RESUMEN
According to the World Health Organization, as of January 3, 2020 to September 13, 2023, there were approximately 23 million confirmed cases of COVID-19 reported in the Russian Federation, about 400 thousand of which were fatal. Considering the high rate of mutation of the RNA-containing virus genome, which inevitably leads to the emergence of new infectious strains (Eris and Pyrola), the search for medicinal antiviral agents remains an urgent task. Moreover, taking into account the actively mutating receptor-binding domain, this task requires fundamentally new solutions. This study proposes a candidate immunoliposomal drug that targets the S protein of SARS-CoV-2 by the monoclonal neutralizing antibody P4A1 and ensures the penetration of a highly active ribonuclease into the virus-infected cell, which degrades, among cellular RNA, viral RNA too. We demonstrate a more than 40-fold increase in the neutralizing activity of the developed drug compared to the free monoclonal neutralizing antibody.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Antivirales/farmacología , Pruebas de Neutralización , Anticuerpos Neutralizantes/farmacología , ARN , Anticuerpos AntiviralesRESUMEN
Nanomedicine has revolutionized the available treatment options during the last decade, but poor selectivity of targeted drug delivery and release is still poses a challenge. In this study, doxorubicin (DOX) and magnetite nanoparticles were encapsulated by freezing-induced loading, coated with polymeric shell bearing two bi-layers of polyarginine/dextran sulphate and finally modified with HER2-specific DARPin proteins. We demonstrated that the enhanced cellular uptake of these nanocarriers predominantly occurs by SKOV-3 (HER2+) cells, in comparison to CHO (HER2-) cells, together with the controlled DOX release using low intensity focused ultrasound (LIFU). In addition, a good ability of DARPin+ capsules to accumulate in the tumor and the possibility of combination therapy with LIFU were demonstrated. A relatively high sensitivity of the obtained nanocarriers to LIFU and their preferential interactions with mitochondria in cancer cells make these carriers promising candidates for cancer treatment, including novel approaches to overcome drug resistance.
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Óxido Ferrosoférrico , Polímeros , Nanomedicina , Doxorrubicina/farmacologíaRESUMEN
The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.
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Antineoplásicos , Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Proteínas Bacterianas/metabolismo , Ribonucleasas/metabolismo , Antineoplásicos/farmacología , Linfocitos T/metabolismoRESUMEN
Development of combined schemes for the treatment of oncological diseases is a promising strategy to improve the effectiveness of antitumor therapy. This paper shows the fundamental possibility of multiplying the antitumor effect by combining targeted and photodynamic therapy. It was demonstrated that sequential treatment of HER-2 positive breast cancer cells with the targeted toxin DARPin-LoPE and the photoactive compound photodithazine leads to a synergistic enhancement of their effect. In the future, this approach is intended to achieve the maximum therapeutic effect while minimizing the risks of negative side effects.
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Neoplasias , Fotoquimioterapia , Receptor ErbB-2 , Línea Celular TumoralRESUMEN
Combining diagnostic and therapeutic functions in one agent is a promising strategy in the development of personalized approaches to the treatment of cancer. The opportunity to combine diagnostics and therapy appeared with the development of nanobiotechnologies and was realized in the concept of theranostics. To date, a number of promising agents based on nanomaterials capable of diagnosing, targeted therapeutic effects, and monitoring the response of tumor cells were obtained within the approach of theranostics. In this work, a new type of theranostic complexes based on upconversion nanoparticles coated with polyethylene glycol and functionalized with the DARPin-LoPE recombinant targeted toxin was developed. Selective binding of complexes to human breast adenocarcinoma cells overexpressing the HER2 receptor and specific toxicity to them were shown.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Luminiscencia , Receptor ErbB-2/metabolismo , Nanomedicina Teranóstica , Animales , Células CHO , Cricetinae , Cricetulus , Femenino , Humanos , Nanopartículas/química , Nanoestructuras/química , Fotoquímica , Polietilenglicoles/química , Unión Proteica , Tulio/química , Iterbio/químicaRESUMEN
Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatotoxicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.
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Antineoplásicos/efectos adversos , Furina/química , Furina/metabolismo , Hígado/efectos de los fármacos , Terapia Molecular Dirigida , Antineoplásicos/uso terapéutico , Sitios de Unión , Línea Celular Tumoral , Células Hep G2 , Humanos , Dominios Proteicos , Transporte de Proteínas/efectos de los fármacosRESUMEN
Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.
