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1.
Nat Commun ; 12(1): 1918, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771997

RESUMO

The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These SFPQ-inhibited CLEs appear preferentially in long introns of genes with neuronal functions and can dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found expressed across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a gene regulation mechanism with wide relevance to human neuropathologies.


Assuntos
Esclerose Amiotrófica Lateral/genética , Códon sem Sentido , Éxons/genética , Fator de Processamento Associado a PTB/genética , Animais , Sequência de Bases , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Hibridização In Situ/métodos , Íntrons/genética , Camundongos , Neurônios/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética
2.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 39(1): 32-37, 2021 Feb 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33723934

RESUMO

OBJECTIVES: A study was conducted to explore the expression pattern and function of ferritin heavy polypeptide gene (fth1b) in zebrafish pharyngeal teeth development and lay the foundation for subsequent research on teeth development and mineralization. METHODS: The zebrafish embryos were harvested at 56, 72, 96, and 120 h after fertilization. The expression of fth1b in zebrafish pharyngeal teeth development was detected by whole embryo in situ hybridization and compared with the known pharyngeal teeth marker dlx2b. The specific knockout of fth1b gene was performed using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology. The development of zebrafish pharyngeal teeth was detected in the fth1b-/- mutant. RESULTS: The expression pattern of fth1b gene was very similar to that of the known zebrafish pharyngeal teeth marker dlx2b and was specifically expressed in the zebrafish pharyngeal teeth during development. After the specific knockout of the gene fth1b, the earliest gene that can be detect in zebrafish pharyngeal teeth-pitx2 was expressed normally during early development. The dlx2b expression was not significantly different from that of wild type zebrafish, but the mineralization of pharyngeal teeth in the mutant was weaker than that of wild type zebrafish. CONCLUSIONS: The gene fth1b is specifically expressed in zebrafish pharyngeal teeth and acts on their early mineralization.


Assuntos
Dente , Peixe-Zebra , Animais , Hibridização In Situ , Odontogênese , Faringe , Peixe-Zebra/genética
3.
Nat Commun ; 12(1): 749, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531476

RESUMO

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFß (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFß signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFß signalling to regulate its pace.


Assuntos
Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Fusão Celular , Galinhas , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/metabolismo , Transdução de Sinais/fisiologia
4.
Ann Pathol ; 41(1): 9-22, 2021 Feb.
Artigo em Francês | MEDLINE | ID: mdl-33446414

RESUMO

The infection due to the SARS-CoV-2 leads lesions mainly observed at the respiratory tract level, but not exclusively. The analyses of these lesions benefited from different autopsy studies. Thus, these lesions were observed in different organs, tissues and cells. These observations allowed us to rapidly improve the knowledge of the pathophysiological mechanisms associated with this emergent infectious disease. The virus can be detected in formalin fixed paraffin embedded tissues using immunohistochemistry, in situ hybridization, molecular biology and/or electron microscopy approaches. However, many uncertainties are still present concerning the direct role of the SARS-CoV-2 on the different lesions observed in different organs, outside the lung, such as the heart, the brain, the liver, the gastrointestinal tract, the kidney and the skin. In this context, it is pivotal to keep going to increase the different tissue and cellular studies in the COVID-19 positive patients aiming to better understanding the consequences of this new infectious disease, notably considering different epidemiological and co-morbidities associated factors. This could participate to the development of new therapeutic strategies too. The purpose of this review is to describe the main histological and cellular lesions associated with the infection due to the SARS-CoV-2.


