Profiling Chromosome Topological Features by Super-Resolution 3D Structured Illumination Microscopy.

Three-dimensional structured illumination microscopy (3D-SIM) and fluorescence in situ hybridization on three-dimensional preserved cells (3D-FISH) have proven to be robust and efficient methodologies for analyzing nuclear architecture and profiling the genome's topological features. These methods have allowed the simultaneous visualization and evaluation of several target structures at super-resolution. In this chapter, we focus on the application of 3D-SIM for the visualization of 3D-FISH preparations of chromosomes in interphase, known as Chromosome Territories (CTs). We provide a workflow and detailed guidelines for sample preparation, image acquisition, and image analysis to obtain quantitative measurements for profiling chromosome topological features. In parallel, we address a practical example of these protocols in the profiling of CTs 9 and 22 involved in the translocation t(9;22) in Chronic Myeloid Leukemia (CML). The profiling of chromosome topological features described in this chapter allowed us to characterize a large-scale topological disruption of CTs 9 and 22 that correlates directly with patients' response to treatment and as a possible potential change in the inheritance systems. These findings open new insights into how the genome structure is associated with the response to cancer treatments, highlighting the importance of microscopy in analyzing the topological features of the genome.

Characterizing Chemotherapy/Radiotherapy-Induced Genome Chaos in Hodgkin's Lymphoma Patients Using M-FISH.

Hodgkin lymphoma (HL) is one of the most common lymphomas, with an incidence of 3 per 100,000 persons. Current treatment uses a cocktail of genotoxic agents, including adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD), along with or without radiotherapy. This treatment regimen has proved to be efficient in killing cancer cells, resulting in HL patients having a survival rate of >90% cancer-free survival at five years. However, this therapy does not have a specific cell target, and it can induce damage in the genome of non-cancerous cells. Previous studies have shown that HL survivors often exhibit karyotypes characterized by complex chromosomal abnormalities that are difficult to analyze by conventional banding. Multicolor fluorescence in situ hybridization (M-FISH) is a powerful tool to analyze complex karyotypes; we used M-FISH to investigate the presence of chromosomal damage in peripheral blood lymphocytes from five healthy individuals and five HL patients before, during, and one year after anti-cancer treatment. Our results show that this anti-cancer treatment-induced genomic chaos that persists in the hematopoietic stem cells from HL patients one year after finishing therapy. This chromosomal instability may play a role in the occurrence of second primary cancers that are observed in 10% of HL survivors. This chapter will describe a protocol for utilizing M-FISH to study treatment-induced genome chaos in Hodgkin's lymphoma (HL) patients, following a brief discussion.

Carica papaya L. sex chromosome review and physical mapping of the serk 2, svp-like and mdar 4 sequences.

Physical mapping evidences the chromosome organization and structure. Despite the data about plant cytogenomics, physical mapping has been conducted from single-copy and/or low-copy genes for few species. Carica papaya cytogenomics has been accomplished from BAC-FISH and repeatome sequences. We aimed to map the serk 2, svp-like and mdar 4 sequences in C. papaya. The sequences were amplified and the amplicons sequenced, showing similarity in relation to serk 2, svp-like and mdar 4 genes. Carica papaya diploidy was confirmed and the mitotic chromosomes characterized. The chromosome 1 exhibited the secondary constriction pericentromeric to the centromere of the long arm. So, we concluded that it is the sex chromosomes. serk 2 was mapped in the long arm interstitial portion of the sex chromosomes, and the interphase nuclei showed two fluorescence signals. Considering these results and the sequencing data from the C. papaya sex chromosomes, svp-like and mdar 4 genes were mapped in the interstitial region of the sex chromosome long arm. Both sequences showed only one fluorescence signal in the interphase nuclei. The procedure adopted here can be reproduced for other single-copy and/or low-copy genes, allowing the construction of cytogenetic maps. In addition, we revisited the cytogenomics data about C. papaya sex chromosomes, presenting a revised point of view about the structure and evolution to these chromosomes.

Karyotypic Reshuffling in the Genus Rhipidomys (Rodentia: Cricetidae: Sigmodontinae) Revealed by Zoo-FISH.

