RESUMO
Imaging-based spatial multi-omics technologies facilitate the analysis of higher-order genomic structures, gene transcription, and the localization of proteins and posttranslational modifications (PTMs) at the single-allele level, thereby enabling detailed observations of biological phenomena, including transcription machinery within cells and tissues. This chapter details the principles of such technologies, with a focus on DNA/RNA/immunofluorescence (IF) sequential fluorescence in situ hybridization (seqFISH). A comprehensive step-by-step protocol for image analysis is provided, covering image preprocessing, spot detection, and data visualization. For practical application, complete Jupyter Notebook codes are made available on GitHub ( https://github.com/Ochiai-Lab/seqFISH_analysis ).
Assuntos
DNA , Imunofluorescência , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , RNA , Software , Hibridização in Situ Fluorescente/métodos , RNA/genética , RNA/análise , RNA/metabolismo , Processamento de Imagem Assistida por Computador/métodos , DNA/genética , Imunofluorescência/métodos , Humanos , AnimaisRESUMO
Telomeres in most somatic cells shorten with each cell division, and critically short telomeres lead to cellular dysfunction, cell cycle arrest, and senescence. Thus, telomere shortening is an important hallmark of human cellular senescence. Quantitative fluorescence in situ hybridization (Q-FISH) using formalin-fixed paraffin-embedded (FFPE) tissue sections allows the estimation of telomere lengths in individual cells in histological sections. In our Q-FISH method, fluorescently labelled peptide nucleic acid (PNA) probes are hybridized to telomeric and centromeric sequences in FFPE human tissue sections, and relative telomere lengths (telomere signal intensities relative to centromere signal intensities) are measured. This chapter describes our Q-FISH protocols for assessing relative telomere lengths in FFPE human tissue sections.
Assuntos
Hibridização in Situ Fluorescente , Inclusão em Parafina , Ácidos Nucleicos Peptídicos , Telômero , Humanos , Hibridização in Situ Fluorescente/métodos , Telômero/genética , Telômero/metabolismo , Ácidos Nucleicos Peptídicos/metabolismo , Ácidos Nucleicos Peptídicos/genética , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Homeostase do Telômero , Centrômero/metabolismo , Centrômero/genéticaRESUMO
In this paper, we have performed an in-depth study of the complete set of the satellite DNA (satDNA) families (i.e. the satellitomes) in the genome of two barley species of agronomic value in a breeding framework, H. chilense (H1 and H7 accessions) and H. vulgare (H106 accession), which can be useful tools for studying chromosome associations during meiosis. The study has led to the analysis of a total of 18 satDNA families in H. vulgare, 25 satDNA families in H. chilense (accession H1) and 27 satDNA families in H. chilense (accession H7) that constitute 46 different satDNA families forming 36 homology groups. Our study highlights different important contributions of evolutionary and applied interests. Thus, both barley species show very divergent satDNA profiles, which could be partly explained by the differential effects of domestication versus wildlife. Divergence derives from the differential amplification of different common ancestral satellites and the emergence of new satellites in H. chilense, usually from pre-existing ones but also random sequences. There are also differences between the two H. chilense accessions, which support genetically distinct groups. The fluorescence in situ hybridization (FISH) patterns of some satDNAs yield distinctive genetic markers for the identification of specific H. chilense or H. vulgare chromosomes. Some of the satellites have peculiar structures or are related to transposable elements which provide information about their origin and expansion. Among these, we discuss the existence of different (peri)centromeric satellites that supply this region with some plasticity important for centromere evolution. These peri(centromeric) satDNAs and the set of subtelomeric satDNAs (a total of 38 different families) are analyzed in the framework of breeding as the high diversity found in the subtelomeric regions might support their putative implication in chromosome recognition and pairing during meiosis, a key point in the production of addition/substitution lines and hybrids.
