RESUMEN

TP53 mutations are associated with short survival and poor treatment response in canine diffuse large B-cell lymphoma (cDLBCL). The expression of TP53 by RNAscope® in situ hybridization and p53 by immunohistochemistry (IHC) was investigated in 37 formalin-fixed paraffin-embedded cDLBCL, to assess their correlation with TP53 mutational status and to evaluate their prognostic value. TP53 was detected in all samples by RNAscope®. Ten of 37 (27%) cases expressed p53 by IHC, with highly variable percentage of positive cells. TP53 RNAscope® scores and p53 IHC results were not correlated. The expression of TP53 by RNAscope® was not influenced by its mutational status. Conversely, p53 IHC and TP53 mutations were significantly associated. p53 IHC predicted TP53 genetic mutations with high accuracy (97.3%). All TP53-mutated samples carrying missense mutations exhibited p53 expression by IHC, while all wild-type cases and a single case with frameshift insertion were negative. In univariable analysis, p53 IHC was associated with shorter time to progression (TTP) and lymphoma-specific survival (LSS). Nevertheless, in multivariable analysis, only treatment significantly affected TTP and LSS. These findings suggest p53 IHC is an accurate, cost-effective tool for predicting TP53 mutations in cDLBCL, unlike TP53 RNAscope®, though its prognostic value requires further validation.

Asunto(s)

Enfermedades de los Perros , Inmunohistoquímica , Hibridación in Situ , Linfoma de Células B Grandes Difuso , Valor Predictivo de las Pruebas , Proteína p53 Supresora de Tumor , Perros , Animales , Enfermedades de los Perros/genética , Inmunohistoquímica/veterinaria , Linfoma de Células B Grandes Difuso/veterinaria , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/mortalidad , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Hibridación in Situ/veterinaria , Masculino , Femenino , Mutación , Pronóstico

RESUMEN

Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in situ expression maps of hundreds of genes. We use direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high-throughput and large multiplexing. We validate the method across a number of species and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.

Asunto(s)

Perfilación de la Expresión Génica , Animales , Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma/genética , Humanos , Hibridación in Situ/métodos , Ratones , Biología Evolutiva/métodos

RESUMEN

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

Asunto(s)

Hibridación in Situ , Ácidos Indolacéticos , Hojas de la Planta , Zea mays , Zea mays/genética , Zea mays/crecimiento & desarrollo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Ácidos Indolacéticos/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Xilema/metabolismo , Xilema/crecimiento & desarrollo , Xilema/citología , Xilema/genética

RESUMEN

Hepatitis B virus (HBV) developed highly intricates mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. With the aid of a series of classical analytical methods such as ultrafiltration, and Southern and Northern blots, a general framework of HBV life cycle has been established. However, this picture still lacks many key histological contexts which involves pathophysiological changes of hepatocytes, non-parenchymal cells, infiltrated leukocytes, and associated extracellular matrix. Here, we describe a CISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in liver tissue of chronic hepatitis B patients. By coupling it with immunohistochemistry and other histological stains, much richer information regarding the HBV-induced pathological changes can be harvested.

Asunto(s)

ADN Viral , Virus de la Hepatitis B , Hibridación in Situ , Hígado , ARN Viral , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Hibridación in Situ/métodos , Hígado/virología , Hígado/metabolismo , ADN Viral/genética , ARN Viral/genética , Hepatitis B Crónica/virología , Compuestos Cromogénicos , Inmunohistoquímica/métodos , ADN Circular/genética , ADN Circular/análisis

RESUMEN

Profiling gene expression while preserving cell locations aids in the comprehensive understanding of cell fates in multicellular organisms. However, simple and flexible isolation of microregions of interest (mROIs) for spatial transcriptomics is still challenging. We present a laser-induced forward transfer (LIFT)-based method combined with a full-length mRNA-sequencing protocol (LIFT-seq) for profiling region-specific tissues. LIFT-seq demonstrated that mROIs from two adjacent sections could reliably and sensitively detect and display gene expression. In addition, LIFT-seq can identify region-specific mROIs in the mouse cortex and hippocampus. Finally, LIFT-seq identified marker genes in different layers of the cortex with very similar expression patterns. These genes were then validated using in situ hybridization (ISH) results. Therefore, LIFT-seq will be a valuable and efficient technique for profiling the spatial transcriptome in various tissues.

