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Fluorescence in situ hybridization (FISH) has revolutionized molecular cytogenetic analysis since the 1980s, enabling precise localization of DNA sequences in cells and tissues. Despite its relevance, applying FISH to formalin-fixed paraffin-embedded (FFPE) tissue samples encounters significant technical challenges. This review addresses the main issues encountered in this context, such as inadequate fixation, contamination, block and slide age, inadequate pretreatment, and FISH technique. Proposed solutions include optimized pretreatment protocols, monitoring of blockage, careful selection of probes, and thorough analysis of results. Implementing good laboratory practices and quality control strategies are essential to ensure reliable results. Additionally, the use of emerging technologies such as artificial intelligence and digital pathology offers new perspectives for improving the efficiency and accuracy of FISH in FFPE samples. This review highlights the importance of a careful and personalized approach to overcome the technical challenges associated with FISH in FFPE samples, strengthening its role in research and clinical diagnosis. RESEARCH HIGHLIGHTS: Few FISH studies on FFPE: The scarcity of studies specifically addressing FISH applications in FFPE tissues highlights a critical gap in the literature. Troubleshooting FISH in FFPE tissues: Identifying and addressing common challenges in FISH techniques when applied to FFPE samples, such as signal quality and hybridization efficiency. Critical aspects of FISH technique: Discuss the main technical considerations crucial for successful FISH in FFPE tissues, including sample preparation, probe selection, and protocol optimization.
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Objective: The molecular pathways underlying hypoxemia-induced alterations in neurodevelopment of infants with congenital heart disease have not been delineated. We used transcriptome analysis to investigate differential gene expression induced by hypoxemia in an ovine artificial-womb model. Methods: Mid-gestation fetal sheep (median [interquartile range] 109 [107-112] days' gestation) were cannulated via the umbilical vessels, attached to a pumpless, low-resistance oxygenator circuit, and incubated in a sterile, fluid environment for 22 [21-23] days. Fetuses were maintained with an oxygen delivery of 20-25 mL/kg/min (normoxemia, n = 3) or 14-16 mL/kg/min (hypoxemia, n = 4). Transcriptional profiling by RNA sequencing was carried out on left frontal brains and hypoxemia-regulated genes were identified by differential gene expression analysis. Results: A total of 228 genes whose expression was up or down regulated by ≥1.5-fold (false discovery rate ≤0.05) were identified. The majority of these genes were induced in hypoxemic animals compared to normoxemic controls, and functional enrichment analysis identified respiratory electron transport as a pathway strongly upregulated in the brain during chronic hypoxemia. Further examination of hypoxemia-induced genes showed robust induction of all 7 subunits of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). Other hypoxemia-induced genes included cytochrome B, a component of complex III, and ATP6, ATP8, both of which are components of complex V. Conclusions: Chronic fetal hypoxemia leads to upregulation of multiple mitochondrial respiratory complex genes critical for energy production and reactive oxygen species generation, including complex I. These data provide valuable insight into potential pathways involved in chronic hypoxemia-induced neuropathology and offers potential therapeutic targets for fetal neuroprotection in fetuses with congenital heart defects.
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Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii. Four species were recently inferred to belong to the P. brasiliensis complex, but the high genetic diversity found in both human and environmental samples have suggested that the number of lineages may be higher. This study aimed to assess the 43-kilodalton glycoprotein genotypes (PbGP43) in paraffin-embedded samples from PCM patients to infer the phylogenetic lineages of the P. brasiliensis complex responsible for causing the infection. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with histopathological diagnosis of PCM were analyzed. DNAs were extracted and amplified for a region of the second exon of the PbGP43 gene. Products were sequenced and aligned with other PbGP43 sequences available. A haplotype network and the phylogenetic relationships among sequences were inferred. Amino acid substitutions were investigated regarding the potential to modify physicochemical properties in the proteins. Six phylogenetic lineages were identified as belonging to the P. brasiliensis complex. Two lineages did not group with any of the four recognized species of the complex, and, interestingly, one of them comprised only FFPE samples. A coinfection involving two lineages was found. Five parsimony-informative sites were identified and three of them showed radical non-synonymous substitutions with the potential to promote changes in the protein. This study expands the knowledge regarding the genetic diversity existing in the P. brasiliensis complex and shows the potential of FFPE samples in species identification and in detecting coinfections.
