Proteínas de Ligação a Tacrolimo/análise , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
The oriented immobilization of sensing molecules (e.g., IgGs, receptors, lectins, and DNA aptamers) on sensor chips is particularly important for maximizing the potential of the sensing molecules, thereby enhancing the sensitivity and target-binding capacity of biosensors. We previously developed â¼30 nm bio-nanocapsules (ZZ-BNCs) consisting of the hepatitis B virus envelope L protein fused with the tandem form of protein A-derived IgG Fc-binding Z domain (ZZ-L protein). ZZ-BNC acts successfully as a scaffold, enhancing both the sensitivity and binding capacity of IgG, a Fc-fused receptor, and Fc-fused lectin to antigens, cytokines, and sugar chains through an oriented immobilization on a biosensor surface. To expand the versatility of ZZ-BNC, we modified ZZ-BNC by replacing the ZZ domain with a DNA-binding single-chain lambda Cro (scCro) domain, thereby developing scCro-BNC. The scCro-BNC was synthesized in yeast cells and homogeneously purified as â¼30 nm sized nanoparticles. In a quartz crystal microbalance, an scCro-BNC-coated sensor chip immobilized with thrombin-binding DNA aptamers showed an â¼5.5-fold higher thrombin-binding capacity and â¼6000-fold higher detection sensitivity than a sensor chip directly coated with DNA aptamers. In addition, the number of bound thrombin molecules per molecule of DNA aptamer increased by â¼7.8-fold with an scCro-BNC coating, consistent with the theoretical thrombin-binding capacity. Collectively, scCro-BNC was shown to perform as an ideal scaffold for maximizing the potential of the DNA aptamer by immobilizing it in an oriented manner. Facilitating a highly sensitive detection of various target molecules, these BNC-based scaffolds are expected to improve a wide range of biosensors while minimizing the number of sensing molecules required.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Nanocápsulas , Imunoglobulina G , Proteína Estafilocócica ARESUMO
Most of the odors that humans perceive daily are complex odors. It is believed that the modulation, enhancement, and suppression of overall complex odors are caused by interactions between odor molecules. In this study, to understand the interaction between odor molecules at the level of human olfactory receptor responses, the effects of 3-octen-2-one, which has been shown to modulate vanilla flavors, were analyzed using a human olfactory receptor sensor that uses all human olfactory receptors (388 types) as sensing molecules. As a result, the response intensity of 1 common receptor (OR1D2) was synergistically enhanced in vanilla flavor with 3-octen-2-one compared with vanilla flavor, and the response of 1 receptor (OR5K1) to vanilla flavor was completely suppressed. These results strongly suggested that the response of human olfactory receptors to complex odors is enhanced or suppressed by relatively few other odor molecules.
Assuntos
Receptores Odorantes , Vanilla , Humanos , Olfato/fisiologia , OdorantesRESUMO
Black tea extracts (BTEs) from four different production areas showed a higher aggregation strength for phosphatidylcholine-based liposomes containing cholesterol used as a viral membrane model. Furthermore, the anti-influenza A virus (IAV) activity of each BTE in vitro demonstrated that although Sri Lanka, Kenya, and Assam had higher anti-IAV activities, Darjeeling had a lower anti-IAV activity, showing a correlation between each BTE and the liposome aggregation strength. Moreover, the antiviral activity strength of BTEs was consistent with the antioxidant activity strength of BTEs, suggesting that the component(s) in black tea that exhibits antioxidant activity would also be the component(s) that accounts for its antiviral activity. Thus, our results propose that BTEs exert their antiviral effects by binding not only hemagglutinin and neuraminidase but also viral membranes directly, especially "cholesterol-rich lipid rafts" and affect the membrane structure, causing the virus to aggregate, thereby inhibiting infection of the host cells.