Assuntos
Cromossomos de Plantas , DNA Satélite , Hordeum , Hibridização in Situ Fluorescente , Hordeum/genética , DNA Satélite/genética , Cromossomos de Plantas/genética , DNA de Plantas/genética , Genoma de Planta/genética , Filogenia , Variação Genética , Meiose/genética , Evolução Molecular , Especificidade da EspécieRESUMO
Supernumerary B chromosomes contribute to intraspecific karyotypic variation. B chromosomes have been detected in more than 2000 organisms; they possess unique and diverse features, including non-Mendelian inheritance. Here, we report one or more B chromosomes in the gynodioecious plant Atractylodes lancea. Among 54 A. lancea lines, 0-2 B chromosomes were detected in both hermaphroditic and female plants, with the B chromosomes appearing as DAPI-bright regions within the nuclei. Genomic in situ hybridization revealed that the B chromosomes had no conserved A chromosome DNA sequences, confirmed by fluorescence in situ hybridization probed with independently dissected B chromosomes. In male meiosis, the B chromosome did not pair with an A chromosome and was therefore eliminated; accordingly, only 20.1% and 18.6% of these univalent B chromosomes remained at the end of meiosis for the 1B lines of KY17-148 and KY17-118, respectively. However, we also found that B chromosomes were transmitted from male parents in 40.8%-44.2% and 47.2% of the next generation; although these transmission rates from male parents were not essentially different from Mendelian inheritance (0.5), the transmission of gametes carrying B chromosomes increased through fertilization or seed development. B chromosomes were transmitted from three of four 1B female parents to 64.3%-92.6% of the next generation, suggesting B chromosome accumulation. We propose that the B chromosome of A. lancea has a specific sequence and persists via non-Mendelian inheritance from female parents. Overall, A. lancea, with its unique characteristics, is a promising model for understanding the structure, evolution, and mechanism of non-Mendelian inheritance of B chromosomes.
Assuntos
Atractylodes , Cromossomos de Plantas , Hibridização in Situ Fluorescente , Meiose , Cromossomos de Plantas/genética , Atractylodes/genética , Meiose/genéticaRESUMO
OBJECTIVE: To establish the diagnostic utility of immunohistochemistry markers p16 along with MDM2 and CDK-4 in confirming the diagnosis of well-differentiated and de-differentiated liposarcoma while taking Fluorescent in situ Hybridisation (FISH) as a gold standard. STUDY DESIGN: A cross-sectional study. Place and Duration of the Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from 30th January 2022 to 30th June 2023. METHODOLOGY: A standard panel of three immunohistochemistry markers p16, MDM2, and CDK4 were applied to 36 cases of atypical lipomatous tumours, well-differentiated liposarcoma (WDLPS), and de-differentiated liposarcoma (DDLPS), on which the gold standard FISH was already performed. The sample size was calculated with the help of a WHO calculator taking prevalence 1-2% in Pakistani population. Qualitative variables such as gender and site of tumour were presented by calculating frequency and percentages and comparison of Immunohistochemistry results with FISH was done by using a 2x2 table. RESULTS: The sensitivity and specificity of this triple marker panel for detecting WDLPS/DDLPS were 43.47% and 15.38%, respectively. The sensitivity and specificity of CDK4 for detecting WDLPS / DDLPS were 82.6% and 15.4%, those of MDM2 were 73.9% and 61.5 %, and those of p16 were 60.9% and 53.8%, respectively. CONCLUSION: Among all three markers, CDK4 was the most sensitive and MDM2 was the most specific marker for detecting WDLPS-DDLPS. It also showed that a combination of these three markers improves the diagnostic credibility of the immunohistochemistry in diagnosing DDLPS and WDLPS but FISH is the most reliable and confirmatory method. KEY WORDS: De-differentiated liposarcoma, Well-differentiated liposarcoma, P16, CDK4, MDM2.