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Paracoccidioides , Paracoccidioidomicosis , Antígenos Fúngicos/genética , Biopsia , Proteínas Fúngicas/genética , Genotipo , Humanos , Paracoccidioides/genética , Paracoccidioidomicosis/diagnóstico , Adhesión en Parafina , FilogeniaRESUMEN
In an era of precision medicine important treatment decisions are dictated by expression of clinically informative tumor protein biomarkers. These biomarkers can be detected by immunohistochemistry (IHC) performed in tumor tissue sections obtained from biopsies or resections. Like all experimental procedures, IHC needs optimization for several of its steps. However, the investigator must avoid optimizing the IHC procedure using valuable human biopsy samples which may be difficult to obtain. Ideally, valuable biopsy samples should only be subjected to IHC once the IHC protocol has been optimized. In this chapter, we describe a procedure for IHC optimization using tri-dimensional (3D) cellular spheroids created from cultured cells. In this approach, cultured cells are pelleted into 3D spheroids, which are then processed just like a tissue sample, namely, fixed, embedded, sectioned, mounted on slides, and stained with IHC just like a human tissue sample. These 3D cellular spheroids have a tissue-like architecture and cellularity resembling a tumor section, and both cellular and antigen structure are preserved. This method is therefore acceptable for IHC optimization before proceeding to the IHC staining of human tumor samples.
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Carcinoma de Pulmón de Células no Pequeñas , Inmunohistoquímica , Neoplasias Pulmonares , Adhesión en Parafina , Esferoides Celulares , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Formaldehído/química , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologíaRESUMEN
In people living with HIV (PLWH), chronic inflammation can lead to cancer initiation and progression, besides driving a dysregulated and diminished immune responsiveness. HIV infection also leads to increased transcription of Human Endogenous Retroviruses (HERVs), which could increase an inflammatory environment and create a tumor growth suppressive environment with high expression of pro-inflammatory cytokines. In order to determine the impact of HIV infection to HERV expression on the breast cancer microenvironment, we sequenced total RNA from formalin-fixed paraffin-embedded (FFPE) breast cancer samples of women HIV-negative and HIV-positive for transcriptome and retrotranscriptome analyses. We performed RNA extraction from FFPE samples, library preparation and total RNA sequencing (RNA-seq). The RNA-seq analysis shows 185 differentially expressed genes: 181 host genes (178 upregulated and three downregulated) and four upregulated HERV transcripts in HIV-positive samples. We also explored the impact of HERV expression in its neighboring breast cancer development genes (BRCA1, CCND1, NBS1/NBN, RAD50, KRAS, PI3K/PIK3CA) and in long non-coding RNA expression (AC060780.1, also known as RP11-242D8.1). We found a significant positive association of HERV expression with RAD50 and with AC060780.1, which suggest a possible role of HERV in regulating breast cancer genes from PLWH with breast cancer. In addition, we found immune system, extracellular matrix organization and metabolic signaling genes upregulated in HIV-positive breast cancer. In conclusion, our findings provide evidence of transcriptional and retrotranscriptional changes in breast cancer from PLWH compared to non-HIV breast cancer, including dysregulation of HERVs, suggesting an indirect effect of the virus on the breast cancer microenvironment.
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In this study, we report the molecular diagnosis and retrospective study of porcine circovirus 3 (PCV3) in frozen and formalin-fixed paraffin-embedded swine tissues (FFPE) collected from 1967 to 2018 in southeastern Brazil (Espírito Santo and Rio de Janeiro states). Frozen tissues from 35 pigs and FFPE tissues from 143 pigs were tested by nested PCR, targeting the PCV3 partial capsid gene. Bidirectional sequencing of 16 positive samples was performed, followed by sequence analysis and haplotype networks. A total of 26/178 samples (14.6%) tested positive for PCV3: 14/35 (40%) frozen tissue and 12/143 (8.4%) FFPE tissue. PCV3 was detected in the 1960s, 1970s, 2000s, and 2010s with the characterization of types PCV3a and PCV3b. A star-like distribution was observed in the grid of haplotypes, with a low haplotype diversity and more recent dispersal of the virus. A total of 40% of asymptomatic animals considered fit for slaughter tested positive for PCV3. In conclusion, PCV3 DNA was detected over 51 years of study, prior to initial reports and, so far, the sample detected in 1967 is the oldest partial capsid sequence described. The circulation of two different genotypes was reported, suggesting more than one introduction event of this virus into Brazil. Moreover, taken together, our studies indicated an ancient origin of PCV3 and its circulation in asymptomatic animals in Brazilian herds.
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Infecciones por Circoviridae/veterinaria , Circovirus/aislamiento & purificación , Enfermedades de los Porcinos/virología , Animales , Brasil , Proteínas de la Cápside/genética , Infecciones por Circoviridae/virología , Circovirus/clasificación , Circovirus/genética , Genotipo , Filogenia , Estudios Retrospectivos , PorcinosRESUMEN
As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.