Assuntos
Antivirais , Camellia sinensis , Antivirais/farmacologia , Chá , Lipossomos , Antioxidantes , Colesterol , Replicação ViralRESUMO
Extracellular vesicles (EVs) have been considered to deliver biological cargos between cells and mediate intercellular communication and potential drug delivery carriers. However, the mechanisms that underlie the biological process of EV uptake and cytoplasmic cargo release in recipient cells are largely unknown. Quantitative and real-time assays for the assessment of cargo delivery efficiency inside recipient cells have not been feasible. In this study, we developed an EV cargo delivery (EVCD) assay using a split luciferase called a NanoBiT system. Recipient cells expressing LgBiT, a large subunit of luciferase, emit luminescence when EV cargo proteins fused with a small luminescence tag (HiBiT tag) that can complement LgBiT are delivered to the cytoplasm of recipient cells. Using the EVCD assay, the cargo delivery efficiency of EVs could be quantitatively measured in real time. This assay was highly sensitive in detecting a single event of cargo delivery per cell. We found that modification of EVs with a virus-derived fusogenic protein significantly enhanced the cytoplasmic cargo delivery; however, in the absence of a fusogenic protein, the cargo delivery efficiency of EVs was below the threshold of the assay. The EVCD assay could assess the effect of entry inhibitors on EV cargo delivery. Furthermore, using a luminescence microscope, the cytoplasmic cargo delivery of EVs was directly visualized in living cells. This assay could reveal the biological mechanism of the cargo delivery processes of EVs.
Assuntos
Vesículas Extracelulares , Luminescência , Transporte Biológico , Comunicação Celular , Citoplasma , Vesículas Extracelulares/metabolismoRESUMO
Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.
Assuntos
Aldosterona/farmacologia , Canais de Cálcio Tipo T/metabolismo , Proteína 2 Inibidora de Diferenciação/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Canais de Cálcio Tipo T/genética , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiologia , Proteína 2 Inibidora de Diferenciação/genética , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacosRESUMO
The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs. A recent study identified neural epidermal growth factor-like (NEL)-like 2 (NELL2) as a novel ligand for Robo3. In this study, we carried out a comprehensive analysis of the interaction between NELL1 and the Robo family of receptors and demonstrated that Robo2 contains a cryptic binding site for both NELL1 and NELL2. NELL1/2 binds to the first fibronectin type III (FNIII) domain of Robo2 but not to intact Robo2. Mutation analysis revealed that several amino acids within the first FNIII domain are critical for NELL1 binding to Robo2 but not to Robo1. The Robo2 deletion mutants without the fourth immunoglobulin domain and single amino acid substitution mutants that can influence the architecture of the ectodomain facilitated binding to NELL1/2. Acidic conditions increased the binding affinity of Robo2 for NELL1. These results suggest that Robo2 functions as a receptor for NELL1/2, particularly under circumstances where Robo2 undergoes proteolytic digestion. If this is not the case, conformational changes of the ectodomain of Robo2 may unmask the binding site for NELL1/2.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Ácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Proteínas de Ligação ao Cálcio , Humanos , Concentração de Íons de Hidrogênio , Mutação , Proteólise , Receptores Imunológicos/química , Receptores Imunológicos/genéticaRESUMO
We report a novel scaffold for clustering and oriented immobilization of human IgG1 Fc-fused lectins on biosensors without chemical modifications. This approach uses a bio-nanocapsule (BNC) displaying a tandem form of IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). Incorporating ZZ-BNC effectively increased both the sensitivity and sugar chain-binding capacity compared with the condition without ZZ-BNC.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Imobilizadas/metabolismo , Fragmentos Fc das Imunoglobulinas/genética , Lectinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Açúcares/análise , Humanos , Proteínas Imobilizadas/química , Imunoglobulina G/imunologia , Lectinas/química , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteína Estafilocócica A/química , Açúcares/químicaRESUMO
The influence of nivolumab on intercellular adhesion forces between T cells and cancer cells was evaluated quantitatively using atomic force microscopy (AFM). Two model T cells, one expressing high levels of programmed cell death protein 1 (PD-1) (PD-1high Jurkat) and the other with low PD-1 expression levels (PD-1low Jurkat), were analyzed. In addition, two model cancer cells, one expressing programmed death-ligand 1 (PD-L1) on the cell surface (PC-9, PD-L1+) and the other without PD-L1 (MCF-7, PD-L1-), were also used. A T cell was attached to the apex of the AFM cantilever using a cup-attached AFM chip, and the intercellular adhesion forces were measured. Although PD-1high T cells adhered strongly to PD-L1+ cancer cells, the adhesion force was smaller than that with PD-L1- cancer cells. After the treatment of PD-1high T cells with nivolumab, the adhesion force with PD-L1+ cancer cells increased to a similar level as with PD-L1- cancer cells. These results can be explained by nivolumab influencing the upregulation of the adhesion ability of PD-1high T cells with PD-L1+ cancer cells. These results were obtained by measuring intercellular adhesion forces quantitatively, indicating the usefulness of single-cell AFM analysis.