Assuntos
Biomarcadores Tumorais , Quinase 4 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Lipossarcoma , Proteínas Proto-Oncogênicas c-mdm2 , Humanos , Lipossarcoma/diagnóstico , Lipossarcoma/patologia , Lipossarcoma/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Masculino , Biomarcadores Tumorais/metabolismo , Estudos Transversais , Pessoa de Meia-Idade , Adulto , Sensibilidade e Especificidade , IdosoRESUMO
OBJECTIVE: We present mosaic distal 13q duplication due to mosaic unbalanced translocation 46,XY,der(14)t(13;14)(q32.2;p13)/46,XY at amniocentesis in a pregnancy associated with a favorable fetal outcome. CASE REPORT: A 37-year-old, gravida 2, para 0, woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 46,XY, add(14) (p13)[17]/46,XY[13] (56.6% mosaicism). Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from cultured amniocytes revealed arr 13q32.2q34 × 2â¼3, consistent with 45% mosaicism for distal 13q duplication. Repeat amniocentesis at 24 weeks of gestation revealed a karyotype of 46,XY,der(14)t(13;14)(q32.2;p13)[14]/46,XY[16] (46.6% mosaicism). The parental karyotypes were normal. aCGH analysis on the DNA extracted from uncultured amniocytes revealed arr 13q32.2q34 × 2.38, consistent with 30-40% mosaicism for distal 13q duplication. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes detected 22.8% (23/101 cells) mosaicism for distal 13q duplication. Prenatal ultrasound findings were unremarkable. At 39 weeks of gestation, a 3616-g phenotypically normal baby was delivered. The karyotypes of cord blood, umbilical cord and placenta were 46,XY,der(14)t(13;14)(q32.2;p13)[20]/46,XY[20] (50% mosaicism), 46,XY,der(14)t(13;14)(q32.2;p13)[14]/46,XY[26] (35% mosaicism) and 46,XY (40/40 cells) (0% mosaicism), respectively. When follow-ups at the age of 4½ months and the age of one year, the peripheral blood had the karyotype of 46,XY,der(14)t(13;14)(q32.2;p13)[18]/46,XY[22] (45% mosaicism). Interphase FISH analysis on buccal mucosal cells at the age of 4½ months revealed 2.7% (3/110 cells) mosaicism for distal 13q duplication, compared with 1% (1/100 cells) in the normal control. The neonate was normal in phenotype and development. CONCLUSIONS: Mosaic unbalanced translocation at amniocentesis can be associated with a favorable fetal outcome, perinatal progressive decrease of the aneuploid cell line and cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes.
Assuntos
Amniocentese , Cromossomos Humanos Par 13 , Mosaicismo , Translocação Genética , Humanos , Feminino , Gravidez , Mosaicismo/embriologia , Adulto , Translocação Genética/genética , Cromossomos Humanos Par 13/genética , Hibridização Genômica Comparativa , Cromossomos Humanos Par 14/genética , Cariotipagem , Aneuploidia , Trissomia/genética , Cariótipo , Resultado da Gravidez/genética , Duplicação Cromossômica/genética , Hibridização in Situ FluorescenteRESUMO
Involved in mitotic condensation, interaction of transcriptional regulatory elements and isolation of structural domains, loop formation has become a paradigm in the deciphering of chromatin architecture and its functional role. Despite the emergence of increasingly powerful genome visualization techniques, the high variability in cell populations and the randomness of conformations still make loop detection a challenge. We introduce an approach for determining the presence and frequency of loops in a collection of experimental conformations obtained by multiplexed super-resolution imaging. Based on a spectral approach, in conjunction with neural networks, this method offers a powerful tool to detect loops in large experimental data sets, both at the population and single-cell levels. The method's performance is confirmed on experimental FISH data where Hi-C and other loop detection results are available. The method is then applied to recently published experimental data, where it provides a detailed and statistically quantified description of the global architecture of the chromosomal region under study.
Assuntos
Cromatina , Hibridização in Situ Fluorescente , Cromatina/metabolismo , Cromatina/genética , Hibridização in Situ Fluorescente/métodos , Humanos , Animais , Redes Neurais de Computação , Conformação de Ácido Nucleico , Cromossomos/genéticaRESUMO
Imaging-based spatial transcriptomics technologies such as Multiplexed error-robust fluorescence in situ hybridization (MERFISH) can capture cellular processes in unparalleled detail. However, rigorous and robust analytical tools are needed to unlock their full potential for discovering subcellular biological patterns. We present Intracellular Spatial Transcriptomic Analysis Toolkit (InSTAnT), a computational toolkit for extracting molecular relationships from spatial transcriptomics data at single molecule resolution. InSTAnT employs specialized statistical tests and algorithms to detect gene pairs and modules exhibiting intriguing patterns of co-localization, both within individual cells and across the cellular landscape. We showcase the toolkit on five different datasets representing two different cell lines, two brain structures, two species, and three different technologies. We perform rigorous statistical assessment of discovered co-localization patterns, find supporting evidence from databases and RNA interactions, and identify associated subcellular domains. We uncover several cell type and region-specific gene co-localizations within the brain. Intra-cellular spatial patterns discovered by InSTAnT mirror diverse molecular relationships, including RNA interactions and shared sub-cellular localization or function, providing a rich compendium of testable hypotheses regarding molecular functions.