Assuntos
/patologia , Autopsia , Fibrose/patologia , Fibrose/virologia , Histocitoquímica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Rim/patologia , Rim/virologia , Fígado/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Pele/patologia , Pele/virologia , Trombose/patologia , Trombose/virologia
5.
J Infect Dis ; 223(5): 752-764, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33502471

RESUMO

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic continues to produce substantial morbidity and mortality. To understand the reasons for the wide-spectrum complications and severe outcomes of COVID-19, we aimed to identify cellular targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and replication in various tissues. METHODS: We evaluated RNA extracted from formalin-fixed, paraffin-embedded autopsy tissues from 64 case patients (age range, 1 month to 84 years; 21 COVID-19 confirmed, 43 suspected COVID-19) by SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR). For cellular localization of SARS-CoV-2 RNA and viral characterization, we performed in situ hybridization (ISH), subgenomic RNA RT-PCR, and whole-genome sequencing. RESULTS: SARS-CoV-2 was identified by RT-PCR in 32 case patients (21 COVID-19 confirmed, 11 suspected). ISH was positive in 20 and subgenomic RNA RT-PCR was positive in 17 of 32 RT-PCR-positive case patients. SARS-CoV-2 RNA was localized by ISH in hyaline membranes, pneumocytes, and macrophages of lungs; epithelial cells of airways; and endothelial cells and vessel walls of brain stem, leptomeninges, lung, heart, liver, kidney, and pancreas. The D614G variant was detected in 9 RT-PCR-positive case patients. CONCLUSIONS: We identified cellular targets of SARS-CoV-2 tropism and replication in the lungs and airways and demonstrated its direct infection in vascular endothelium. This work provides important insights into COVID-19 pathogenesis and mechanisms of severe outcomes.


Assuntos
/virologia , Endotélio Vascular/virologia , Sistema Respiratório/virologia , Replicação Viral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , Criança , Pré-Escolar , Feminino , Humanos , Hibridização In Situ , Lactente , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , /isolamento & purificação , Tropismo Viral , Sequenciamento Completo do Genoma , Adulto Jovem
6.
Methods Mol Biol ; 2209: 307-319, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201477

RESUMO

The mechanisms involved in the posttranscriptional control of the replicative cycle of the human immunodeficiency virus (HIV), specifically the molecular events which allow the interaction between the viral genomic RNA (gRNA) and the cellular machinery for the transport, translation, or intracellular packaging, have not been yet elucidated. In this chapter, we describe the in situ hybridization-proximity ligation assay (ISH-PLA) to characterize interactions between the genomic RNA (gRNA) of HIV-1 and viral proteins or host proteins involved in nuclear export and translation initiation. We also present data that validate the ISH-PLA as a simple and useful tool to study HIV-1 gRNA-protein interactions within cells.


Assuntos
HIV-1/genética , Hibridização In Situ/métodos , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células HeLa , Humanos , Ligação Proteica
7.
Methods Mol Biol ; 2209: 387-401, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201482

RESUMO

Ribonucleases remodel RNAs to render them functional or to send them on their way toward degradation. In our laboratory, we study these pathways in detail using a plethora of different techniques. These can range from the isolation of RNAs in various RNase mutants to determine their implication in maturation or decay pathways by Northern blot, to proving their direct roles in RNA cleavage reactions using purified enzymes and transcribed substrates in vitro. In this chapter, we provide in-depth protocols for the techniques we use daily in the laboratory to assay RNase activity in vivo, with detailed notes on how to get these methods to work optimally. This chapter complements Chapter 25 on assays of ribonuclease action in vitro.


Assuntos
Bacillus subtilis/enzimologia , Ensaios Enzimáticos/métodos , Hibridização In Situ/métodos , RNA Bacteriano/metabolismo , Ribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , Cinética
8.
Methods Mol Biol ; 2222: 381-394, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301103

RESUMO

Genomic in situ hybridization (GISH) is an invaluable cytogenetic technique which enables the visualization of whole genomes in hybrids and polyploidy taxa. Total genomic DNA from one or two different species/genomes is used as a probe, labeled with a fluorochrome, and directly detected on mitotic chromosomes from root tip meristems. In sugarcane and sugarcane hybrids, we were able to characterize interspecific hybrids of two closely related species as well as intergeneric hybrids of two closely related genera.