Assuntos
Adesão Celular/efeitos dos fármacos , Microscopia de Força Atômica , Nivolumabe/farmacologia , Linfócitos T/citologia , Antígeno B7-H1 , Humanos , Células Jurkat , Células MCF-7 , Receptor de Morte Celular Programada 1 , Análise EspectralRESUMO
Ongoing aortic wall degeneration and subsequent aneurysm exclusion failure are major concerns after an endovascular aneurysm repair with a stent-graft. An ideal solution would be a drug therapy that targets the aortic wall and inhibits wall degeneration. Here, we described a novel drug delivery system, which allowed repetitively charging a graft with therapeutic drugs and releasing them to the aortic wall in vivo. The system was composed of a targeted graft, which was labeled with a small target molecule, and the target-recognizing nanocarrier, which contained suitable drugs. We developed the targeted graft by decorating a biotinylated polyester graft with neutravidin. We created the target-recognizing nanocarrier by conjugating drug-containing liposomes with biotinylated bio-nanocapsules. We successfully demonstrated that the target-recognizing nanocarriers could bind to the targeted graft, both in vitro and in blood vessels of live mice. Moreover, the drug released from our drug delivery system reduced the expression of matrix metalloproteinase-9 in mouse aortas. Thus, this hybrid system represents a first step toward an adjuvant therapy that might improve the long-term outcome of endovascular aneurysm repair.
Assuntos
Aorta/efeitos dos fármacos , Aneurisma Aórtico/terapia , Prótese Vascular , Sistemas de Liberação de Medicamentos/métodos , Metaloproteinase 9 da Matriz/metabolismo , Quinolinas/administração & dosagem , Animais , Aorta/metabolismo , Aorta/patologia , Avidina/química , Portadores de Fármacos/química , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Nanoestruturas/química , Desenho de Prótese , Quinolinas/química , Resultado do TratamentoRESUMO
BACKGROUND: The uterus is an organ that is directly accessible via the transvaginal route, whereas the drug delivery system and the gene delivery system (GDS) for the uterus are very limited, even in animal models. In the present study, we optimized a bionanocapsule (BNC) comprising a hepatitis B virus envelope L-protein particle, for which a structurally similar particle has been used as an immunogen of a conventional HB vaccine worldwide for more than 30 years, as a local uterine GDS using a mouse model. METHODS: To display various antibodies for re-targeting to different cells other than hepatic cells, the pre-S1 region of BNC was replaced with a tandem form of the protein A-derived immunoglobulin G Fc-interacting region (Z domain, ZZ-BNC). To induce strong cell adhesion after local administration into the uterine cavity, ZZ-BNC was modified with a transactivator of transcription (TAT) peptide. RESULTS: Gene transfer using TAT-modified ZZ-BNC is approximately 5000- or 18-fold more efficient than the introduction of the same dose of naked DNAs or the use of the cationic liposomes, respectively. TAT-modified ZZ-BNC was rapidly eliminated from the uterus and had no effect on the pregnancy rate, litter size or fetal growth. CONCLUSIONS: TAT-modified ZZ-BNC could be a useful GDS for uterine endometrial therapy via local uterine injection.
Assuntos
Técnicas de Transferência de Genes , Nanopartículas , Peptídeos , Útero/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Animais , Feminino , Expressão Gênica , Genes Reporter , Imuno-Histoquímica , Camundongos , Nanopartículas/química , Peptídeos/química , Gravidez , Transgenes , Proteínas do Envelope Viral/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
It has been reported that phospholipid nanoparticles (PNPs) including liposomes and exosomes could efficiently induce lipid droplets (LDs) in macrophages. However, in non-macrophage cells, the effects of PNPs on the induction of LDs have not been thoroughly investigated. In this report, we directly compared non-macrophage and macrophage cell lines in terms of LD induction by various formulation of liposomes containing phosphatidylserine and exosomes. All non-macrophage cell lines as well as macrophage cell lines tested in this study showed evident LD induction in response to these PNPs, though the efficacy of LD induction in non-macrophage cell lines varied considerably. Our results suggest that LD formation is a common and crucial response to PNPs in mammalian cells not only in macrophages but also in non-macrophage cells.