Asunto(s)

Perfilación de la Expresión Génica , Transcriptoma , Animales , Perfilación de la Expresión Génica/métodos , Ratones , Rayos Láser , Hipocampo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Hibridación in Situ/métodos , Corteza Cerebral/metabolismo , Análisis de Secuencia de ARN/métodos

RESUMEN

BACKGROUND: MYB RNA in situ hybridization (ISH) has emerged as a reliable and accessible marker to support adenoid cystic carcinoma (ACC) diagnosis, though still not well studied. Here, we report our results in a validation and prospective cohort to improve MYB RNA ISH diagnostic accuracy. METHODS: 79 cases (23 retrospective and 56 prospective) underwent MYB RNA ISH testing (44 ACC and 35 non-ACC). MYB RNA ISH results were initially interpreted based on previously established (original) scoring criteria. Weighted "i-scores", percent positive tumor cells, percent tumor cells with large signals (% LS), and staining pattern (abluminal, diffuse, focal non-patterned, or negative) were inputs for logistic regression models. Final model performance characteristics were compared with original scoring criteria and MYB::NFIB FISH results. RESULTS: An abluminal pattern was characteristic and exclusive to ACC. All i-scores, % LS, and percent positive were significantly higher in ACC. Original scoring criteria yielded a 95.5% sensitivity (Sn), 68.6% specificity (Sp), and 83.5% accuracy. MYB::NFIB FISH yielded a 42.9% sensitivity, 100% specificity, and 60% accuracy. Optimizing for performance, simplicity, and minimal collinearity, our final model was defined as: abluminal pattern and/or % LS > 16.5%, which resulted in a 93.2% Sn, 97.1% Sp, and 94.9% accuracy for ACC diagnosis. False negatives included an ACC with striking tubular eosinophilia and a MYBL1::NFIB translocated ACC. One false positive exclusive to the final model was a nasopharyngeal carcinoma with MYB amplification. CONCLUSIONS: MYB RNA ISH has a higher Sn than MYB::NFIB FISH while retaining high Sp. Our model provides improvements to specificity compared to original scoring criteria and highlight the importance of abluminal staining pattern and % LS. Nonetheless, alternate fusions remain key false negatives while rare non-ACC with other mechanisms of MYB activation may present as false positives.

Asunto(s)

Biomarcadores de Tumor , Carcinoma Adenoide Quístico , Proteínas Proto-Oncogénicas c-myb , Sensibilidad y Especificidad , Humanos , Carcinoma Adenoide Quístico/diagnóstico , Carcinoma Adenoide Quístico/genética , Carcinoma Adenoide Quístico/patología , Proteínas Proto-Oncogénicas c-myb/genética , Femenino , Persona de Mediana Edad , Masculino , Anciano , Adulto , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Estudios Retrospectivos , Hibridación in Situ/métodos , Estudios Prospectivos , Anciano de 80 o más Años , Hibridación Fluorescente in Situ/métodos , Adulto Joven

