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1.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806574

RESUMO

It is well established that miR-9 contributes to retinal neurogenesis. However, little is known about its presence and effects in the postnatal period. To expand our knowledge, miRNA-small RNA sequencing and in situ hybridization supported by RT-qPCR measurement were carried out. Mir-9 expression showed two peaks in the first three postnatal weeks in Wistar rats. The first peak was detected at postnatal Day 3 (P3) and the second at P10, then the expression gradually decreased until P21. Furthermore, we performed in silico prediction and established that miR-9 targets OneCut2 or synaptotagmin-17. Another two microRNAs (mir-135, mir-218) were found from databases which also target these proteins. They showed a similar tendency to mir-9; their lowest expression was at P7 and afterwards, they showed increase. We revealed that miR-9 is localized mainly in the inner retina. Labeling was observed in ganglion and amacrine cells. Additionally, horizontal cells were also marked. By dual miRNA-in situ hybridization/immunocytochemistry and qPCR, we revealed alterations in their temporal and spatial expression. Our results shed light on the significance of mir-9 regulation during the first three postnatal weeks in rat retina and suggest that miRNA could act on their targets in a stage-specific manner.


Assuntos
MicroRNAs/metabolismo , Retina/metabolismo , Animais , Hibridização In Situ/métodos , Cuidado Pós-Natal , Ratos , Ratos Wistar , Células Ganglionares da Retina/metabolismo , Fatores de Transcrição/metabolismo
2.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799739

RESUMO

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição/genética , Transcrição Genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Hibridização In Situ/métodos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
3.
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
4.
Cancer Sci ; 112(5): 2020-2032, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33675098

RESUMO

KRAS is the most frequently mutated in ovarian endometriosis. However, it is unclear whether the KRAS mutant allele's mRNA is expressed and plays a biological role in ovarian endometriosis. Here, we performed mutation-specific RNA in situ hybridization to evaluate mutant allele expression of KRAS p.G12V, the most frequently detected mutation in ovarian endometriosis in our previous study, in formalin-fixed paraffin-embedded tissue (FFPE) samples of ovarian endometriosis, cancer cell lines, and ovarian cancers. First, we verified that mutant or wild-type allele of KRAS were expressed in all 5 cancer cell lines and 9 ovarian cancer cases corresponding to the mutation status. Next, we applied this assay to 26 ovarian endometriosis cases, and observed mutant allele expression of KRAS p.G12V in 10 cases. Mutant or wild-type allele of KRAS were expressed in line with mutation status in 12 available endometriosis cases for which KRAS gene sequence was determined. Comparison of clinical features between ovarian endometriosis with KRAS p.G12V mutant allele expression and with KRAS wild-type showed that KRAS p.G12V mutant allele expression was significantly associated with inflammation in ovarian endometriosis. Finally, we assessed the spatial distribution of KRAS mutant allele expression in 5 endometriosis cases by performing multiregional sampling. Intratumor heterogeneity of KRAS mutant allele expression was observed in two endometriosis cases, whereas the spatial distribution of KRAS p.G12V mutation signals were diffuse and homogenous in ovarian cancer. In conclusion, evaluation of oncogene mutant expression will be useful for clarifying the biological significance of oncogene mutations in benign tumors.


Assuntos
Alelos , Endometriose/genética , Expressão Gênica , Genes ras , Hibridização In Situ/métodos , Mutação , Doenças Ovarianas/genética , Adulto , Linhagem Celular , Endometriose/patologia , Feminino , Humanos , Microdissecção e Captura a Laser , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Doenças Ovarianas/patologia , Neoplasias Ovarianas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
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
14.
Methods Mol Biol ; 2174: 89-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813246

RESUMO

With the advances in sequencing technology and transcriptome analysis, it is estimated that up to 75% of the human genome is transcribed into RNAs. This finding prompted intensive investigations on the biological functions of noncoding RNAs and led to very exciting discoveries of microRNAs as important players in disease pathogenesis and therapeutic applications. Research on long noncoding RNAs (lncRNAs) is in its infancy, yet a broad spectrum of biological regulations has been attributed to lncRNAs. Here, we provide a collection of detailed experimental protocols for lncRNA studies, including lncRNA immunoprecipitation, lncRNA pull-down, lncRNA northern blot analysis, lncRNA in situ hybridization, and lncRNA knockdown. We hope that the information included in this chapter can speed up research on lncRNAs biology and eventually lead to the development of clinical applications with lncRNA as novel prognostic markers and therapeutic targets.


