RESUMEN
OBJECTIVE: The aim of the study was to assess inflammatory markers and clinical outcomes in adult patients admitted to hospital with mild-to-moderate COVID-19 and treated with a combination of standard-of-care (SOC) and targeted immunosuppressive therapy including anti-IL-17A (netakimab), anti-IL-6R (tocilizumab), or JAK1/JAK2 inhibitor (baricitinib) or with a standard-of-care therapy alone. METHODS: The observational cohort study included 154 adults hospitalized between February and August, 2020 with RT-PCR-confirmed SARS-CoV-2 with National Early Warning Score2 (NEWS2) < 7 and C-reactive protein (CRP) levels ≤ 140 mg/L on the day of the start of the therapy or observation. Patients were divided into the following groups: I) 4 mg baricitinib, 1 or 2 times a day for an average of 5 days (n = 38); II) 120 mg netakimab, one dose (n = 48); III) 400 mg tocilizumab, one dose (n = 34), IV) SOC only: hydroxychloroquine, antiviral, antibacterial, anticoagulant, and dexamethasone (n = 34). RESULTS: CRP levels significantly decreased after 72 h in the tocilizumab (p = 1 x 10-5) and netakimab (p = 8 x 10-4) groups and remained low after 120 h. The effect was stronger with tocilizumab compared to other groups (p = 0.028). A significant decrease in lactate dehydrogenase (LDH) levels was observed 72 h after netakimab therapy (p = 0.029). NEWS2 scores significantly improved 72 h after tocilizumab (p = 6.8 x 10-5) and netakimab (p = 0.01) therapy, and 120 h after the start of tocilizumab (p = 8.6 x 10-5), netakimab (p = 0.001), or baricitinib (p = 4.6 x 10-4) therapy, but not in the SOC group. Blood neutrophil counts (p = 6.4 x 10-4) and neutrophil-to-lymphocyte ratios (p = 0.006) significantly increased 72 h after netakimab therapy and remained high after 120 h. The percentage of patients discharged 5-7 days after the start of therapy was higher in the tocilizumab (44.1%) and netakimab (41.7%) groups than in the baricitinib (31.6%) and SOC (23.5%) groups. Compared to SOC (3 of the 34; 8.8%), mortality was lower in netakimab (0 of the 48; 0%, RR = 0.1 (95% CI: 0.0054 to 1.91)), tocilizumab (0 of the 34; 0%, RR = 0.14 (95% CI: 0.0077 to 2.67)), and baricitinib (1 of the 38; 2.6%, RR = 0.3 (95% CI: 0.033 to 2.73)) groups. CONCLUSION: In hospitalized patients with mild-to-moderate COVID-19, the combination of SOC with anti-IL-17A or anti-IL-6R therapy were superior or comparable to the combination with JAK1/JAK2 inhibitor, and all three were superior to SOC alone. Whereas previous studies did not demonstrate significant benefit of anti-IL-17A therapy for severe COVID-19, our data suggest that such therapy could be a rational choice for mild-to-moderate disease, considering the generally high safety profile of IL-17A blockers. The significant increase in blood neutrophil count in the netakimab group may reflect efflux of neutrophils from inflamed tissues. We therefore hypothesize that neutrophil count and neutrophil-to-lymphocyte ratio could serve as markers of therapeutic efficiency for IL-17A-blocking antibodies in the context of active inflammation.
