RESUMO
Hematopoietic stem cells (HSCs) sustain blood formation throughout life and are the functional units of bone marrow transplantation. We show that transient expression of six transcription factors Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37 imparts multilineage transplantation potential onto otherwise committed lymphoid and myeloid progenitors and myeloid effector cells. Inclusion of Mycn and Meis1 and use of polycistronic viruses increase reprogramming efficacy. The reprogrammed cells, designated induced-HSCs (iHSCs), possess clonal multilineage differentiation potential, reconstitute stem/progenitor compartments, and are serially transplantable. Single-cell analysis revealed that iHSCs derived under optimal conditions exhibit a gene expression profile that is highly similar to endogenous HSCs. These findings demonstrate that expression of a set of defined factors is sufficient to activate the gene networks governing HSC functional identity in committed blood cells. Our results raise the prospect that blood cell reprogramming may be a strategy for derivation of transplantable stem cells for clinical application.
Assuntos
Reprogramação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Meis1 , Proteína Proto-Oncogênica N-Myc , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Análise de Célula Única , TranscriptomaRESUMO
Despite measures taken world-wide, the coronavirus disease 2019 (COVID-19) pandemic continues. Because efficient antiviral drugs are not yet widely available, vaccination is the best option to control the infection rate. Although this option is obvious in the case of COVID-19-naive individuals, it is still unclear when individuals who have recovered from a previous SARS-CoV-2 infection should be vaccinated and whether the vaccination raises immune responses against the coronavirus and its novel variants. In this study, we collected peripheral blood from 84 healthy human donors of different COVID-19 status who were vaccinated with the Sputnik Light vaccine and measured the dynamics of the Ab and T cell responses, as well as the virus-neutralizing activity (VNA) in serum, against two SARS-CoV-2 variants, B.1.1.1 and B.1.617.2. We showed that vaccination of individuals previously exposed to the virus considerably boosts the existing immune response. In these individuals, receptor-binding domain (RBD)-specific IgG titers and VNA in serum were already elevated on the 7th day after vaccination, whereas COVID-19-naive individuals developed the Ab response and VNA mainly 21 d postvaccination. Additionally, we found a strong correlation between RBD-specific IgG titers and VNA in serum, and according to these data vaccination may be recommended when the RBD-specific IgG titers drop to 142.7 binding Ab units/ml or below. In summary, the results of the study demonstrate that vaccination is beneficial for both COVID-19-naive and recovered individuals, especially since it raises serum VNA against the B.1.617.2 variant, one of the five SARS-CoV-2 variants of concern.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , SARS-CoV-2/imunologia , Vacinas Sintéticas/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/imunologia , Federação Russa , Linfócitos T/imunologia , VacinaçãoRESUMO
In contrast to the standard enzyme-replacement therapy, administered from once per 7-14 days to 2-3 times a week in patients with severe hemophilia B, as a result of a single injection, gene therapy can restore F9 gene expression and maintain it for a prolonged time. In clinical research, the approach of delivering a functional copy of a gene using adeno-associated viral (AAV) vectors is widely used. The scientific community is actively researching possible modifications to improve delivery efficiency and expression. In preclinical studies, the possibility of genome editing using CRISPR/Cas9 technology for the treatment of hemophilia B is also being actively studied.