Assuntos
Genoma de Planta , Genômica , Hibridização Genética , Hibridização In Situ/métodos , Cruzamentos Genéticos , Genômica/métodos , Hibridização in Situ Fluorescente/métodos , Raízes de Plantas , Saccharum/genética
9.
Methods Mol Biol ; 2245: 85-92, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315196

RESUMO

The RNA in situ hybridization assay is essential in many studies to evaluate gene expression in vivo. It consists of generating tissue sections and subsequently hybridizing these sections with RNA probes. Keeping RNA intact is a challenge while harvesting tissue samples, processing through embedding, sectioning them, and conditioning the sections for hybridization. These challenges are particularly strong for adult skeletal tissues due to their copious, dense, and mineralized extracellular matrices. Here, we describe a method optimized to successfully hybridize RNA species, even of low abundance, in adult mouse bone and cartilage samples. This method involves tissue fixation with paraformaldehyde, demineralization with Morse's solution and paraffin embedding, all of which can be completed in 4 days. Sections are then generated and hybridized using a 1-day standard protocol. Sections prepared using this method are compatible with immunostaining and standard staining procedures for skeletal tissues.


Assuntos
Osso e Ossos/metabolismo , Hibridização In Situ/métodos , RNA , Animais , Osso e Ossos/citologia , Cartilagem/citologia , Cartilagem/metabolismo , Análise de Dados , Expressão Gênica , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos
10.
Methods Mol Biol ; 2245: 93-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315197

RESUMO

MicroRNA (miRNA) in situ hybridization (ISH) is a highly sensitive method that allows for the detection of expression and distribution of miRNAs in fixed paraffin-embedded tissues. MiRNA ISH requires time-consuming optimization based on the tissue type analyzed, method of tissue fixation, and miRNA detection probe. Here, we provide the optimized miRNA ISH protocol for human cartilage and mouse whole knee joints that also entails the necessary steps for sample collection, processing, and preparation for high-quality ISH staining.


Assuntos
Cartilagem Articular/metabolismo , Hibridização In Situ , Articulação do Joelho/metabolismo , MicroRNAs/genética , Animais , Humanos , Hibridização In Situ/métodos , Camundongos , Oligonucleotídeos , Inclusão em Parafina , Fixação de Tecidos
11.
Hum Pathol ; 109: 69-79, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33321162

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was demonstrated in the placenta; however, the data on the prevalence of placental infection and associated histopathology are limited. To identify the frequency and features of SARS-CoV-2 involvement, we performed a clinicopathologic analysis of 75 placental cases from women infected at the time of delivery and 75 uninfected controls. Placental samples were studied with anti-SARS-CoV-2 immunohistochemistry and/or in situ hybridization. Positive results were confirmed by electron microscopy and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). During delivery, only one woman had symptoms of coronavirus disease 2019, six women reported previous symptoms, and 68 women were asymptomatic. All neonates tested negative for SARS-CoV-2 as per nasopharyngeal swab PCR results. Obstetric histories were unremarkable in 29 of 75 SARS-CoV-2-positive and 8 of 75 SARS-CoV-2-negative women. Placental examination was normal in 12 of 75 infected and 3 of 75 uninfected subjects, respectively. In the remaining cases, placental pathology correlated with obstetric comorbidities without significant differences between SARS-CoV-2-positive and SARS-CoV-2-negative women. SARS-CoV-2 was identified in one placenta of an infected, but asymptomatic, parturient. Viral staining was predominantly localized to the syncytiotrophoblast (STB) which demonstrated marked damage accompanied by perivillous fibrin deposition and mixed intervillositis. A significant decrease of viral titers was detected in the attached umbilical cord compared with the villous parenchyma as per qRT-PCR. SARS-CoV-2 is seldom identified in placentas of infected women. Placental involvement by the virus is characterized by STB damage disrupting the placental barrier and can be seen in asymptomatic mothers without evidence of vertical transmission.


Assuntos
/virologia , Placenta/patologia , Trofoblastos/patologia , Trofoblastos/virologia , Adulto , Feminino , Humanos , Hibridização In Situ/métodos , Placenta/virologia , Gravidez , RNA Viral , Trofoblastos/química , Carga Viral
12.
Methods Mol Biol ; 2170: 143-154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797457

RESUMO

MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. Therefore, in situ detection of mature miRNAs is important for understanding the functions for both miRNAs and their targets. The construction of promoter-reporter fusions and examination of their in planta expression has been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs, because the mature miRNAs are too short (~21-nucleotides) to exhibit stable hybridization signals. In recent years, the Locked nucleic acid (LNA) modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.