Assuntos
Biologia Molecular/métodos , Neoplasias/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , Biomarcadores Tumorais/genética , Northern Blotting/métodos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Genoma Humano , Humanos , Imunoprecipitação/métodos , Hibridização In Situ/métodos , Neoplasias/patologia , Oncogenes , RNA Longo não Codificante/metabolismo , Transdução de Sinais
15.
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
16.
PLoS One ; 15(9): e0239467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970731

RESUMO

Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcript molecules at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We instead multiplex within a single transcript, using probes targeted to the 5' and 3' regions of muscle dystrophin mRNA. Our approach shows this method can reveal transcriptional dynamics in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs in quantitative fashion (with the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggest this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.


Assuntos
Distrofina/genética , Expressão Gênica/genética , Hibridização In Situ/métodos , Animais , Distrofina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Multiplex/métodos , Músculos/metabolismo , RNA Mensageiro/genética , Transcrição Genética/genética
17.
Arch Virol ; 165(10): 2373-2377, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761270
18.
Lab Invest ; 100(11): 1485-1489, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32647285

RESUMO

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes.


Assuntos
Betacoronavirus/isolamento & purificação , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Feminino , Humanos , Indicadores e Reagentes , Rim/virologia , Pulmão/virologia , Inclusão em Parafina , Placenta/virologia , Gravidez
19.
Ann Diagn Pathol ; 48: 151565, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32659620

RESUMO

Infection by SARS-CoV-2 commonly begins in the nasopharynx, and the cytologic and molecular correlates are not characterized. Fifty-eight cytologic preps (20 oral and 38 from the nasopharynx) were obtained from ten patients and analyzed in a blinded fashion for SARS-CoV-2 spike and envelope protein by immunohistochemistry and viral RNA by in situ hybridization. qRTPCR identified three positive cases and seven controls; the three cases reported mild symptoms that resolved in 2-3 days. Blinded analyses confirmed the presence of the SARS-CoV-2 spike and envelope proteins and viral RNA in the three cases and viral absence in the seven controls. A signal for the positive cases was evident in each nasopharyngeal and none of the oral samples. Viral RNA/proteins localized exclusively to glandular cells and was present in high copy number. Blinded analysis of the cytology documented that the glandular cells infected by SARS-CoV-2 showed marked degeneration with ciliocytophthoria; viral inclusions were not evident. Co-expression analysis showed viral infected cells had increased apoptosis, marked by strong expression of activated caspase 3. Weekly serial testing of two of the cases showed persistence of productive viral infection for up to 2 weeks after symptom onset. It is concluded that the target cell of SARS-CoV-2 in the head and neck region is the glandular cell of the nasal passages, that viral infection is lytic and associated with high copy number that facilitates viral spread. The method outlines a simple, rapid test for productive SARS-CoV-2 based on immunohistochemistry or in situ hybridization of the glandular cells from the nasopharynx.


Assuntos
Infecções por Coronavirus/diagnóstico , Citodiagnóstico/métodos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Nasofaringe/virologia , Pneumonia Viral/diagnóstico , Betacoronavirus , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral/análise , Proteínas Virais/análise
20.
Nucleic Acids Res ; 48(15): e86, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32544240

RESUMO

Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.


Assuntos
DNA/isolamento & purificação , Citometria de Fluxo/métodos , Imagem Individual de Molécula/métodos , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Humanos , Hibridização In Situ/métodos , Linfócitos/química , Reação em Cadeia da Polimerase/métodos
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