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Tratamiento Farmacológico de COVID-19 , Adulto , Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales , Anticuerpos Monoclonales Humanizados , Azetidinas , Humanos , Purinas , Pirazoles , SARS-CoV-2 , Sulfonamidas , Resultado del TratamientoRESUMEN
The interplay between T- and B-cell compartments during naïve, effector and memory T cell maturation is critical for a balanced immune response. Primary B-cell immunodeficiency arising from X-linked agammaglobulinemia (XLA) offers a model to explore B cell impact on T cell subsets, starting from the thymic selection. Here we investigated characteristics of naïve and effector T cell subsets in XLA patients, revealing prominent alterations in the corresponding T-cell receptor (TCR) repertoires. We observed immunosenescence in terms of decreased diversity of naïve CD4+ and CD8+ TCR repertoires in XLA donors. The most substantial alterations were found within naïve CD4+ subsets, and we have investigated these in greater detail. In particular, increased clonality and convergence, along with shorter CDR3 regions, suggested narrower focused antigen-specific maturation of thymus-derived naïve Treg (CD4+CD45RA+CD27+CD25+) in the absence of B cells - normally presenting diverse self and commensal antigens. The naïve Treg proportion among naïve CD4 T cells was decreased in XLA patients, supporting the concept of impaired thymic naïve Treg selection. Furthermore, the naïve Treg subset showed prominent differences at the transcriptome level, including increased expression of genes specific for antigen-presenting and myeloid cells. Altogether, our findings suggest active B cell involvement in CD4 T cell subsets maturation, including B cell-dependent expansion of the naïve Treg TCR repertoire that enables better control of self-reactive T cells.
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Agammaglobulinemia/inmunología , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Agammaglobulinemia/genética , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , Estudios de Casos y Controles , Regiones Determinantes de Complementariedad/genética , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Inmunosenescencia/genética , Inmunosenescencia/inmunología , Masculino , Células T de Memoria/inmunología , Persona de Mediana Edad , Modelos Inmunológicos , Transcriptoma , Adulto JovenRESUMEN
We present protein-PAINT - the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes.
RESUMEN
Single-molecule localization microscopy relies on either controllable photoswitching of fluorescent probes or their robust blinking. We have found that blinking of monomeric red fluorescent proteins TagRFP, TagRFP-T, and FusionRed occurs at moderate illumination power and matches well with camera acquisition speed. It allows for super-resolution image reconstruction of densely labelled structures in live cells using various algorithms.
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Proteínas Luminiscentes/química , Algoritmos , Células HeLa , Humanos , Microscopía Fluorescente , Proteína Fluorescente RojaRESUMEN
Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7-mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis.
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Muerte Celular , Lisosomas , Microscopía Fluorescente/métodos , Optogenética/métodos , Fármacos Fotosensibilizantes/metabolismo , Oxígeno Singlete/farmacología , Caspasa 3/análisis , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Células HeLa , Humanos , Luz , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lisosomas/genética , Lisosomas/metabolismo , Fármacos Fotosensibilizantes/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Oxígeno Singlete/metabolismo , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice. We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor.
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Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Ligando OX40/metabolismo , Traslado Adoptivo/métodos , Animales , Carcinoma/inmunología , Carcinoma/terapia , Línea Celular , Neoplasias del Colon/inmunología , Neoplasias del Colon/terapia , Femenino , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Memoria Inmunológica/inmunología , Memoria Inmunológica/fisiología , Inmunoterapia/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ligando OX40/inmunología , Receptores OX40/inmunología , Receptores OX40/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
The development of tumor therapies based on the activation of antitumor immunity requires tumor models that are highly immunogenic. The immunologic response to fluorescent proteins, green fluorescent protein (GFP), or enhanced GFP (EGFP) was demonstrated in different cancer models. However, for live animal imaging, red and far-red fluorescent proteins are preferable, but their immunogenicity has not been studied. We assessed the immunogenicity of the red fluorescent protein, KillerRed (KR), in CT26 murine colon carcinoma. We showed a slower growth and a lower tumor incidence of KR-expressing tumors in comparison with nonexpressing ones. We found that KR-expressing lung metastases and rechallenged tumors were not formed in mice that had been surgically cured of KR-expressing primary tumors. The effect of low-dose cyclophosphamide (CY) treatment was also tested, as this is known to activate antitumor immune responses. The low-dose CY therapy of CT26-KR tumors resulted in inhibition of tumor growth and improved mouse survival. In summary, we have established a highly immunogenic tumor model that could be valuable for investigations of the mechanisms of antitumor immunity and the development of new therapeutic approaches.