RESUMEN

BACKGROUND: HER2-targeted therapies have recently emerged as an option in the management of metastatic colorectal cancer (mCRC) overexpressing HER2. However, data regarding HER2 status in primary CRC and its corresponding liver metastases are limited, potentially influencing clinical decisions. Therefore, the aim of this study was to compare the HER2 status in primary CRC and paired liver metastases. METHODS: Patients with mCRC who were operated from their primary colorectal cancer and their corresponding synchronous or metachronous liver metastases, in the digestive surgery department of Besançon University Hospital, between April 1999 and October 2021, were included. Tissue microarrays were constructed from matched primary CRC and liver metastastic tissue samples. HER2 status was assessed by immunohistochemistry and in situ hybridization according to Valtorta's criteria. RESULTS: A series of 108 paired primary CRC and liver metastases, including a series of multiple liver metastases originating from the same patients (n = 24), were assessed. Among the primary CRC, 89 (82.4%), 17 (15.8%) and 2 (1.8%) cases were scored 0, 1 + and 2 + respectively. In liver metastases, 99 (91.7%), 7 (6.5%) and 2 (1.8%) were scored 0, 1 + and 2, respectively. Overall, there was a 19% discrepancy rate in HER2 status between primary CRC and metastases, which increased to 21% in cases with multiple synchronous or metachronous liver metastases in a given patient. No significant difference was found between metachronous and synchronous metastases regarding the HER2 status (p = 0.237). CONCLUSIONS: Our study highlights the temporal and spatial heterogeneity of HER2 status between primary CRC and corresponding liver metastases. These findings raise the question of a sequential evaluation of the HER2 status during disease progression, to provide the most suitable treatment strategy.

Asunto(s)

Biomarcadores de Tumor , Neoplasias Colorrectales , Neoplasias Hepáticas , Receptor ErbB-2 , Humanos , Neoplasias Colorrectales/patología , Receptor ErbB-2/análisis , Receptor ErbB-2/metabolismo , Femenino , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Anciano , Biomarcadores de Tumor/análisis , Inmunohistoquímica , Adulto , Anciano de 80 o más Años , Hibridación in Situ , Análisis de Matrices Tisulares

RESUMEN

BACKGROUND: The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. METHODS: We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). FINDINGS: We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. INTERPRETATION: RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. FUNDING: Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

Asunto(s)

Antígenos Bacterianos , Mycobacterium tuberculosis , ARN Mensajero , Humanos , Mycobacterium tuberculosis/genética , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Hibridación in Situ , Tuberculosis/microbiología , ARN Bacteriano/genética , Inmunohistoquímica , Granuloma/microbiología , Granuloma/metabolismo , Pulmón/microbiología , Pulmón/patología , Pulmón/metabolismo

RESUMEN

Dietary administration of a copper chelator, cuprizone (CPZ), has long been reported to induce intense and reproducible demyelination of several brain structures such as the corpus callosum. Despite the widespread use of CPZ as an animal model for demyelinating diseases such as multiple sclerosis (MS), the mechanism by which it induces demyelination and then allows robust remyelination is still unclear. An intensive mapping of the cell dynamics of oligodendrocyte (OL) lineage during the de- and remyelination course would be particularly important for a deeper understanding of this model. Here, using a panel of OL lineage cell markers as in situ hybridization (ISH) probes, including Pdgfra, Plp, Mbp, Mog, Enpp6, combined with immunofluorescence staining of CC1, SOX10, we provide a detailed dynamic profile of OL lineage cells during the entire course of the model from 1, 2, 3.5 days, 1, 2, 3, 4,5 weeks of CPZ treatment, as well as after 1, 2, 3, 4 weeks of recovery from CPZ treatment. The result showed an unexpected early death of mature OLs and response of OL progenitor cells (OPCs) in vivo upon CPZ challenge, and a prolonged upregulation of myelin-forming OLs compared to the intact control even 4 weeks after CPZ withdrawal. These data may serve as a basic reference system for future studies of the effects of any intervention on de- and remyelination using the CPZ model, and imply the need to optimize the timing windows for the introduction of pro-remyelination therapies in demyelinating diseases such as MS.