Assuntos
Algoritmos , Encéfalo , Perfilação da Expressão Gênica , Hibridização in Situ Fluorescente , Transcriptoma , Perfilação da Expressão Gênica/métodos , Humanos , Hibridização in Situ Fluorescente/métodos , Animais , Encéfalo/metabolismo , Camundongos , Biologia Computacional/métodos , RNA/genética , RNA/metabolismo , Software , Linhagem CelularRESUMO
17p13 deletions including TP53 and other genes represent a common cause for reduced/lost p53 function in tumor cells. In this study, we analyzed the impact of 17p13 (TP53) deletions and p53 expression on tumor aggressiveness and patient prognosis in urothelial carcinoma. The 17p13 copy number status was analyzed by fluorescence in situ hybridization (FISH) on more than 2700 urothelial bladder carcinomas in a tissue microarray format. 17p13 deletion data were compared to p53 expression data measured by immunohistochemistry (IHC) in a previous study. Different types of p53 alterations were compared with tumor phenotype and clinical outcome data. Deletions of 17p13 occurred in 23% of 2185 analyzable carcinomas. The fraction of tumors with 17p13 deletions increased from pTa G2 low (9%) to pTa G3 (24%, p < 0.0001). In muscle-invasive carcinomas, 17p13 deletions were associated with advanced pT stage (p = 0.0246), but unrelated to patient prognosis (p > 0.5). 17p13 deletions were significantly related to p53 immunostaining (p = 0.0375). 17p13 deletions were most common in tumors with complete lack of p53 staining (31%), which supports the concept that many of these tumors have a complete loss of p53 function (p53 null phenotype). 17p13 deletions were also increased in tumors with high p53 staining (25%). In conclusion, 17p13 deletions were most commonly seen in p53 negative cancers, supporting their role as a cause for the p53 null phenotype in urothelial cancer. The association of 17p13 deletions with high grade and advanced pT stage may reflect increasing genomic instability going along with stage and grade progression.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 17 , Fenótipo , Proteína Supressora de Tumor p53 , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Prognóstico , Cromossomos Humanos Par 17/genética , Proteína Supressora de Tumor p53/genética , Masculino , Feminino , Hibridização in Situ Fluorescente , Idoso , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is a high risk form of ALL associated with dismal outcomes in the pre-tyrosine kinase inhibitor (TKI) era. Addition of a TKI to chemotherapy improves outcomes. Therefore, testing for the presence of the Philadelphia chromosome by at least two methods at the time of diagnosis is critical. Diagnostic testing may include karyotype, fluorescent in situ hybridisation (FISH), and RT-PCR for the BCR::ABL1 transcript. The significance of low-level BCR::ABL1 transcript by RT-PCR in the absence of the Philadelphia chromosome on karyotype or by FISH is unknown. METHODS: This is a retrospective review of children diagnosed with acute leukemia at our institution from 2010 to 2020. Those positive for the BCR::ABL1 transcript by qualitative RT-PCR, and negative for t(9;22) by karyotype or FISH were analyzed for demographics, cytogenetic and molecular features at diagnosis and relapse, treatment and outcomes. The Kaplan-Meier method was used to estimate event-free and overall survival. RESULTS: Forty-seven of 306 (15%) patients with Ph- ALL had low-level BCR::ABL1 detected by RT-PCR. Most (77%) had B-cell ALL. The e1a2 transcript was detected most frequently, in 43 (91%) patients. BCR::ABL1 was quantifiable in 12/43 (28%) patients, with a median of 0.0008% (range 0.0003-0.095%). Seven patients (15%) relapsed. No patient with low-level BCR::ABL1 at diagnosis developed Ph + ALL at relapse. There was no difference in 5-year event-free (77% versus 81%, p = 0.407) or overall survival (86% versus 91%, p = 0.3) between children with low-level BCR::ABL1 (n = 47) and those without (n = 259). CONCLUSION: BCR::ABL1 low-level positivity in children with newly diagnosed Ph- ALL is a relatively common finding and did not adversely affect outcome for patients treated using a contemporary risk-adapted approach.