INTRODUCTION: Rhipidomys is the second most specious and the most widespread genus of the tribe Thomasomyini. Chromosomal data have been an important tool in the taxonomy of the group that presents low variability of diploid number (2n) and highly variable fundamental numbers (FNs). Despite such diversity, the genus has been studied mainly by classical and banding cytogenetic techniques. METHODS: This study performed a comparative study between R. emiliae (2n = 44, FN = 52), R. macrurus (2n = 44, FN = 49), R. nitela (2n = 50, FN = 71), and R. mastacalis (2n = 44, FN = 72) using chromosome painting probes of two Oryzomyini species. RESULTS: Our analysis revealed pericentric inversion as the main rearrangement involved in the karyotype evolution of the group, although tandem fusions/fissions were also detected. In addition, we detected eight syntenic associations exclusive of the genus Rhipidomys, and three syntenic associations shared between species of the tribe Thomasomyini and Oryzomyini. CONCLUSION: Comparative cytogenetic analysis by ZOO-FISH on genus Rhipidomys supports a pattern of chromosomal rearrangement already suggested by comparative G-banding. However, the results suggest that karyotype variability in the genus could also involve the occurrence of an evolutionary new centromere.

DBD-FISH Using Specific Chromosomal Region Probes for the Study of Cervical Carcinoma Progression.

Genomic instability is an important biomarker in the progression of cervical carcinoma. DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) is a sensitive method that detects strand breaks, alkali-labile sites, and incomplete DNA excision repair in cells of the cervical epithelium. This technique integrates the microgel immersion of cells from a vaginal lesion scraping and the DNA unwinding treatment with the capacity of FISH integrated into digital image analysis. Cells captured within an agarose matrix are lysed and submerged in an alkaline unwinding solution that generates single-stranded DNA motifs at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with DNA-labeled probes. The quantity of a hybridized probe at a target sequence corresponds to the measure of the single-stranded DNA produced during the unwinding step, which is equivalent to the degree of local DNA breakage. DNA damage does not show uniformly throughout the entire DNA of a cell; rather, it is confined to specific chromosomal sites. In this chapter, an overview of the technique is supplied, focusing on its ability for assessing the association between DNA damage in specific sequences and in the progressive stages of cervical carcinoma.

Discordant results for ALK based on immunohistochemistry versus fluorescence in situ hybridization in a cohort of patients diagnosed with lung adenocarcinoma.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) rearrangement located on the short arm of chromosome 2, region 2 and band 3 is frequent in lung cancer patients who respond to targeted therapies with ALK inhibitors Therefore, their identification has become a standard diagnostic test in patients with advanced NSCLS, as such chromosomal alterations may lead to the activation of important signalling pathways involved in cell survival and proliferation. METHODS: To investigate the ALK gene status, we performed FISH and IHC assays in 18 lung adenocarcinoma patients, 12 women and 6 men, aged between 29 and 85 years. Paraffin-embedded samples were analyzed in the Pathology Department of the Hospital Universitario San Ignacio. RESULTS: Results between the two techniques in 5 patients showed discordant patterns, being positive for FISH and negative for IHC. The borderline to define ALK positivity was set at 15%, These results present experimental evidence that the techniques differ in specific situations. CONCLUSIONS: Our findings show that it is advisable to investigate the ALK gene status in patients with suspected lung cancer using both FISH and IHC in combination.

Identification of a Small Supernumerary Marker Chromosome in a Turner Syndrome Patient with Karyotype mos 46,X,+mar/45,X.

Turner Syndrome is characterized by a normal X chromosome and the partial or complete absence of a second sexual chromosome. Small supernumerary marker chromosomes are present in 6.6% of these patients. Because of the wide range of Turner syndrome karyotypes, it is difficult to establish a relationship with the phenotype of the patients. We present the case of a female patient with Turner syndrome, insulin resistance, type 2 diabetes, and intellectual disability. The karyotype revealed the presence of mosaicism with a monosomy X cell line and a second line with a small marker chromosome. FISH of two different tissues was used to identify the marker chromosome with probes for X and Y centromeres. Both tissues presented mosaicism for a two X chromosome signal, differing in the percentage of the monosomy X cell percentage. Comparative genomic hybridization with the CytoScanTMHD assay was performed in genomic DNA from peripheral blood, allowing us to determine the size and breakage points of the small marker chromosome. The patient presents a phenotype that combines classic Turner syndrome features and unlikely ones as intellectual disability. The size, implicated genes, and degree of inactivation of the X chromosome influence the broad spectrum of phenotypes resulting from these chromosomes.

Hybridization Chain Reaction Fluorescence In Situ Hybridization (HCR-FISH) in Ambystoma mexicanum Tissue.