Asunto(s)

Linaje de la Célula , Cuprizona , Enfermedades Desmielinizantes , Oligodendroglía , Cuprizona/toxicidad , Animales , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Oligodendroglía/metabolismo , Modelos Animales de Enfermedad , Hibridación in Situ/métodos , Ratones Endogámicos C57BL , Ratones , Remielinización/efectos de los fármacos , Remielinización/fisiología , Masculino , Quelantes/toxicidad , Quelantes/farmacología , Vaina de Mielina/patología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo

RESUMEN

Aging is a major risk factor for the development or the worsening of retinal degenerative conditions. The intricate network of the neural retina determined that the retinal aging is a complicated process. The aim of this study is to delineate the transcriptomic changes of major retinal neurons during aging in C57BL/6 mice at single-cell level. We analyzed the transcriptional profiles of the photoreceptor, bipolar, amacrine, and Müller glial cells of 1.5-2 and 24-30 months old mice using single-cell RNA sequencing technique. We selectively confirmed the differences in gene expression using immunofluorescence staining and RNA in situ hybridization analysis. We found that each retinal cell type had unique changes upon aging. However, they all showed signs of dysregulated glucose and energy metabolism, and perturbed proteostasis. In particular, old Müller glia exhibited the most profound changes, including the upregulation of cell metabolism, stress-responses, antigen-presentation and immune responses and metal ion homeostasis. The dysregulated gliogenesis and differentiation was confirmed by the presence of Müller glia expressing rod-specific genes in the inner nuclear layer and the outer plexiform layer of the old retina. We further pinpointed the specific loss of GABAergic amacrine cells in old retina. Our study emphasized changes of amacrine and Müller glia during retinal aging, provided resources for further research on the molecular and cellular regulatory mechanisms underlying aging-associated retinal deterioration.

Asunto(s)

Envejecimiento , Células Amacrinas , Metabolismo Energético , Ratones Endogámicos C57BL , Proteostasis , Animales , Células Amacrinas/metabolismo , Ratones , Envejecimiento/fisiología , Metabolismo Energético/fisiología , Células Ependimogliales/metabolismo , Retina/metabolismo , Neuronas GABAérgicas/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/genética , Hibridación in Situ , Homeostasis/fisiología

RESUMEN

OBJECTIVES: There is a paucity of data on North American cohorts of patients with penile squamous cell carcinoma (pSCC). Herein, we aimed to assess the sensitivity of various modalities to identify human papillomavirus (HPV) status, determine the prevalence of high-risk HPV-positivity, and evaluate the prognostic impact of relevant clinicopathologic variables. METHODS: Patients with pSCC (n = 121) consecutively treated with partial/total penectomy (2000-2022) at a single institution were included. HPV status (based on immunohistochemistry [IHC], in situ hybridization [ISH], and panviral metagenomic sequencing [PMS]), histologic features, and outcomes were reviewed. Outcome events included death due to disease and progression. RESULTS: The majority of patients were white (105/121, 86.8%). Thirty-seven (30.6%) were high-risk HPV-positive, and morphologic evaluation had a sensitivity of 97.3% (95% confidence interval [CI], 86.2-99.5) for predicting high-risk HPV status compared to IHC/ISH/PMS. Disease progression was more common among high-risk HPV-negative compared to high-risk HPV-positive patients (HR 2.74, CI 1.12-8.23, P = 0.03). Moreover, among high-risk HPV-negative patients, those with moderate-poorly differentiated tumors had increased disease-specific mortality (32.6%, CI 17.1-48.1) compared to those with well-differentiated tumors (0%). Among high-risk HPV-positive patients, those with basaloid morphology had lower disease-specific mortality (0% vs 14.4%, CI 0.0-33.1). CONCLUSIONS: We demonstrate high-risk HPV-positivity in approximately one-third of patients with pSCC. Morphologic evaluation alone had a high sensitivity in correctly determining HPV status. Our results suggest that high-risk HPV status and morphologic features (differentiation in high-risk HPV-negative, and basaloid subtype in high-risk HPV-positive pSCC) may have prognostic value.