Assuntos
Proteínas de Fusão bcr-abl , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Criança , Masculino , Feminino , Proteínas de Fusão bcr-abl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Pré-Escolar , Adolescente , Estudos Retrospectivos , Hibridização in Situ Fluorescente , Lactente , Cromossomo FiladélfiaRESUMO
BACKGROUND: Jasminum sambac, a widely recognized ornamental plant prized for its aromatic blossoms, exhibits three flora phenotypes: single-petal ("SP"), double-petal ("DP"), and multi-petal ("MP"). The lack of detailed characterization and comparison of J. sambac mitochondrial genomes (mitogenomes) hinders the exploration of the genetic and structural diversity underlying the varying floral phenotypes in jasmine accessions. RESULTS: Here, we de novo assembled three mitogenomes of typical phenotypes of J. sambac, "SP", "DP", and "MP-hutou" ("HT"), with PacBio reads and the "HT" chloroplast (cp) genome with Illumina reads, and verified them with read mapping and fluorescence in situ hybridization (FISH). The three mitogenomes present divergent sub-genomic conformations, with two, two, and four autonomous circular chromosomes ranging in size from 35.7 kb to 405.3 kb. Each mitogenome contained 58 unique genes. Ribosome binding sites with conserved AAGAAx/AxAAAG motifs were detected upstream of uncanonical start codons TTG, CTG and GTG. The three mitogenomes were similar in genomic content but divergent in structure. The structural variations were mainly attributed to recombination mediated by a large (~ 5 kb) forward repeat pair and several short repeats. The three jasmine cp. genomes showed a well-conserved structure, apart from a 19.9 kb inversion in "HT". We identified a 14.3 kb "HT"-specific insertion on Chr7 of the "HT" nuclear genome, consisting of two 7 kb chloroplast-derived fragments with two intact ndhH and rps15 genes, further validated by polymerase chain reaction (PCR). The well-resolved phylogeny suggests faster mitogenome evolution in J. sambac compared to other Oleaceae species and outlines the mitogenome evolutionary trajectories within Lamiales. All evidence supports that "DP" and "HT" evolved from "SP", with "HT" being the most recent derivative of "DP". CONCLUSION: The comprehensive characterization of jasmine organelle genomes has added to our knowledge of the structural diversity and evolutionary trajectories behind varying jasmine traits, paving the way for in-depth exploration of mechanisms and targeted genetic research.
Assuntos
Genoma Mitocondrial , Genoma de Planta , Jasminum , Jasminum/genética , Genoma de Cloroplastos , Cloroplastos/genética , Hibridização in Situ FluorescenteRESUMO
The individual detection of human immunodeficiency virus (HIV) virions and resolution from extracellular vesicles (EVs) during analysis is a difficult challenge. Infectious enveloped virions and nonviral EVs are released simultaneously by HIV-infected host cells, in addition to hybrid viral EVs containing combinations of HIV and host components but lacking replicative ability. Complicating the issue, EVs and enveloped virions are both delimited by a lipid bilayer and share similar size and density. The feature that distinguishes infectious virions from host and hybrid EVs is the HIV genomic RNA (gRNA), which allows the virus to replicate. Single-particle analysis techniques, which provide snapshots of single biological nanoparticles, could resolve infectious virions from EVs. However, current single-particle analysis techniques focus mainly on protein detection, which fail to resolve hybrid EVs from infectious virions. A method to simultaneously detect viral protein and internal gRNA in the same particle would allow resolution of infectious HIV from EVs and noninfectious virions. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor with single-molecule fluorescence in situ hybridization. Using SPIRFISH, we detect HIV-1 envelope protein gp120 and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNAs within EVs. This may have major utility for EV therapeutics, which are increasingly focused on EV-mediated RNA delivery. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles.