In situ hybridization is a standard procedure for visualizing mRNA transcripts in tissues. The recent adoption of fluorescent probes and new signal amplification methods have facilitated multiplexed RNA imaging in tissue sections and whole tissues. Here we present protocols for multiplexed hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) staining, imaging, cell segmentation, and mRNA quantification in regenerating axolotl tissue sections. We also present a protocol for whole-mount staining and imaging of developing axolotl limbs.

Fluorescence In Situ Hybridization of DNA Probes on Mitotic Chromosomes of the Mexican Axolotl.

Fluorescence in situ hybridization (FISH) is used extensively for visual localization of specific DNA fragments (and RNA fragments) in broad applications on chromosomes or nuclei at any stage of the cell cycle: metaphase, anaphase, or interphase. The cytogenetic slides that serve as a target for the labeled DNA probe might be prepared using any approach suitable for obtaining cells with appropriate morphology for imaging and analysis. In this chapter, we focus on the application of molecular cytogenetic methods such as DNA labeling, slide preparation, and in situ hybridization related to cells from Mexican axolotl.

An outlook on fluorescent in situ hybridization coupled to flow cytometry as a versatile technique to evaluate the effects of foods and dietary interventions on gut microbiota.

The increasing interest in the effects of the gut microbiota on host health has stimulated the investigation of the composition of this microbial community and the factors affecting these microorganisms. This review discusses the recent advances and progress applications in the use of the fluorescent in situ hybridization (FISH) coupled to flow cytometry (FC) technique (FISH-FC) in studies evaluating the gut microbiota published in the last 10 years, with particular emphasis on the effects of foods and dietary interventions. These studies have shown that FISH-FC technique is capable of detecting and quantifying several groups of bacteria found as part of the gut microbiota. FISH-FC can be considered an effective, versatile, and rapid technique to evaluate alterations in gut microbiota composition caused by different foods as assessed in studies in vitro, in vivo, and in clinical trials. Some specific probes have been most used to represent the general gut microbiota, such as those specific to Lactobacillus spp./Enterococcus spp., Bacteroidaceae/Prevotellaceae, Clostridium histolyticum, and Bifidobacterium spp. FISH-FC technique could have an important opportunity for application in studies with next-generation probiotics belonging to the gut microbiota. Optimizations of FISH-FC protocols could allow more discoveries about the gut microbiota, including the development of new probes targeting microorganisms still not explored, the analysis of individual portions of the intestine, and the proposition of novel quantitative approaches.

Chromosomal Analysis of Ctenolucius hujeta Valenciennes, 1850 (Characiformes): A New Piece in the Chromosomal Evolution of the Ctenoluciidae.

Ctenoluciidae (Characiformes), a family of freshwater fishes, comprises 2 genera, Ctenolucius and Boulengerella, with 7 recognized species. Up to now, only species of the genus Boulengerella have been subjected to cytogenetic studies. Here, we investigated the karyotype and other cytogenetic features of pike characin, Ctenolucius hujeta, using conventional (Giemsa staining, C-banding, Ag-NOR staining) and molecular (rDNA, telomeric sequences, and fiber-FISH mapping) procedures. This species has a diploid chromosome number of 2n = 36, and a karyotype composed of 12m + 20sm + 4a and FN = 68, similar to that found in Boulengerella species. However, differences regarding the number and distribution of several chromosomal markers support a distinct generic status. Colocalization of the 18S and 5S rDNA genes is an exclusive characteristic of the C. hujeta genome, with an interspersed distribution in the chromosomal fiber, an unusual phenomenon among eukaryotes. Additionally, our results support the view that Ctenoluciidae and Lebiasinidae families are closely related.

Concordance Analysis of ALK Gene Fusion Detection Methods in Patients with Non-Small-Cell Lung Cancer from Chile, Brazil, and Peru.

About 4% to 7% of the non-small-cell lung cancer patients have anaplastic lymphoma kinase (ALK) rearrangements, and specific targeted therapies improve patients' outcomes significantly. ALK gene fusions are detected by immunohistochemistry or fluorescent in situ hybridization as gold standards in South America. Next-generation sequencing-based assays are a reliable alternative, able to perform simultaneous detection of multiple events from a single sample. We analyzed 4240 non-small-cell lung cancer samples collected in 37 hospitals from Chile, Brazil, and Peru, where ALK rearrangements were determined as part of their standard of care (SofC) using either immunohistochemistry or fluorescent in situ hybridization. A subset of 1450 samples was sequenced with the Oncomine Focus Assay (OFA), and the concordance with the SofC tests was measured. An orthogonal analysis was performed using a real-time quantitative PCR echinoderm microtubule-associated protein-like 4-ALK fusion detection kit. ALK fusion prevalence is similar for Chile (3.67%; N = 2142), Brazil (4.05%; N = 1013), and Peru (4.59%; N = 675). Although a comparison between OFA and SofC assays showed similar sensitivity, OFA had significantly higher specificity and higher positive predictive value, which opens new opportunities for a more specific determination of ALK gene rearrangements.