Asunto(s)

Carcinoma de Células Escamosas , Infecciones por Papillomavirus , Neoplasias del Pene , Humanos , Masculino , Neoplasias del Pene/virología , Neoplasias del Pene/patología , Neoplasias del Pene/mortalidad , Persona de Mediana Edad , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/patología , Carcinoma de Células Escamosas/virología , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/mortalidad , Anciano , Inmunohistoquímica , Adulto , Hibridación in Situ , Papillomaviridae/aislamiento & purificación , Papillomaviridae/genética , Estudios Retrospectivos , Anciano de 80 o más Años , Factores de Riesgo , Pronóstico , Progresión de la Enfermedad , Valor Predictivo de las Pruebas , Virus del Papiloma Humano

RESUMEN

The pecten is a fold-structured projection at the ocular fundus in bird eyes, showing morphological diversity between the diurnal and nocturnal species. However, its biological functions remain unclear. This study investigated the morphological and histological characteristics of pectens in wild birds. Additionally, the expression of non-visual opsin genes was studied in chicken pectens. These genes, identified in the chicken retina and brain, perceive light periodicity regardless of visual communication. Similar pleat numbers have been detected among bird taxa; however, pecten size ratios in the ocular fundus showed noticeable differences between diurnal and nocturnal birds. The pectens in nocturnal brown hawk owl show extremely poor vessel distribution and diameters compared with that of diurnal species. RT-PCR analysis confirmed the expression of Opn5L3, Opn4x, Rrh and Rgr genes. In situ hybridization analysis revealed the distribution of Rgr-positive reactions in non-melanotic cells around the pecten vessels. This study suggests a novel hypothesis that pectens develop dominantly in diurnal birds as light acceptors and contribute to continuous visual function or the onset of periodic behaviour.

Asunto(s)

Hibridación in Situ , Opsinas , Retina , Animales , Opsinas/genética , Opsinas/metabolismo , Retina/fisiología , Pollos/fisiología , Pollos/genética , Aves/fisiología , Ritmo Circadiano/fisiología , Encéfalo/metabolismo

RESUMEN

RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.

Asunto(s)

Hibridación in Situ , ARN , Hibridación in Situ/métodos , ARN/genética , ARN/análisis , Humanos , Compuestos Cromogénicos/química , Colorimetría/métodos , Imagen Individual de Molécula/métodos

RESUMEN

AIM: This study aimed to demonstrate the role of Epstein-Barr Virus (EBV) in papillary thyroid carcinomas (PTC) developing on the background of Hashimoto's thyroiditis (HT). METHODS: The presence of EBV in tumoral tissue, lymphocytes, and peritumoral normal thyroid tissue was investigated using the in situ hybridization method in paraffin blocks. The subtypes of PTC, tumor diameter, TNM stage, multifocality, invasion of thyroid capsule, perineural invasion, and muscular tissue invasion were identified and compared according to EBV involvement. RESULTS: Eighty-one patients with HT diagnosis, with 93.8% (n=76) female and 6.2% (n=5) male, were included in the study. Papillary microcarcinoma was the pathological diagnosis in 24.2% (n=15) of the cases. EBV was identified in 58.06% (n=36) of the tumor cells nuclei, 58.06% (n=36) in the tumor cell cytoplasm, 16.12% (n=10) in tumor infiltrative lymphocytes, and 53.2% (n=33) in normal parenchymal follicle epithelial cells (NPFEC). In the T2 stage, the rate of EBV nuclear positivity in patients was significantly higher (p=0.034). The classic variant of papillary carcinoma was accompanied by a significantly higher rate of EBV-negative NPFEC (67.6%, p=0.049). In multifocal tumors, EBV positivity was found to be significantly higher in lymphocytes in the surrounding tissues (58.3%, p=0.034). CONCLUSION: A significant increase in EBV positivity in the surrounding tissue lymphocytes was observed in multifocal PTC developing on a background of HT. This suggests a possible association between HT and EBV.