Assuntos
Vesículas Extracelulares , HIV-1 , RNA Viral , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Humanos , RNA Viral/genética , RNA Viral/metabolismo , HIV-1/genética , Hibridização in Situ Fluorescente , Proteínas Virais/metabolismo , Proteínas Virais/químicaRESUMO
Microbes are important components of the tumor microenvironment and have a close relationship with tumors. However, there is still a lack of research on the intratumoral microbiota in bladder cancer and its impact on the tumor immune microenvironment. In this study, we used fluorescence in situ hybridization (FISH) and observed a substantial presence of microbiota in bladder cancer tissues, with greater abundance compared to that in normal bladder tissues. Based on the BIC database, we found that the microbiome of bladder cancer is highly diverse and its structure is significantly different from that of other tumors. To investigate the relationships among the intratumoral microbiota, tumor immunity, and prognosis in bladder cancer patients, we analyzed bladder cancer-specific differentially expressed immune- and antimicrobial-related genes from the ImmPort, TISIDB, and TCGA databases. We identified 11 hub genes and constructed a prognostic risk model. Further analysis revealed differences at the family and genus levels between distinct groups. Using LEfSe analysis, we identified six hub biomarkers and developed a novel microbial-based scoring system. The scoring system allows subgrouping of bladder cancer patients, with significant differences in prognosis, immune cell infiltration, tumor mutation burden, and immune checkpoints among different groups. Further FISH and immunofluorescence co-staining experiments initially verified that the specific distribution of microorganisms and M2 macrophages in bladder cancer may be closely related to the poor prognosis of patients. In conclusion, this study revealed the characteristics of the intratumoral microbiota in bladder cancer and identified potential prognostic targets for clinical application.
Assuntos
Microbiota , Microambiente Tumoral , Neoplasias da Bexiga Urinária , Neoplasias da Bexiga Urinária/microbiologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Humanos , Prognóstico , Microbiota/genética , Microambiente Tumoral/imunologia , Hibridização in Situ Fluorescente , Biomarcadores Tumorais/genética , Feminino , Masculino , Pessoa de Meia-Idade , IdosoRESUMO
Gametogenesis produces gametes as a piece of genetic information transmitted to the offspring. While during sexual reproduction, progeny inherits a mix of genetic material from both parents, asexually reproducing organisms transfer a copy of maternal or paternal DNA to the progeny clonally. Parthenogenetic, gynogenetic and hybridogenetic animals have developed various mechanisms of gametogenesis, however, their inheritance is not fully understood. Here, we focused on the inheritance of asexual gametogenesis in hybrid Pelophylax esculentus (RL), emerging after crosses of P. lessonae (LL) and P. ridibundus (RR). To understand the mechanisms of gametogenesis in hybrids, we performed three-generation experiments of sexual P. ridibundus females and hybrids from all-male hybrid populations. Using fluorescent in situ hybridization, micronuclei analysis, flow cytometry and genotyping, we found that most adult hybrid males simultaneously produced two types of clonal sperm. Also, most male tadpole progeny in two successive backcrossed generations simultaneously eliminated L and R parental genomes, while some progeny produced only one type of sperm. We hypothesize that the reproductive variability of males producing two kinds of sperm is an adaptive mechanism to reproduce in mixed populations with P. ridibundus and may explain the extensive distribution of the all-male lineage across the European River Basin.