Assuntos
Sondas de DNA/química , MicroRNAs/análise , MicroRNAs/química , Oligonucleotídeos/química , RNA de Plantas/análise , RNA de Plantas/química , Hibridização In Situ
13.
Methods Mol Biol ; 2219: 181-194, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33074541

RESUMO

The Porifera are one of the best candidates as the sister group to all other metazoans. Studies on this phylum are therefore expected to shed light on the origin and early evolution of key animal features. Transcriptomic or genomic data acquired during the last 10 years have highlighted the conservation of most of the main genes and pathways involved in the development of the other metazoans. The next step is to determine how similar genetic tool boxes can result in widely dissimilar body plan organization, dynamics, and life histories. To answer these questions, three main axes of research are necessary: (1) conducting more gene expression studies; (2) developing knockdown protocols; and (3) reinterpreting sponge cell biology using modern tools. In this chapter we focus on the in situ hybridization (ISH) technique, needed to establish the spatiotemporal expression of genes, both on whole mount individuals and paraffin sections, and at different stages of development (adults, embryos, larvae, buds) of the homoscleromorph sponge Oscarella lobularis.


Assuntos
Hibridização In Situ/métodos , Poríferos/genética , Animais , Microscopia/métodos , Poríferos/citologia , Poríferos/ultraestrutura , Inclusão do Tecido/métodos , Fixação de Tecidos/métodos
14.
Am J Surg Pathol ; 45(1): 14-24, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32826529

RESUMO

Coronavirus disease-19 (COVID-19) is caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although SARS-CoV-2 is visualized on electron microscopy, there is an increasing demand for widely applicable techniques to visualize viral components within tissue specimens. Viral protein and RNA can be detected on formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. Herein, we evaluate the staining performance of ISH for SARS-CoV-2 and an IHC directed at the SARS-CoV nucleocapsid protein and compare these results to a gold standard, tissue quantitative real-time polymerase chain reaction (qRT-PCR). We evaluated FFPE sections from 8 COVID-19 autopsies, including 19 pulmonary and 39 extrapulmonary samples including the heart, liver, kidney, small intestine, skin, adipose tissue, and bone marrow. We performed RNA-ISH for SARS-CoV-2 on all cases with IHC for SARS-CoV and SARS-CoV-2 qRT-PCR performed on selected cases. Lungs from 37 autopsies performed before the COVID-19 pandemic served as negative controls. The ISH and IHC slides were reviewed by 4 observers to record a consensus opinion. Selected ISH and IHC slides were also reviewed by 4 independent observers. Evidence of SARS-CoV-2 was identified on both the IHC and ISH platforms. Within the postmortem lung, detected viral protein and RNA were often extracellular, predominantly within hyaline membranes in patients with diffuse alveolar damage. Among individual cases, there was regional variation in the amount of detectable virus in lung samples. Intracellular viral RNA and protein was localized to pneumocytes and immune cells. Viral RNA was detected on RNA-ISH in 13 of 19 (68%) pulmonary FFPE blocks from patients with COVID-19. Viral protein was detected on IHC in 8 of 9 (88%) pulmonary FFPE blocks from patients with COVID-19, although in 5 cases the stain was interpreted as equivocal. From the control cohort, FFPE blocks from all 37 patients were negative for SARS-CoV-2 RNA-ISH, whereas 5 of 13 cases were positive on IHC. Collectively, when compared with qRT-PCR on individual tissue blocks, the sensitivity and specificity for ISH was 86.7% and 100%, respectively, while those for IHC were 85.7% and 53.3%, respectively. The interobserver variability for ISH ranged from moderate to almost perfect, whereas that for IHC ranged from slight to moderate. All extrapulmonary samples from COVID-19-positive cases were negative for SARS-CoV-2 by ISH, IHC, and qRT-PCR. SARS-CoV-2 is detectable on both RNA-ISH and nucleocapsid IHC. In the lung, viral RNA and nucleocapsid protein is predominantly extracellular and within hyaline membranes in some cases, while intracellular locations are more prominent in others. The intracellular virus is detected within pneumocytes, bronchial epithelial cells, and possibly immune cells. The ISH platform is more specific, easier to analyze and the interpretation is associated with the improved interobserver agreement. ISH, IHC, and qRT-PCR failed to detect the virus in the heart, liver, and kidney.