Assuntos
Hemofilia A , Hemofilia B , Humanos , Hemofilia B/terapia , Hemofilia B/tratamento farmacológico , Fator IX/genética , Fator IX/uso terapêutico , Fator IX/metabolismo , Vetores Genéticos/genética , Terapia Genética , Hemofilia A/genética , Dependovirus/genética , Dependovirus/metabolismoRESUMO
Tissue autofluorescence of fixed tissue sections is a major concern of fluorescence microscopy. The adrenal cortex emits intense intrinsic fluorescence that interferes with signals from fluorescent labels, resulting in poor-quality images and complicating data analysis. We used confocal scanning laser microscopy imaging and lambda scanning to characterize the mouse adrenal cortex autofluorescence. We evaluated the efficacy of tissue treatment methods in reducing the intensity of the observed autofluorescence, such as trypan blue, copper sulfate, ammonia/ethanol, Sudan Black B, TrueVIEWTM Autofluorescence Quenching Kit, MaxBlockTM Autofluorescence Reducing Reagent Kit, and TrueBlackTM Lipofuscin Autofluorescence Quencher. Quantitative analysis demonstrated autofluorescence reduction by 12-95%, depending on the tissue treatment method and excitation wavelength. TrueBlackTM Lipofuscin Autofluorescence Quencher and MaxBlockTM Autofluorescence Reducing Reagent Kit were the most effective treatments, reducing the autofluorescence intensity by 89-93% and 90-95%, respectively. The treatment with TrueBlackTM Lipofuscin Autofluorescence Quencher preserved the specific fluorescence signals and tissue integrity, allowing reliable detection of fluorescent labels in the adrenal cortex tissue. This study demonstrates a feasible, easy-to-perform, and cost-effective method to quench tissue autofluorescence and improve the signal-to-noise ratio in adrenal tissue sections for fluorescence microscopy.
Assuntos
Córtex Suprarrenal , Lipofuscina , Camundongos , Animais , Corantes , Fluorescência , Microscopia de Fluorescência , Azul Tripano , Indicadores e Reagentes , Microscopia Confocal/métodosRESUMO
The adrenal glands are important endocrine organs that play a major role in the stress response. Some adrenal glands abnormalities are treated with hormone replacement therapy, which does not address physiological requirements. Modern technologies make it possible to develop gene therapy drugs that can completely cure diseases caused by mutations in specific genes. Congenital adrenal hyperplasia (CAH) is an example of such a potentially treatable monogenic disease. CAH is an autosomal recessive inherited disease with an overall incidence of 1:9500-1:20,000 newborns. To date, there are several promising drugs for CAH gene therapy. At the same time, it remains unclear how new approaches can be tested, as there are no models for this disease. The present review focuses on modern models for inherited adrenal gland insufficiency and their detailed characterization. In addition, the advantages and disadvantages of various pathological models are discussed, and ways of further development are suggested.
Assuntos
Hiperplasia Suprarrenal Congênita , Recém-Nascido , Humanos , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Hiperplasia Suprarrenal Congênita/terapia , Glândulas Suprarrenais , Mutação , IncidênciaRESUMO
Gender bias and the role of sex hormones in autoimmune diseases are well established. In specific pathogen-free nonobese diabetic (NOD) mice, females have 1.3-4.4 times higher incidence of type 1 diabetes (T1D). Germ-free (GF) mice lost the gender bias (female-to-male ratio 1.1-1.2). Gut microbiota differed in males and females, a trend reversed by male castration, confirming that androgens influence gut microbiota. Colonization of GF NOD mice with defined microbiota revealed that some, but not all, lineages overrepresented in male mice supported a gender bias in T1D. Although protection of males did not correlate with blood androgen concentration, hormone-supported expansion of selected microbial lineages may work as a positive-feedback mechanism contributing to the sexual dimorphism of autoimmune diseases. Gene-expression analysis suggested pathways involved in protection of males from T1D by microbiota. Our results favor a two-signal model of gender bias, in which hormones and microbes together trigger protective pathways.