Comparative Cytogenetic Analysis of Three Eumeninae Species (Hymenoptera, Vespidae).

Eumeninae represents the largest subfamily within Vespidae, with 3,600 species described. Of these, only 18 have been cytogenetically analysed. In the present study, we used both classical and molecular techniques to characterise and compare the karyotypes of 3 Eumeninae species, namely, Ancistrocerus sp., Pachodynerus grandis, and Pachodynerus nasidens. Ancistrocerus sp. presented a haploid chromosome number of n = 12, with the first 2 chromosomes of the karyotype being almost entirely heterochromatic and much larger than the remaining chromosomes. The 2 Pachodynerus species presented the same chromosome number (n = 11 and 2n = 22) but displayed different karyotypic formulae. Additionally, chromosomal polymorphisms were observed in the analysed P. nasidens female. In the 3 species, heterochromatin was located in one of the chromosome arms. Fluorochrome staining revealed a balanced composition of AT and GC bases within the chromatin for each of the 3 species, except for few regions that were visibly GC-rich. All species had a single 18S rDNA site that co-localised with GC-rich regions; however, this localisation varied from species to species and not all GC-rich regions corresponded to ribosomal genes. Based on the cytogenetic data obtained here, we discuss the possible numerical/structural rearrangements that may be involved in the karyotypic evolution of the 3 studied species. In addition to the first description of the molecular cytogenetic characteristics of the Eumeninae subfamily and the genus Pachodynerus, this study also provides a relevant contribution towards the discussion of chromosomal evolution in Eumeninae wasps.

Cytogenetic Analysis of Panaqolus tankei Cramer & Sousa, 2016 (Siluriformes, Loricariidae), an Ornamental Fish Endemic to Xingu River, Brazil.

Despite conservation of the diploid number, a huge diversity in karyotype formulae is found in the Ancistrini tribe (Loricariidae, Hypostominae). However, the lack of cytogenetic data for many groups impairs a comprehensive understanding of the chromosomal relationships and the impact of chromosomal changes on their evolutionary history. Here, we present for the first time the karyotype of Panaqolus tankei Cramer & Sousa, 2016. We focused on the chromosomal characterization, using conventional and molecular cytogenetic techniques to unravel the evolutionary trends of this tribe. P. tankei, as most species of its sister group Pterygoplichthini, also possessess a conserved diploid number of 52 chromosomes. We observed heterochromatin regions in the centromeres of many chromosomes; pairs 5 and 6 presented interstitial heterochromatin regions, whereas pairs 23 and 24 showed extensive heterochromatin regions in their q arms. In situ localization of 18S rDNA showed hybridization signals correlating with the nucleolus organizer regions, which are located in the q arms of pair 5. However, the 5S rDNA was detected in the centromeric and terminal regions of the q arms of pair 8. (TTAGGG)n hybridized only in the terminal regions of all chromosomes. Microsatellite in situ localization showed divergent patterns, (GA)15 repeated sequences were restricted to the terminal regions of some chromosomes, whereas (AC)15 and (GT)15 showed a scattered hybridization pattern throughout the genome. Intraspecific comparative genomic hybridization was performed on the chromosomes of P. tankei to verify the existence of sex-specific regions. The results revealed only a limited number of overlapping hybridization signals, coinciding with the heterochromatin in centromeric regions without any sex-specific signals in both males and females. Our study provides a karyotype description of P. tankei, highlighting extensive differences in the karyotype formula, the heterochromatin regions, and sites of 5S and 18S rDNA, as compared with data available for the genus.

Universal fluorescence in situ hybridization (FISH) protocol for mapping repetitive DNAs in insects and other arthropods.