Assuntos
/diagnóstico , Imuno-Histoquímica , Hibridização In Situ , Pulmão/virologia , RNA Viral/análise , /genética , /virologia , Humanos , Fosfoproteínas/análise , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes
15.
Diabetes ; 70(3): 759-771, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33310740

RESUMO

The causes of the increased risk of severe coronavirus disease 2019 (COVID-19) in people with diabetes are unclear. It has been speculated that renin-angiotensin system (RAS) blockers may promote COVID-19 by increasing ACE2, which severe acute respiratory syndrome coronavirus 2 uses to enter host cells, along with the host protease TMPRSS2. Taking a reverse translational approach and by combining in situ hybridization, primary cell isolation, immunoblotting, quantitative RT-PCR, and liquid chromatography-tandem mass spectrometry, we studied lung and kidney ACE2 and TMPRSS2 in diabetic mice mimicking host factors linked to severe COVID-19. In healthy young mice, neither the ACE inhibitor ramipril nor the AT1 receptor blocker telmisartan affected lung or kidney ACE2 or TMPRSS2, except for a small increase in kidney ACE2 protein with ramipril. In contrast, mice with comorbid diabetes (aging, high-fat diet, and streptozotocin-induced diabetes) had heightened lung ACE2 and TMPRSS2 protein levels and increased lung ACE2 activity. None of these parameters were affected by RAS blockade. ACE2 was similarly upregulated in the kidneys of mice with comorbid diabetes compared with aged controls, whereas TMPRSS2 (primarily distal nephron) was highest in telmisartan-treated animals. Upregulation of lung ACE2 activity in comorbid diabetes may contribute to an increased risk of severe COVID-19. This upregulation is driven by comorbidity and not by RAS blockade.


Assuntos
/genética , Diabetes Mellitus Experimental/metabolismo , Dieta Hiperlipídica , Rim/metabolismo , Pulmão/metabolismo , Serina Endopeptidases/genética , Fatores Etários , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , /metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Immunoblotting , Hibridização In Situ , Rim/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Ramipril/farmacologia , /genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Telmisartan/farmacologia
16.
Nucleic Acids Res ; 49(2): 657-673, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367834

RESUMO

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.


Assuntos
Sistema Nervoso Central/química , Camundongos/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , Primatas/metabolismo , Ratos/metabolismo , Animais , Sistema Nervoso Central/citologia , Feminino , Hibridização In Situ , Injeções Intraventriculares , Injeções Espinhais , Macaca fascicularis , Masculino , Neuroglia/química , Neurônios/química , Oligonucleotídeos Antissenso/administração & dosagem , Especificidade de Órgãos , RNA Longo não Codificante/análise , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , Ratos Sprague-Dawley , Ribonuclease H , Distribuição Tecidual
17.
Methods Mol Biol ; 2230: 115-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197012

RESUMO

The biological signals that coordinate the three-dimensional outgrowth and patterning of the vertebrate limb bud have been well delineated. These include a number of vital embryonic signaling pathways, including the fibroblast growth factor, WNT, transforming growth factor, and hedgehog. Collectively these signals converge on multiple progenitor populations to drive the formation of a variety of tissues that make up the limb musculoskeletal system, such as muscle, tendon, cartilage, stroma, and bone. The basic mechanisms regulating the commitment and differentiation of diverse limb progenitor populations has been successfully modeled in vitro using high density primary limb mesenchymal or micromass cultures. However, this approach is limited in its ability to more faithfully recapitulate the assembly of progenitors into organized tissues that span the entire musculoskeletal system. Other biological systems have benefitted from the development and availability of three-dimensional organoid cultures which have transformed our understanding of tissue development, homeostasis and regeneration. Such a system does not exist that effectively models the complexity of limb development. However, limb bud organ cultures while still necessitating the use of collected embryonic tissue have proved to be a powerful model system to elucidate the molecular underpinning of musculoskeletal development. In this methods article, the derivation and use of limb bud organ cultures from murine limb buds will be described, along with strategies to manipulate signaling pathways, examine gene expression and for longitudinal lineage tracking.