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Proteínas Luminiscentes/biosíntesis , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Clonación Molecular , Femenino , Expresión Génica , Humanos , Proteínas Luminiscentes/genética , Transfección , Proteína Fluorescente RojaRESUMEN
Fibroblasts isolated from skin biopsy specimens from patients with genetic forms of Parkinson's disease, carriers of mutations in LRRK2 and PARK2 genes, and from a healthy volunteer were reprogrammed using lentiviral vectors into induced pluripotent stem cells (iPSC). iPSC were differentiated into neuron-like cells using a cocktail of differentiation factors (N2, B27, and Noggin). The iPSC lines derived from patients with different mutations and from a healthy volunteer cultured under the same conditions were characterized by different proportion of neuronal precursors and differentiated neurons. Control Po2 line contained 56% precursors, while B15 line with LRRK2 gene mutation (G2019S) contained 35% precursor cells. Similar regularities were characteristic of Tr5 culture carrying compound heterozygous mutations in PARK2 gene (del202-203AG and IVS1+1G/A) and containing 4% neuronal precursors. Further comparative studies of iPSC carrying various mutations and comparison with normal human cells will help to understand the molecular pathogenesis of some genetic variants of Parkinson's disease.
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Reprogramación Celular/fisiología , Fibroblastos/patología , Neuronas/fisiología , Enfermedad de Parkinson , Proteínas Serina-Treonina Quinasas/genética , Ubiquitina-Proteína Ligasas/genética , Diferenciación Celular/genética , Células Cultivadas , Fibroblastos/fisiología , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/fisiología , Lentivirus/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mutación , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fenotipo , Piel/patología , TransfecciónRESUMEN
Reaction of the glutathione system of Jurkat tumor cells and blood lymphocytes was evaluated under conditions of culturing with 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole (KRIBB3), a selective inhibitor of heat shock protein Hsp27. The results indicated the regulatory role of Hsp27 in the maintenance of the functional activities of glutathione reductase, glutathione peroxidase, and realization of apoptotic death of Jurkat cells and blood lymphocytes. Inhibition of Hsp27 in Jurkat tumor cells led to imbalance of the glutathione system and increase of the share of annexin-positive cells.
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Glutatión/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Adulto , Animales , Anisoles/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Femenino , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Proteínas de Choque Térmico HSP27/antagonistas & inhibidores , Proteínas de Choque Térmico HSP27/genética , Humanos , Isoxazoles/farmacología , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacos , Adulto JovenRESUMEN
We have used an original chromatography/mass spectrometry technique to study the pharmacokinetics of dipeptide carnosine in C57 Black/6 mice after intra-peritoneal administration of the drug at a dose of 1 g/kg. The basic pharmacokinetic characteristics of carnosine were measured the in the blood and brain. The obtained concentration-time curve has a biexponential character. It is shown that the maximum concentration of carnosine in the blood plasma is Cmax = 1081.75 ± 124.24 µg/mL and it is achieved in a time interval of Tmax = 0.25 h. We showed that i.p. administration of exogenous carnosine could significantly increase the concentration of that substance in the brain. Tissue availability of dipeptide carnosine for brain tissue is relatively good and constitutes 59% from the total amount of blood carnosine. It was found that the maximum concentration of carnosine in the brain occurs at the sixth hour after i.p. administration when the concentration of drug in the blood is minimal.
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Química Encefálica/efectos de los fármacos , Encéfalo/metabolismo , Carnosina/farmacología , Carnosina/farmacocinética , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
We studied the effects of histone deacetylase inhibitor that stimulates transcriptional activity via histone hyperacetylation on memory formation. Sodium butyrate and sodium valproate enhanced memory in chicks following "weak" training with memory transfer into long-term state. Quantitative analysis of c-Fos and ZENK transcriptional factor gene expression in six structures of chick brain revealed induction of these genes in the structures involved in this type of learning. Sodium valproate administration did not increase this induction, but even reduced it. These findings suggest that sodium butyrate and sodium valproate exert cognitive stimulating action in the "weak" memory formation paradigm, and that this effect is not mediated via enhanced expression of transcriptional factors, which are traditionally considered as "molecular switcher" for memory transfer into long-term state.
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Reacción de Prevención/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Inmediatos-Precoces/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Memoria/efectos de los fármacos , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Butiratos/farmacología , Pollos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/genética , Técnicas Histológicas , Estadísticas no Paramétricas , Factores de Tiempo , Ácido Valproico/farmacologíaRESUMEN
Three-dimensional gel-based microchip allowing simultaneous quantitative detection of total (PSAtot) and free (PSAfree) forms of prostate specific antigen in human serum (in a format "one patient-one biochip") was developed. A method, which doesn't require preliminary construction of calibration curves when performing an assay, was applied for quantitative determination of PSAtot and PSAfree. Gel elements with immobilized antigen (PSA) in different concentration, forming an internal calibration curve, were included in a structure of the microchip, in addition to the elements with immobilized antibodies specific against PSAtot and PSAfree. The specialized software "ImaGelAssay" was used for data processing and interpretation. The sensitivity of the assay performed on biochips was 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. Variation coefficient for the measurements inside one series of microchips didn't exceed 10%. Correlation coefficient between the results of measurements in human sera obtained on biochips and by the standard ELISA method was 0.988 for PSAtot and 0.987 for PSAfree.