Repetitive DNAs comprise large portion of eukaryote genomes. In genome projects, the assembly of repetitive DNAs is challenging due to the similarity between repeats, which generate ambiguities for alignment. Fluorescence in situ hybridization (FISH) is a powerful technique for the physical mapping of various sequences on chromosomes. This technique is thus very helpful in chromosome-based genome assemblies, providing information on the fine architecture of genomes and their evolution. However, various protocols are currently used for FISH mapping, most of which are relatively laborious and expensive, or work properly only with a specific type of probes or sequences, and there is a need for a universal and affordable FISH protocol. Here we tested a FISH protocol for mapping of different DNA repeats, such as multigene families (rDNAs, U snDNAs, histone genes), satellite DNAs, microsatellites, transposable elements, DOP-PCR products, and telomeric motif (TTAGG)n, on the chromosomes of various insects and other arthropods. Different cell types and stages obtained from diverse tissues were used. The FISH procedure proved high quality and reliable results in all experiments performed. We obtained data on the chromosomal distribution of DNA repeats in representatives of insects and other arthropods. Thus, our results allow us to conclude that the protocol is universal and requires only time adjustment for chromosome/DNA denaturation. The use of this FISH protocol will facilitate studies focused on understanding the evolution and role of repetitive DNA in arthropod genomes.

Citogenética de las hemopatías malignas en la era de la secuenciación / Cytogenetics of malignant hemopathies in the sequencing era

Las neoplasias hematológicas se caracterizan por un gran número y complejidad de alteraciones genéticas, desde la formación de genes de fusión a partir de translocaciones e inversiones cromosómicas hasta mutaciones génicas y alteraciones epigenéticas que han permitido la identificación de nuevos oncogenes y genes supresores de tumores responsables de su etiología. Al abordar el estudio genético de las leucemias se utilizan múltiples técnicas como la citogenética convencional, citogenética molecular (hibridaciónin situ por fluorescencia (FISH), esta última con una mayor sensibilidad, especificidad y rapidez que permiten el diagnóstico, la estratificación pronóstica y seguimiento de la enfermedad. Las técnicas anteriores se integran con técnicas de biología molecular, secuenciación génica, entre otras, que permiten el hallazgo de nuevos marcadores genéticos con una mejor caracterización de las hemopatías malignas y la posibilidad del desarrollo de nuevos fármacos específicos que actúen sobre la diana molecular. El objetivo fue revisar la utilidad de la citogenética y la secuenciación génica en el estudio de la leucemia mieloide aguda y la leucemia linfocítica crónica. Ante las ventajas, desventajas y limitaciones de estas técnicas genéticas es necesario utilizarlas de forma complementaria y nunca excluyente(AU)

Hematological neoplasms are characterized by a large number and great complexity of genetic disorders, from the formation of fusion genes after chromosomal translocations and inversions to gene mutation and epigenetic disorders that have permitted the identification of new oncogenes and tumor-suppressing genes responsible for their etiology. When addressing the genetic study of leukemias, multiple techniques are used, such as conventional cytogenetics, molecular cytogenetics, and fluorescence in situ hybridization (FISH), the latter having the higher degree of sensitivity, specificity and speed, which allow diagnosis, prognostic stratification and follow-up of the disease. The previous techniques are integrated with molecular biology techniques, gene sequencing, among others, which allow discovery of new genetic markers with better characterization of malignant hemopathies and the possibility of developing new specific drugs against the molecular target. The objective was to review the usefulness of cytogenetics and gene sequencing in the study of acute myeloid leukemia and chronic lymphocytic leukemia. Given the advantages, disadvantages and limitations of these genetic techniques, it is necessary to use them in as complementary but never exclusive management ways(AU)

Pilocytic Astrocytoma: A Review of General, Clinical, and Molecular Characteristics.

Pilocytic astrocytomas are the primary tumors most frequently found in children and adolescents, accounting for approximately 15.6% of all brain tumors and 5.4% of all gliomas. They are mostly found in infratentorial structures such as the cerebellum and in midline cerebral structures such as the optic nerve, hypothalamus, and brain stem. The present study aimed to list the main characteristics about this tumor, to better understand the diagnosis and treatment of these patients, and was conducted on search of the published studies available in NCBI, PubMed, MEDLINE, Scielo, and Google Scholar. It was possible to define the main histologic findings observed in these cases, such as mitoses, necrosis, and Rosenthal fibers. We described the locations usually most affected by tumor development, and this was associated with the most frequent clinical features. The comparison between the molecular diagnostic methods showed great use of fluorescent in situ hybridization, polymerase chain reaction (PCR), and reverse transcriptase-PCR, important techniques for the detection of BRAF V600E mutation and BRAF-KIAA1549 fusion, characteristic molecular alterations in pilocytic astrocytomas.