Assuntos
Androgênios/metabolismo , Doenças Autoimunes/imunologia , Autoimunidade , Infecções Bacterianas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/microbiologia , Animais , Autoimunidade/imunologia , Castração , Feminino , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Interferon gama/biossíntese , Ativação Linfocitária , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Metagenoma , Camundongos , Camundongos Endogâmicos NOD , Caracteres SexuaisRESUMO
MYB is a key regulator of definitive hematopoiesis and it is dispensable for the development of primitive hematopoietic cells in vertebrates. To delineate definitive versus primitive hematopoiesis during differentiation of human embryonic stem cells, we have introduced reporters into the MYB locus and inactivated the gene by bi-allelic targeting. To recapitulate the early developmental events more adequately, the mutant and wild type human embryonic stem cell lines were differentiated in defined culture conditions without the addition of hematopoietic cytokines. The differentiation of the reporter cell lines demonstrated that MYB is specifically expressed throughout emerging hematopoietic cell populations. Here we show that the disruption of the MYB gene leads to severe defects in the development and proliferation of primitive hematopoietic progenitors while the emergence of primitive blood cells is not affected. We also provide evidence that MYB is essential for neutrophil and T cell development and the upregulation of innate immunity genes during hematopoietic differentiation. Our results suggest that the endothelial origin of primitive blood cells is direct and does not include the intermediate step of primitive hematopoietic progenitors.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Animais , Células Sanguíneas , Diferenciação Celular , Linhagem Celular , Hematopoese/genética , HumanosRESUMO
Deletion of the innate immune adaptor myeloid differentiation primary response gene 88 (MyD88) in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) results in microbiota-dependent protection from the disease: MyD88-negative mice in germ-free (GF), but not in specific pathogen-free conditions develop the disease. These results could be explained by expansion of particular protective bacteria ("specific lineage hypothesis") or by dominance of negative (tolerizing) signaling over proinflammatory signaling ("balanced signal hypothesis") in mutant mice. Here we found that colonization of GF mice with a variety of intestinal bacteria was capable of reducing T1D in MyD88-negative (but not wild-type NOD mice), favoring the balanced signal hypothesis. However, the receptors and signaling pathways involved in prevention or facilitation of the disease remained unknown. The protective signals triggered by the microbiota were revealed by testing NOD mice lacking MyD88 in combination with knockouts of several critical components of innate immune sensing for development of T1D. Only MyD88- and TIR-domain containing adapter inducing IFN ß (TRIF) double deficient NOD mice developed the disease. Thus, TRIF signaling (likely downstream of Toll-like receptor 4, TLR4) serves as one of the microbiota-induced tolerizing pathways. At the same time another TLR (TLR2) provided prodiabetic signaling by controlling the microbiota, as reduction in T1D incidence caused by TLR2 deletion was reversed in GF TLR2-negative mice. Our results support the balanced signal hypothesis, in which microbes provide signals that both promote and inhibit autoimmunity by signaling through different receptors, including receptors of the TLR family.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/microbiologia , Microbiota , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Anti-Infecciosos/metabolismo , Bactérias/metabolismo , Vida Livre de Germes , Ilhotas Pancreáticas/patologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Fator 88 de Diferenciação Mieloide/metabolismo , FilogeniaRESUMO
BACKGROUND: The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. RESULTS: We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. CONCLUSIONS: Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Introdução de Genes/métodos , RNA não Traduzido/genética , Animais , Clonagem Molecular , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , MicroinjeçõesAssuntos
Oxigenação por Membrana Extracorpórea , Pulmão/diagnóstico por imagem , Transplante de Células-Tronco Mesenquimais/métodos , Respiração Artificial , Síndrome do Desconforto Respiratório/terapia , Adulto , Seguimentos , Volume Expiratório Forçado , Nível de Saúde , Humanos , Pulmão/fisiopatologia , Masculino , Saúde Mental , Pessoa de Meia-Idade , Desempenho Físico Funcional , Qualidade de Vida , Retorno ao Trabalho , Índice de Gravidade de Doença , Teste de CaminhadaRESUMO
Type 1 diabetes (T1D) is a debilitating autoimmune disease that results from T-cell-mediated destruction of insulin-producing beta-cells. Its incidence has increased during the past several decades in developed countries, suggesting that changes in the environment (including the human microbial environment) may influence disease pathogenesis. The incidence of spontaneous T1D in non-obese diabetic (NOD) mice can be affected by the microbial environment in the animal housing facility or by exposure to microbial stimuli, such as injection with mycobacteria or various microbial products. Here we show that specific pathogen-free NOD mice lacking MyD88 protein (an adaptor for multiple innate immune receptors that recognize microbial stimuli) do not develop T1D. The effect is dependent on commensal microbes because germ-free MyD88-negative NOD mice develop robust diabetes, whereas colonization of these germ-free MyD88-negative NOD mice with a defined microbial consortium (representing bacterial phyla normally present in human gut) attenuates T1D. We also find that MyD88 deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of specific pathogen-free MyD88-negative NOD donors attenuates T1D in germ-free NOD recipients. Together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a critical epigenetic factor modifying T1D predisposition.