Assuntos
Hibridização Genética , Reprodução Assexuada , Animais , Masculino , Feminino , Reprodução Assexuada/genética , Padrões de Herança/genética , Espermatozoides/fisiologia , Ranidae/genética , Ranidae/fisiologia , Rana esculenta/genética , Genótipo , Hibridização in Situ FluorescenteRESUMO
BACKGROUND: Evaluation of indeterminate pulmonary nodules (IPNs) often creates a diagnostic conundrum which may delay the early detection of lung cancer. Rare circulating genetically abnormal cells (CGAC) have previously demonstrated utility as a biomarker for discriminating benign from malignant small IPNs in the LungLB assay. CGAC are identified using a unique 4-color fluorescence in-situ hybridization (FISH) assay and are thought to reflect early cell-based events in lung cancer pathogenesis and the anti-tumor immune response. LungLB is a prognostic tool that combines the CGAC biomarker and clinical features to aid in IPN evaluation by improving the stratification of patient risk of malignancy. METHODS: Herein we describe the analytical performance of the LungLB blood test. Analytical validation was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with adaptations for rare cell-based assays. Multiple operators, reagent lots, and assay runs were tested to examine accuracy, precision, reproducibility, and interfering factors. RESULTS: The FISH probes used in the LungLB assay demonstrate 100% sensitivity and specificity for their intended chromosomal loci (3q29, 3p22.1, 10q22.3 and 10cen). LungLB demonstrates analytical sensitivity of 10 CGAC per 10,000 lymphocytes analyzed, 100% analytical specificity, and high linearity (R2 = 0.9971). Within run measurements across 100 samples demonstrated 96% reproducibility. Interfering factors normally found in blood (lipemia, biotin) and exposure to adverse temperatures (-20ºC or 37ºC) did not interfere with results. Sample stability was validated to 96 hours. CONCLUSION: The analytical performance of LungLB in this validation study successfully demonstrates it is robust and suitable for everyday clinical use.
Assuntos
Hibridização in Situ Fluorescente , Neoplasias Pulmonares , Nódulos Pulmonares Múltiplos , Humanos , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Reprodutibilidade dos Testes , Nódulos Pulmonares Múltiplos/diagnóstico , Nódulos Pulmonares Múltiplos/genética , Nódulos Pulmonares Múltiplos/patologia , Biomarcadores Tumorais/genética , Sensibilidade e Especificidade , Células Neoplásicas Circulantes/patologiaRESUMO
Eukaryotic genomes exhibit a dynamic interplay between single-copy sequences and repetitive DNA elements, with satellite DNA (satDNA) representing a substantial portion, mainly situated at telomeric and centromeric chromosomal regions. We utilized Illumina next-generation sequencing data from Adalia bipunctata to investigate its satellitome. Cytogenetic mapping via fluorescence in situ hybridization was performed for the most abundant satDNA families. In silico localization of satDNAs was carried out using the CHRISMAPP (Chromosome In Silico Mapping) pipeline on the high-fidelity chromosome-level assembly already available for this species, enabling a meticulous characterization and localization of multiple satDNA families. Additionally, we analyzed the conservation of the satellitome at an interspecific scale. Specifically, we employed the CHRISMAPP pipeline to map the satDNAs of A. bipunctata onto the genome of Adalia decempunctata, which has also been sequenced and assembled at the chromosome level. This analysis, along with the creation of a synteny map between the two species, suggests a rapid turnover of centromeric satDNA between these species and the potential occurrence of chromosomal rearrangements, despite the considerable conservation of their satellitomes. Specific satDNA families in the sex chromosomes of both species suggest a role in sex chromosome differentiation. Our interspecific comparative study can provide a significant advance in the understanding of the repeat genome organization and evolution in beetles.