Assuntos
Hibridização In Situ/métodos , Mesoderma/crescimento & desenvolvimento , Desenvolvimento Musculoesquelético/genética , Técnicas de Cultura de Órgãos/métodos , Animais , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/genética , Humanos , Botões de Extremidades/crescimento & desenvolvimento , Botões de Extremidades/metabolismo , Mesoderma/metabolismo , Camundongos , Transdução de Sinais/genética
18.
Methods Mol Biol ; 2230: 259-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197019

RESUMO

A method for preparing frozen sections with an adhesive film is described. In order to observe fine structures and weak fluorescence of samples, new types of adhesive films [Cryofilm type 3C(16UF) and 4D(16UF)] are used. The adhesive film is made with very clear and very low autofluorescence. For gene analysis, a very thin adhesive film (LMD film) is used to cut by means of the laser microdissection (LMD). For MALDI mass spectrometry imaging (MALDI-MSI), a conductive adhesive film (Cryofilm type MS) is used to avoid electric charge of the sample. A biological sample is frozen quickly and freeze-embedded. The frozen sample is cut with a very sharp disposable blade made from fine tungsten carbide. The combination of the adhesive films and the blade can generate 3 micrometer thick sections from samples including bone, while it is also possible to generate 1 µm thick sections. The morphology of bone and soft tissues are preserved using this method. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly observed with an oil immersion lens at high magnification. Sections generated using the Cryofilm type 3C(16UF) shows weak fluorescent signals more clearly than sections generated with the previously reported adhesive films [Cryofilm type 2C(9) and 2C(10)]. Furthermore fluorescence of the fine structures in cells is clearly shown using a super-high-resolution microscope. Several staining and experimental methods such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization can be performed on these sections. This method is also useful for preparing frozen sections of large sample such as a whole-body mouse and rat. In gene analysis, gene quality of sample collected from the section made with the LMD film is superior to that of sample made by a conventional method. The Cryofilm type MS makes almost complete section from tissues including hard tissues and large samples. The satisfactory signals are detected from the section with MALDI-MSI.


Assuntos
Osso e Ossos/ultraestrutura , Secções Congeladas/métodos , Histocitoquímica/métodos , Microtomia/métodos , Animais , Criopreservação/métodos , Fibroblastos/ultraestrutura , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Camundongos , Microscopia/métodos , Osteoblastos/ultraestrutura , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
19.
Methods Mol Biol ; 2230: 367-376, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197026

RESUMO

Whole mount in situ hybridization is a sensitive method used to characterize the spatial and temporal expression of RNA transcripts throughout an entire tissue. This method is an excellent tool for studying gene expression during embryonic development. Here, we describe a procedure for digoxigenin labeled in situ hybridization on whole embryos.


Assuntos
Embrião de Mamíferos/ultraestrutura , Desenvolvimento Embrionário/efeitos dos fármacos , Hibridização In Situ/métodos , Sondas RNA/farmacologia , Animais , Digoxigenina/farmacologia , Embrião de Mamíferos/diagnóstico por imagem , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Gravidez , Sondas RNA/isolamento & purificação
20.
Sci Rep ; 10(1): 21894, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318594

RESUMO

The rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.


Assuntos
Antígenos Virais/imunologia , Imuno-Histoquímica , /isolamento & purificação , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , /virologia , Chlorocebus aethiops , Furões , Hibridização In Situ , Sondas RNA/imunologia , Vírus da SARS/isolamento & purificação , Células Vero
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