Assuntos
Bactérias/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/microbiologia , Imunidade Inata/imunologia , Intestinos/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/genética , Feminino , Imunidade Inata/genética , Interferon gama/imunologia , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide/genética , Filogenia , Organismos Livres de Patógenos Específicos , Fatores de TempoRESUMO
X-linked adrenoleukodystrophy is a metabolic disease associated with mutations in the ABCD1 gene (ATP-binding cassette subfamily D). Numerous pathogenic variants in this gene lead to a wide spectrum of symptoms, including adrenal insufficiency, slowly progressive dying-back axonopathy and demyelination of the central nervous system in specific phenotypes. The induced pluripotent stem cell line was derived from a patient diagnosed with x-ALD. Due to the complexity of developing working therapy based on animal models, it's crucial to obtain the cell model directly from patients. Peripheral blood mononuclear cells (PBMCs) isolated from the donor's whole blood were reprogrammed into induced pluripotent stem cells and then characterized. Expression of pluripotency markers SSEA4, TRA-1-60, SOX2, OCT4 is proven quantitatively and qualitatively, iPSCs demonstrate the ability to differentiate into three germ layers and the absence of Sendai virus expression factors.
Assuntos
Adrenoleucodistrofia , Animais , Humanos , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/metabolismo , Leucócitos Mononucleares/metabolismo , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/metabolismo , Mutação , FenótipoRESUMO
Recently, high-throughput sequencing of influenza A viruses has become a routine test. It should be noted that the extremely high diversity of the influenza A virus complicates the task of determining the sequences of all eight genome segments. For a fast and accurate analysis, it is necessary to select the most suitable reference for each segment. At the same time, there is no standardized method in the field of decoding sequencing results that allows the user to update the sequence databases to which the reads obtained by virus sequencing are compared. The IAVCP (influenza A virus consensus and phylogeny) was developed with the goal of automatically analyzing high-throughput sequencing data of influenza A viruses. Its goals include the extraction of a consensus genome directly from paired raw reads. In addition, the pipeline enables the identification of potential reassortment events in the evolutionary history of the virus of interest by analyzing the topological structure of phylogenetic trees that are automatically reconstructed.
Assuntos
Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Influenza A , Filogenia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Vírus da Influenza A/genética , Vírus da Influenza A/classificação , Humanos , Genômica/métodos , Influenza Humana/virologia , Biologia Computacional/métodosRESUMO
CDC73-related disorders are inherited in an autosomal dominant manner. An individual with a CDC73-related disorder may have inherited the disorder from an affected parent or developed it as the result of a de novo pathogenic variant of CDC73. The iPSC line was obtained by reprogramming the PBMCs of a patient with a heterozygous type mutation of the CDC73 gene. This cell line could be useful to scrutinize and study the development of CDC73-associated parathyroid carcinoma.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias das Paratireoides , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Supressoras de Tumor/genética , Mutação/genética , Neoplasias das Paratireoides/genética , Neoplasias das Paratireoides/metabolismo , Neoplasias das Paratireoides/patologia , Fatores de Transcrição/genéticaRESUMO
Pathogenic variants in the KCNV2 gene can cause a rare retinal dystrophy that can be inherited recessively, known as cone dystrophy with supernormal rod response (CDSRR). CDSRR leads to specific changes in photoreceptors' electroretinogram response, especially in the rods, poor visual acuity, photophobia, and even maculopathy. The derived iPSC lines from patients with CDSRR may pave the way for apprehension of the pathogenetic mechanism and drug development using in vitro models. PBMCs were established into induced pluripotent stem cells and then characterized by confirming the expression of pluripotency markers, demonstrating the ability to differentiate into the three germ layers, and obtaining normal karyotyping.