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Biomarcadores de Tumor/sangre , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/diagnóstico , Análisis por Matrices de Proteínas/métodos , Análisis por Matrices de Proteínas/normas , Calibración , Humanos , Inmunohistoquímica , Masculino , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
DNA analysis is a key procedure in genetic engineering. Nowadays the analysis is often done by PCR with Taq DNA polymerase. Although the last enzyme price is quite low, demand for numerous analyses results in much money expenditure which are not affordable for many laboratories. In a meanwhile, many screening tasks do not require the highly purified enzyme. Taking into account the enzyme unique properties it makes possible to marginally simplify its production without resorting to costly or lengthy techniques such as column chromatography and/or dialysis. Here the data of routine usage of Taq DNA polymerase prepared according to the protocol developed in our laboratory is presented. The protocol takes only several hours to realize and does not need qualified personnel or expensive equipment. Yet it gives the enzyme preparation suitable for most screening purposes. The isolated Taq DNA polymerase stock can be stored as ammonium sulfate suspension in a refrigerator for prolonged period, not less than 6 months. The working enzyme solution is prepared from the stock suspension on demand, not more than once in a month and can be stored also in a refrigerator.
RESUMEN
iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed, iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro, phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation, validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data, typically by relating the obtained levels of gene expression to the levels of expression of a "house keeping gene", a gene whose expression is presumed not to change during manipulation of the cells. In the literature, typically only one such reference gene is used and its stability of expression during cell manipulation is not demonstrated. We are not aware of any study systematically looking at the expression of such genes in human iPSC or during their differentiation into neurons. Here we compare the expression of 16 reference genes in iPSC, neural stem cells (NSC) and neurons derived from iPSC. The applications GeNorm and NormFinder were used to identify the most suitable reference genes. We showed that ACTb, C1orf43, PSMB4, GAPDH and HMBS have the most stable expression. The use of these reference genes allows an accurate normalization of qRT-PCR results in all the cell types discussed above. We hope that this report will help to enable the performance of proper qRT-PCR results normalization in studies with iPSC-derived cells and in disease-modeling reports.
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Células Madre Pluripotentes Inducidas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Actinas/genética , Actinas/metabolismo , Células Cultivadas , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Humanos , Hidroximetilbilano Sintasa/genética , Hidroximetilbilano Sintasa/metabolismo , Células Madre Pluripotentes Inducidas/citología , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estándares de ReferenciaRESUMEN
Parkinson's disease is caused by the degeneration of midbrain dopaminergic neurons. A rare recessive form of the disease may be caused by a mutation in the PARK2 gene, whose product, Parkin, controls mitophagy and programmed cell death. The level of pro- and anti-apoptotic factors of the Bcl-2 family was determined in dopaminergic neurons derived from the induced pluripotent stem cells of a healthy donor and a Parkinson's disease patient bearing PARK2 mutations. Western blotting was used to study the ratios of Bax, Bak, Bcl-2, Bcl-XL, and Bcl-W proteins. The pro-apoptotic Bak protein level in PARK2-neurons was shown to be two times lower than that in healthy cells. In contrast, the expression of the anti-apoptotic factors Bcl-XL, Bcl-W, and Bcl-2 was statistically significantly higher in the mutant cells compared to healthy dopaminergic neurons. These results indicate that PARK2 mutations are accompanied by an imbalance in programmed cell death systems in which non-apoptotic molecular mechanisms play the leading role.
RESUMEN
Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.
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Hidrogeles , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Proteínas/análisis , Proteínas/clasificación , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodosRESUMEN
An original method for life-time diagnosis of Greutzfeldt-Jakob's disease, a neurodegenerative disease belonging to transmissive spongiform encephalopathies, is proposed. It consists in inoculation of the examined biological materials (blood serum and clot) into a continuous culture of the rat Gasser's node neurinoma, followed by passages and study of the inoculated culture by morphological methods and electron microscopy.
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Técnicas de Laboratorio Clínico , Síndrome de Creutzfeldt-Jakob/diagnóstico , Animales , Técnicas de Cultivo , Humanos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , RatasRESUMEN
Optimal regimens of low-frequency alternating magnetic field generated by Polius-1 apparatus were developed during treatment of 195 chronic pancreatitis sufferers. When sinusoidal and continuous in induction 17.5 mT, the field proved beneficial in the condition studied as shown by laboratory findings and long-term therapeutic response.