Assuntos
Exossomos/fisiologia , Gotículas Lipídicas/metabolismo , Lipossomos/farmacologia , Macrófagos/ultraestrutura , Animais , Linhagem Celular , Humanos , Lipossomos/química , Nanopartículas/química , Fosfatidilserinas , FosfolipídeosRESUMO
BACKGROUND: Various nanocarriers have been used to deliver subunit vaccines specifically to dendritic cells (DCs) for the improvement of immunogenicity. However, due to their insufficient DC priming ability, these vaccines could not elicit effective innate immunity. We have recently developed a DC-targeting bio-nanocapsule (BNC) by displaying anti-CD11c IgGs via protein A-derived IgG Fc-binding Z domain on the hepatitis B virus envelope L protein particles (α-DC-ZZ-BNC). RESULTS: After the chemical modification with antigens (Ags), the α-DC-ZZ-BNC-Ag complex could deliver Ags to DCs efficiently, leading to effective DC maturation and efficient endosomal escape of Ags, followed by Ag-specific T cell responses and IgG productions. Moreover, the α-DC-ZZ-BNC modified with Japanese encephalitis virus (JEV) envelope-derived D3 Ags could confer protection against 50-fold lethal dose of JEV injection on mice. CONCLUSION: The α-DC-ZZ-BNC-Ag platform was shown to induce humoral and cellular immunities effectively without any adjuvant.
Assuntos
Antígeno CD11c/imunologia , Células Dendríticas/imunologia , Imunogenicidade da Vacina , Vacinas contra Encefalite Japonesa/imunologia , Nanocápsulas/química , Animais , Antígenos Virais/administração & dosagem , Antígenos Virais/imunologia , Linhagem Celular , Células Dendríticas/metabolismo , Vírus da Encefalite Japonesa (Espécie)/química , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Humanos , Imunidade Celular , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Vacinas contra Encefalite Japonesa/administração & dosagem , Camundongos Endogâmicos BALB C , Ovalbumina/química , Tamanho da Partícula , Proteína Estafilocócica A/química , Proteínas do Envelope Viral/químicaRESUMO
Bio-nanocapsules (BNCs) consisting of hepatitis B virus surface antigen (HBsAg) L proteins and phospholipids are used as efficient non-viral carriers for liver-specific delivery of genes and drugs. Considering the administration to HB vaccinees and HB patients, endogenous anti-HBsAg immunoglobulins (HBIGs) may reduce the delivery efficacy and prevent repetitive administration. Therefore, low immunogenic BNCs were generated by inserting two point mutations in the HBsAg L protein, which were found in HBV escape mutants. Escape mutant-type BNC (emBNC) showed 50% lower HBIG binding capacity than that of parental BNC (wtBNC). It induced HBIG production to a lesser extent than that associated with wtBNC in BALB/c mice. The emBNC could accumulate into human hepatocyte-derived tumor in mice pre-treated with HBIGs. The complex of emBNC and cationic liposomes could deliver plasmid DNA to HepG2 cells efficiently in the presence of HBIGs. Thus, emBNC could evade HBIG-neutralizing antibodies, expanding the clinical utility of BNC-based nanomedicine.
Assuntos
Sistemas de Liberação de Medicamentos , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Mutação , Nanocápsulas/administração & dosagem , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Lipossomos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanocápsulas/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have developed an automated robot that facilitates non-invasive isolation of a single cell with the most favorable properties from arrays containing >105 cells, thus allowing the establishment of new cell screening methods for bio-medicines. In this chapter, an outline of the proposed automated single-cell analysis and isolation system (hereafter called 'single-cell robot') is reviewed by comparison with a conventional fluorescence-activated cell sorter (FACS). The single-cell robot could perform high-throughput screening for both mammalian cells secreting the highest amount of bio-medicines (e.g. Chinese hamster ovary (CHO) cells or hybridomas), and stem cells with the highest pluripotency (e.g., embryonic stem (ES) cells), from huge number of cell libraries based on the recently proposed concept of "single cell-based breeding". The rational screening method for the de novo agonist design could also be performed using yeast cells expressing functional mammalian cytokine receptors (e.g., epidermal growth factor receptor (EGFR), somatostatin G protein-coupled receptor (SSTR5), and interleukin 5 receptor (IL5R)). Furthermore, the single-cell robot could comprehensively analyze the reaction between olfactory sensory neurons and specific odorants, which will shed light on how odorants are recognized by olfactory receptors. Taken together, these unique features of the proposed single-cell robot will contribute to the high-throughput development of forthcoming bio-medicines.
Assuntos
Automação/métodos , Pesquisa Biomédica/métodos , Separação Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Análise de Célula Única/métodos , Animais , Humanos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.