RESUMO
Influenza A viruses (IAVs) pose a serious threat to global health. On the one hand, these viruses cause seasonal flu outbreaks in humans. On the other hand, they are a zoonotic infection that has the potential to cause a pandemic. The most important natural reservoir of IAVs are waterfowl. In this study, we investigated the occurrence of IAV in birds in the Republic of Buryatia (region in Russia). In 2020, a total of 3018 fecal samples were collected from wild migratory birds near Lake Baikal. Of these samples, 11 were found to be positive for the H13N8 subtype and whole-genome sequencing was performed on them. All samples contained the same virus with the designation A/Unknown/Buryatia/Arangatui-1/2020. To our knowledge, virus A/Unknown/Buryatia/Arangatui-1/2020 is the first representative of the H13N8 subtype collected on the territory of Russia, the sequence of which is available in the GenBank database. An analysis of reassortments based on the genome sequences of other known viruses has shown that A/Unknown/Buryatia/Arangatui-1/2020 arose as a result of reassortment. In addition, a reassortment most likely occurred several decades ago between the ancestors of the viruses recently collected in China, the Netherlands, the United States and Chile. The presence of such reassortment emphasizes the ongoing evolution of the H13N8 viruses distributed in Europe, North and East Asia, North and South America and Australia. This study underscores the importance of the continued surveillance and research of less-studied influenza subtypes.
Assuntos
Aves , Genoma Viral , Vírus da Influenza A , Influenza Aviária , Filogenia , Vírus Reordenados , Sequenciamento Completo do Genoma , Animais , Vírus Reordenados/genética , Vírus Reordenados/classificação , Vírus Reordenados/isolamento & purificação , Influenza Aviária/virologia , Influenza Aviária/epidemiologia , Federação Russa/epidemiologia , Aves/virologia , Vírus da Influenza A/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Fezes/virologia , Animais Selvagens/virologiaRESUMO
Codon optimization has evolved to enhance protein expression efficiency by exploiting the genetic code's redundancy, allowing for multiple codon options for a single amino acid. Initially observed in E. coli, optimal codon usage correlates with high gene expression, which has propelled applications expanding from basic research to biopharmaceuticals and vaccine development. The method is especially valuable for adjusting immune responses in gene therapies and has the potenial to create tissue-specific therapies. However, challenges persist, such as the risk of unintended effects on protein function and the complexity of evaluating optimization effectiveness. Despite these issues, codon optimization is crucial in advancing gene therapeutics. This study provides a comprehensive review of the current metrics for codon-optimization, and its practical usage in research and clinical applications, in the context of gene therapy.
RESUMO
Cone dystrophy with supernormal rod response (CDSRR) is associated with pathogenic variants of the KCNV2 gene that result in severe symptoms, including color vision defects, decreased visual acuity, and specific changes in electroretinogram responses. Two iPSC lines were obtained from two patients in the same family with different types of mutations in the KCNV2 gene. These lines could serve as a useful model for studying the pathogenetic mechanism and treatment development for CDSRR. PBMCs from donors have been reprogrammed into iPSC lines. Derived clones were characterized with mutation sequencing, analysis of common pluripotency-associated markers at the protein levels, and in vitro differentiation studies.
Assuntos
Distrofia de Cones , Células-Tronco Pluripotentes Induzidas , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Humanos , Células Fotorreceptoras Retinianas Bastonetes , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genéticaRESUMO
The adeno-associated virus (AAV) is one of the most potent vectors in gene therapy. The experimental profile of this vector shows its efficiency and accepted safety, which explains its increased usage by scientists for the research and treatment of a wide range of diseases. These studies require using functional, pure, and high titers of vector particles. In fact, the current knowledge of AAV structure and genome helps improve the scalable production of AAV vectors. In this review, we summarize the latest studies on the optimization of scalable AAV production through modifying the AAV genome or biological processes inside the cell.
RESUMO
Recently, the mRNA platform has become the method of choice in vaccine development to find new ways to fight infectious diseases. However, this approach has shortcomings, namely that mRNA vaccines require special storage conditions, which makes them less accessible. This instability is due to the fact that the five-prime and three-prime ends of the mRNA are a substrate for the ubiquitous exoribonucleases. To address the problem, circular mRNAs have been proposed for transgene delivery as they lack these ends. Notably, circular RNAs do not have a capped five-prime end, which makes it impossible to initiate translation canonically. In this review, we summarize the current knowledge on cap-independent translation initiation methods and discuss which approaches might be most effective in developing vaccines and other biotechnological products based on circular mRNAs.