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Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/farmacología , Inmunidad Innata/inmunología , Modelos Inmunológicos , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Animales , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/genética , Inmunidad Innata/efectos de los fármacos , RatonesRESUMEN
BACKGROUND: Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2. METHODS: A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice. RESULTS: Using SypHer2, pHi was demonstrated to be 7.34±0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas. CONCLUSIONS: Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models. GENERAL SIGNIFICANCE: We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution.
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Técnicas Biosensibles , Concentración de Iones de Hidrógeno , Neoplasias/metabolismo , Animales , Hipoxia de la Célula , Ingeniería Genética , Células HeLa , Humanos , Ratones , Neoplasias/patología , Esferoides CelularesRESUMEN
The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light-dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm(2) and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). Irradiation of KillerRed-expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP-like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed. Representative fluorescence image of two KillerRed-expressing spheroids before and immediately after CW irradiation.
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Terapia Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Fotoblanqueo , Fotoquimioterapia/métodos , Fototerapia/métodos , Neoplasias del Cuello Uterino/terapia , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Técnicas de Cultivo de Célula , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Femenino , Células HeLa , Humanos , Rayos Láser , Necrosis , Imagen Óptica , Fototerapia/instrumentación , Andamios del TejidoRESUMEN
Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.
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Degradación de ARNm Mediada por Codón sin Sentido/genética , Análisis de la Célula Individual/métodos , Regiones no Traducidas 3'/genética , Animales , Embrión no Mamífero/metabolismo , Citometría de Flujo , Genes Reporteros , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Microscopía Fluorescente , Empalme del ARN/genética , Xenopus laevisRESUMEN
A novel class of fluorescent dyes based on conformationally locked GFP chromophore is reported. These dyes are characterized by red-shifted spectra, high fluorescence quantum yields and pH-independence in physiological pH range. The intra- and intermolecular mechanisms of radiationless deactivation of ABDI-BF2 fluorophore by selective structural locking of various conformational degrees of freedom were studied. A unique combination of solvatochromic and lipophilic properties together with "infinite" photostability (due to a dynamic exchange between free and bound dye) makes some of the novel dyes promising bioinspired tools for labeling cellular membranes, lipid drops and other organelles.
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Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Aminación , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Microscopía Confocal , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solventes/química , Espectrometría de FluorescenciaRESUMEN
KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.
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Apoptosis , Proteínas Fluorescentes Verdes/química , Lisosomas/química , Necrosis , Fármacos Fotosensibilizantes/química , Animales , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Fibroblastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/química , Lisosomas/metabolismo , Estrés Oxidativo , Fotoquimioterapia/instrumentación , Fotoquimioterapia/métodos , Ratas , Proteínas de Unión al GTP rab/química , Proteínas de Unión a GTP rab7 , Proteína Fluorescente RojaRESUMEN
Tumor-targeted delivery of cytotoxins presents considerable advantages over their passive transport. Chemical conjugation of cytotoxic module to antibody is limited due to insufficient reproducibility of synthesis, and recombinant immunotoxins are aimed to overcome this disadvantage. We obtained genetically encoded immunophotosensitizer 4D5scFv-miniSOG and evaluated its photocytotoxic effect in vitro. A single-chain variable fragment (scFv) of humanized 4D5 antibody was used as a targeting vehicle for selective recognition of the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) overexpressed in many human carcinomas. As a phototoxic module we used a recently described photoactivated fluorescent flavoprotein miniSOG. We found that recombinant protein 4D5scFv-miniSOG exerts a highly specific photo-induced cytotoxic effect on HER2/neu-positive human breast adenocarcinoma SK-BR-3 cells (IC50= 160 nM). We demonstrated that the 4D5scFv-miniSOG specifically binds to HER2-positive cells and internalizes via receptor-mediated endocytosis. Co-treatment of breast cancer cells with 4D5scFv-miniSOG and Taxol or junction opener protein JO-1 produced remarkable additive effects.
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Antineoplásicos/farmacología , Flavoproteínas/farmacología , Inmunotoxinas/farmacología , Terapia Molecular Dirigida/métodos , Neoplasias/terapia , Fármacos Fotosensibilizantes/farmacología , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/farmacología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Flavoproteínas/genética , Humanos , Inmunotoxinas/genética , Concentración 50 Inhibidora , Luz , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismoRESUMEN
BACKGROUND: Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells. METHODS: HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo. RESULTS: Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG. CONCLUSIONS: miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. GENERAL SIGNIFICANCE: This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.