Asunto(s)

Infecciones por Virus de Epstein-Barr , Enfermedad de Hashimoto , Herpesvirus Humano 4 , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , Masculino , Femenino , Enfermedad de Hashimoto/virología , Enfermedad de Hashimoto/patología , Herpesvirus Humano 4/aislamiento & purificación , Adulto , Neoplasias de la Tiroides/virología , Neoplasias de la Tiroides/patología , Cáncer Papilar Tiroideo/virología , Cáncer Papilar Tiroideo/patología , Persona de Mediana Edad , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Hibridación in Situ , Anciano , Carcinoma Papilar/virología , Carcinoma Papilar/patología

RESUMEN

Overlaying omics data onto spatial biological dimensions has been a promising technology to provide high-resolution insights into the interactome and cellular heterogeneity relative to the organization of the molecular microenvironment of tissue samples in normal and disease states. Spatial omics can be categorized into three major modalities: (a) next-generation sequencing-based assays, (b) imaging-based spatially resolved transcriptomics approaches including in situ hybridization/in situ sequencing, and (c) imaging-based spatial proteomics. These modalities allow assessment of transcripts and proteins at a cellular level, generating large and computationally challenging datasets. The lack of standardized computational pipelines to analyze and integrate these nonuniform structured data has made it necessary to apply artificial intelligence and machine learning strategies to best visualize and translate their complexity. In this review, we summarize the currently available techniques and computational strategies, highlight their advantages and limitations, and discuss their future prospects in the scientific field.

Asunto(s)

Proteómica , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , Proteómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biología Computacional/métodos , Aprendizaje Automático , Genómica/métodos , Hibridación in Situ/métodos , Transcriptoma

RESUMEN

Amelogenesis imperfecta (AI) is a diverse group of inherited diseases featured by various presentations of enamel malformations that are caused by disturbances at different stages of enamel formation. While hypoplastic AI suggests a thickness defect of enamel resulting from aberrations during the secretory stage of amelogenesis, hypomaturation AI indicates a deficiency of enamel mineralization and hardness established at the maturation stage. Mutations in ENAM, which encodes the largest enamel matrix protein, enamelin, have been demonstrated to cause generalized or local hypoplastic AI. Here, we characterized 2 AI families with disparate hypoplastic and hypomaturation enamel defects and identified 2 distinct indel mutations at the same location of ENAM, c588+1del and c.588+1dup. Minigene splicing assays demonstrated that they caused frameshifts and truncation of ENAM proteins, p.Asn197Ilefs*81 and p.Asn197Glufs*25, respectively. In situ hybridization of Enam on mouse mandibular incisors confirmed its restricted expression in secretory stage ameloblasts and suggested an indirect pathogenic mechanism underlying hypomaturation AI. In silico analyses indicated that these 2 truncated ENAMs might form amyloid structures and cause protein aggregation with themselves and with wild-type protein through the added aberrant region at their C-termini. Consistently, protein secretion assays demonstrated that the truncated proteins cannot be properly secreted and impede secretion of wild-type ENAM. Moreover, compared to the wild-type, overexpression of the mutant proteins significantly increased endoplasmic reticulum stress and upregulated the expression of unfolded protein response (UPR)-related genes and TNFRSF10B, a UPR-controlled proapoptotic gene. Caspase, terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays further revealed that both truncated proteins, especially p.Asn197Ilefs*81, induced cell apoptosis and decreased cell survival, suggesting that the 2 ENAM mutations cause AI through ameloblast cell pathology and death rather than through a simple loss of function. This study demonstrates that an ENAM mutation can lead to generalized hypomaturation enamel defects and suggests proteinopathy as a potential pathogenesis for ENAM-associated AI.

Asunto(s)

Amelogénesis Imperfecta , Animales , Femenino , Humanos , Masculino , Ratones , Ameloblastos/patología , Amelogénesis Imperfecta/genética , Apoptosis/genética , Proteínas del Esmalte Dental/genética , Proteínas de la Matriz Extracelular , Hibridación in Situ , Mutación , Linaje

RESUMEN

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. In situ hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4.