Assuntos
Centrômero , Besouros , DNA Satélite , Hibridização in Situ Fluorescente , Animais , Besouros/genética , DNA Satélite/genética , Centrômero/genética , Hibridização in Situ Fluorescente/métodos , Mapeamento Cromossômico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Masculino , Cromossomos de Insetos/genética , Cromossomos Sexuais/genética , Sintenia , Feminino , Especificidade da EspécieRESUMO
Multiple sex chromosomes usually arise from chromosomal rearrangements which involve ancestral sex chromosomes. There is a fundamental condition to be met for their long-term fixation: the meiosis must function, leading to the stability of the emerged system, mainly concerning the segregation of the sex multivalent. Here, we sought to analyze the degree of differentiation and meiotic pairing properties in the selected fish multiple sex chromosome system present in the wolf-fish Hoplias malabaricus (HMA). This species complex encompasses seven known karyotype forms (karyomorphs) where the karyomorph C (HMA-C) exhibits a nascent XY sex chromosomes from which the multiple X1X2Y system evolved in karyomorph HMA-D via a Y-autosome fusion. We combined genomic and cytogenetic approaches to analyze the satellite DNA (satDNA) content in the genome of HMA-D karyomorph and to investigate its potential contribution to X1X2Y sex chromosome differentiation. We revealed 56 satDNA monomers of which the majority was AT-rich and with repeat units longer than 100 bp. Seven out of 18 satDNA families chosen for chromosomal mapping by fluorescence in situ hybridization (FISH) formed detectable accumulation in at least one of the three sex chromosomes (X1, X2 and neo-Y). Nine satDNA monomers showed only two hybridization signals limited to HMA-D autosomes, and the two remaining ones provided no visible FISH signals. Out of seven satDNAs located on the HMA-D sex chromosomes, five mapped also to XY chromosomes of HMA-C. We showed that after the autosome-Y fusion event, the neo-Y chromosome has not substantially accumulated or eliminated satDNA sequences except for minor changes in the centromere-proximal region. Finally, based on the obtained FISHpatterns, we speculate on the possible contribution of satDNA to sex trivalent pairing and segregation.
Assuntos
Caraciformes , DNA Satélite , Hibridização in Situ Fluorescente , Cromossomos Sexuais , Animais , DNA Satélite/genética , Cromossomos Sexuais/genética , Masculino , Caraciformes/genética , Feminino , Evolução Molecular , Meiose/genética , Cariótipo , Cromossomo Y/genéticaRESUMO
Candidatus Patescibacteria, also known as candidate phyla radiation (CPR), including the class-level uncultured clade JAEDAM01 (formerly a subclass of Gracilibacteria/GN02/BD1-5), are ubiquitous in activated sludge. However, their characteristics and relationships with other organisms are largely unknown. They are believed to be episymbiotic, endosymbiotic or predatory. Despite our understanding of their limited metabolic capacity, their precise roles remain elusive due to the difficulty in cultivating and identifying them. In previous research, we successfully recovered high-quality metagenome-assembled genomes (MAGs), including a member of JAEDAM01 from activated sludge flocs. In this study, we designed new probes to visualize the targeted JAEDAM01-associated MAG HHAS10 and identified its host using fluorescence in situ hybridization (FISH). The FISH observations revealed that JAEDAM01 HHAS10-like cells were located within dense clusters of Zoogloea, and the fluorescence brightness of zoogloeal cells decreased in the vicinity of the CPR cells. The Zoogloea MAGs possessed genes related to extracellular polymeric substance biosynthesis, floc formation and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The JAEDAM01 MAG HHAS10 possessed genes associated with type IV pili, competence protein EC and PHA degradation, suggesting a Zoogloea-dependent lifestyle in activated sludge flocs. These findings indicate a new symbiotic relationship between JAEDAM01 and Zoogloea.
Assuntos
Esgotos , Simbiose , Águas Residuárias , Zoogloea , Esgotos/microbiologia , Zoogloea/genética , Zoogloea/metabolismo , Águas Residuárias/microbiologia , Hibridização in Situ Fluorescente , Metagenoma , FilogeniaRESUMO
In situ imaging of genes of pathogenic bacteria can profile cellular heterogeneity, such as the emergence of drug resistance. Fluorescence in situ hybridization (FISH) serves as a classic approach to image mRNAs inside cells, but it remains challenging to elucidate genomic DNAs and relies on multiple fluorescently labeled probes. Herein, we present a dead Cas12a (dCas12a)-labeled polymerase chain reaction (CasPCR) assay for high-contrast imaging of cellular drug-resistant genes. We employed a syncretic dCas12a-green fluorescent protein (dCas12a-GFP) to tag the amplicons, thereby enabling high-contrast imaging and avoiding multiple fluorescently labeled probes. The CasPCR assay can quantify quinolone-resistant Salmonella enterica in mixed populations and identify them isolated from poultry farms.
Assuntos
Reação em Cadeia da Polimerase , Salmonella enterica , Salmonella enterica/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Hibridização in Situ Fluorescente/métodos , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Animais , Quinolonas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Sistemas CRISPR-Cas/genéticaRESUMO
Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (-) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.