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Flavoproteínas/genética , Terapia Genética/métodos , Oxígeno/metabolismo , Fármacos Fotosensibilizantes/metabolismo , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Muerte Celular/genética , Línea Celular , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Dermatitis Fototóxica/etiología , Dermatitis Fototóxica/genética , Dermatitis Fototóxica/metabolismo , Femenino , Flavoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Luz/efectos adversos , Ratones , Ratones Desnudos , Mitocondrias/genética , Mitocondrias/metabolismo , Riboflavina/genética , Riboflavina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.
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Proteínas Luminiscentes/farmacología , Fármacos Fotosensibilizantes/farmacología , Animales , Transformación Celular Neoplásica , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Cromatina/efectos de la radiación , Femenino , Células HeLa , Histonas/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Imagen Molecular , Fármacos Fotosensibilizantes/metabolismo , Transporte de Proteínas , Proteína Fluorescente RojaRESUMEN
Cyan fluorescent proteins (CFP) with tryptophan66-based chromophore are widely used for live cell imaging. In contrast to green and red fluorescent proteins, no charged states of the CFP chromophore have been described. Here, we studied synthetic CFP chromophore and found that its indole group can be deprotonated rather easily (pKa 12.4).We then reproduced this effect in the CFP mCerulean by placing basic amino acids in the chromophore microenvironment. As a result, green-emitting variant with an anionic chromophore and key substitution Val61Lys was obtained. This is the first evidence strongly suggesting that tryptophan-based chromophores in fluorescent proteins can exist in an anionic charged state. Switching between protonated and deprotonated Trp66 in fluorescent proteins represents a new unexplored way to control their spectral properties.
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Aniones , Proteínas Fluorescentes Verdes/química , Triptófano/química , Proteínas Fluorescentes Verdes/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
Circular permutation of fluorescent proteins provides a substrate for the design of molecular sensors. Here we describe a systematic exploration of permutation sites for mCherry and mKate using a tandem fusion template approach. Circular permutants retaining more than 60% (mCherry) and 90% (mKate) brightness of the parent molecules are reported, as well as a quantitative evaluation of the fluorescence from neighboring mutations. Truncations of circular permutants indicated essential N- and C-terminal segments and substantial flexibility in the use of these molecules. Structural evaluation of two cp-mKate variants indicated no major conformational changes from the previously reported wild-type structure, and cis conformation of the chromophores. Four cp-mKates were identified with over 80% of native fluorescence, providing important new building blocks for sensor and complementation experiments.
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Proteínas Luminiscentes/química , Secuencia de Aminoácidos , Color , Cristalografía por Rayos X , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Proteína Fluorescente RojaRESUMEN
A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA normalization, may significantly increase the efficacy of random sequencing and is essential for rare gene discovery. Duplex-specific nuclease (DSN) normalization allows the generation of normalized full-length-enriched cDNA libraries to permit a high gene discovery rate. The method is based on the unique properties of DSN from the Kamchatka crab and involves denaturation-reassociation of cDNA, degradation of the ds-fraction formed by abundant transcripts by DSN, and PCR amplification of the remaining ss-DNA fraction. The method has been evaluated in various plant and animal models.
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ADN Complementario/análisis , ADN Complementario/genética , Biblioteca de Genes , Animales , Anomuros/enzimología , Anomuros/genética , ADN Complementario/metabolismo , ADN de Cadena Simple/genética , Desoxirribonucleótidos/metabolismo , Electroforesis en Gel de Agar/métodos , Endonucleasas/metabolismo , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Análisis de SecuenciaRESUMEN
The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses.
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ADN Complementario/genética , Biblioteca de Genes , Secuencia de Bases , Clonación Molecular/métodos , ADN Complementario/metabolismo , Desoxirribonucleasas/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa/métodosRESUMEN
A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.