Asunto(s)

Aletas de Animales , Proteína Morfogenética Ósea 4 , Factores de Crecimiento de Fibroblastos , Receptores de Calcitriol , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/embriología , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Aletas de Animales/embriología , Aletas de Animales/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/genética , Técnicas de Silenciamiento del Gen , Transducción de Señal , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ

RESUMEN

Histamine H1 receptor (H1R) in the central nervous system plays an important role in various functions, including learning and memory, aggression, feeding behaviors, and wakefulness, as evidenced by studies utilizing H1R knockout mice and pharmacological interventions. Although previous studies have reported the widespread distribution of H1R in the brains of rats, guinea pigs, monkeys, and humans, the detailed distribution in the mouse brain remains unclear. This study provides a comprehensive description of the distribution of H1R mRNA in the mouse brain using two recently developed techniques: RNAscope and in situ hybridization chain reaction, both of which offer enhanced sensitivity and resolution compared to traditional methodologies such as radioisotope labeling, which were used in previous studies. The H1R mRNA expression was observed throughout the entire brain, including key regions implicated in sleep-wake regulatory functions, such as the pedunculopontine tegmental nucleus and dorsal raphe. Additionally, strong H1R mRNA signals were identified in the paraventricular hypothalamus and ventromedial hypothalamus, which may explain the potential mechanisms underlying histamine-mediated feeding regulation. Notably, we identified strong H1R mRNA expression in previously unreported cerebral regions, such as the dorsal endopiriform nucleus, bed nucleus of the accessory olfactory tract, and postsubiculum. These findings significantly contribute to our understanding of the multifaceted roles of H1R in diverse brain functions.

Asunto(s)

Mapeo Encefálico , Encéfalo , ARN Mensajero , Receptores Histamínicos H1 , Animales , Masculino , Ratones , Encéfalo/metabolismo , Mapeo Encefálico/métodos , Hibridación in Situ , Ratones Endogámicos C57BL , Receptores Histamínicos H1/metabolismo , ARN Mensajero/metabolismo

RESUMEN

The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of A. aegypti embryos for immunohistochemical and in situ hybridization studies. The processing of A. aegypti embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100-150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27°C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at protocols.io to clarify the protocol and provide easy access and training to anyone interested in the preparation of A. aegypti embryos for biological studies.

Asunto(s)

Aedes , Hibridación in Situ , Animales , Aedes/embriología , Hibridación in Situ/métodos , Embrión no Mamífero , Fijación del Tejido/métodos , Inmunohistoquímica , Femenino

RESUMEN

The thymus of fishes is located as a dual organ in a rostrodorsal projection within the gill chamber and is covered by the operculum. The histological organization of the teleost fish thymus displays considerable diversity, particularly in salmonids where a clear distinction between the thymus cortex and medulla is yet to be defined. Recent interest has focused on the role of B cells in thymic function, but the presence of these cells within the salmon thymus remains poorly understood. In this morphological study, we applied in situ hybridization to investigate developing Atlantic salmon thymi for the expression of recombination activating (Rag) genes 1 and 2. We identified the location of the cortex, aligning with the previously described inner zone. Expression of IgM and IgD transcripts was predominantly observed in cells within the outer and subcapsular zones, with lesser expression in the cortex and inner zone. IgT expression was confined to a limited number of cells in the inner zone and capsule. The location of the thymus medulla could not be established. Our results are discussed in the context of the recently identified lymphoid organs, namely the intrabranchial lymphoid tissue (ILT) and the salmon bursa.

Asunto(s)

Salmo salar , Timo , Animales , Salmo salar/genética , Salmo salar/inmunología , Timo/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Inmunoglobulinas/genética , Hibridación in Situ/veterinaria