Caso atípico de leucemia mieloide aguda con coexistencia de NPM1-A e inversión del cromosoma 16 / A rare case of acute myeloid leukemia with coexistence of NPM1-A mutation and chromosome 16 inversion

Introducción: La leucemia mieloide aguda (LMA) es un grupo heterogéneo de desórdenes clonales con una gran variabilidad en términos de patogénesis, características morfológicas, genéticas e inmunofenotípicas. Las mutaciones en el gen NPM1 representan una de las más comunes en las LMA y está asociada con una respuesta clínica favorable. Por citogenética, la inversión del cromosoma 16 define el subgrupo de las LMA de factor de unión al grupo con un pronóstico favorable. Objetivo: Describir un caso con diagnóstico de LMA en los cuales el estudio molecular del gen NPM1 y de la inv(16) fueron positivos. Caso clínico: A nivel molecular, la hibridación in situ fluorescente fue positivo a la inv(16) y por biología molecular fue positivo tanto a la inv(16) como al gen NPM1-A, elementos de baja frecuencia de aparición. Se le administró a la paciente un esquema de poliquimioterapia no intensiva para mejorarla clínicamente. Después de una mejoría clínica inicial, la paciente comenzó con complicaciones y falleció. Conclusiones: La coexistencia de estas dos mutaciones es muy poco frecuente en pacientes con LMA, y a pesar de ser de buen pronóstico la paciente falleció a los pocos días de tratamiento(AU)

Introduction: Acute myeloid leukemia (AML) is a heterogeneous group of clonal disorders with great variability in terms of pathogenesis, morphological, genetic and immunophenotypic characteristics. NPM1 mutations represent one of the most common in AML and are associated with favorable clinical response. By cytogenetics, chromosome 16 inversion defines, with a favorable prognosis, the core‐binding factor for the subgroup of AMLs Objective: To describe a AML case in which the molecular study of the NPM1 gene and the chromosome 16 inversion were positive. Clinical case: At the molecular level, fluorescent in situ hybridization was positive for chromosome 16 inversion and, by molecular biology, it was positive for both chromosome 16 inversion and for the NPM1-A gene, elements with a low frequency of appearance. The patient was administered a non-intensive combination as part of a chemotherapy regimen to improve her clinical status. After initial clinical improvement, the patient began with complications and died. Conclusions: The coexistence of these two mutations is very rare in patients with AML. Despite presenting a good prognosis, the patient died after a few days of treatment(AU)

Características del diagnóstico prenatal por hibridación fluorescente in situ en Cuba / Features of the prenatal diagnosis by fluorescence in situ hybridization in Cuba

Introducción: El diagnóstico prenatal mediante la hibridación fluorescente in situ disminuye el tiempo de diagnóstico al no ser necesario el cultivo celular. Objetivo: Describir las características y experiencias del diagnóstico prenatal por hibridación fluorescente in situ en Cuba. Método: En amniocitos in situ se aplicaron sondas CEP y LSI para la detección de aneuploidías de los cromosomas 21,18,13, X y Y y sondas LSI para la detección de deleciones asociadas a síndromes de microdeleción. Resultados: Se remitieron al Centro Nacional de Genética Médica 629 casos de alto riesgo genético. Prevaleció la indicación de alteraciones fetales detectadas por ecografía. En 612 (97 por ciento) casos se obtuvo un diagnóstico satisfactorio, entre ellos, 50 (8,1 por ciento) casos positivos, con predominio del síndrome Down en 26 casos. Se corroboraron por citogenética convencional 312 casos con 98 por ciento de concordancia con los resultados obtenidos por hibridación fluorescente in situ. Se utilizó el líquido amniótico refrigerado para corroborar casos de diagnóstico dudoso obtenido por citogenética y se detectaron 3 fetos con mosaicos cromosómicos, el origen de un cromosoma marcador y la definición del sexo fetal en un caso. Conclusiones: Con la tecnología por hibridación fluorescente in situ el diagnóstico prenatal logra una segura opción de análisis en aquellos casos de embarazos de alto riesgo genético. Debido a limitaciones tecnológicas, la prueba por hibridación fluorescente in situ en células amnióticas en interfase, se ha adaptado a nuestras condiciones, para lograr siempre un diagnóstico seguro con el menor perjuicio posible a la embarazada, el feto y su familia(AU)

Introduction: Prenatal diagnosis by fluorescent in situ hybridization decreases the time of diagnosis not being necessary the cell culture. Objective: To describe the characteristics and experiences of prenatal diagnosis by fluorescent in situ hybridization in Cuba. Method: In in situ amniocytes CEP catheters were applied and LSI for the detection of aneuploidies of the 21,18,13, X and Y chromosomes, and LSI catheters for the detection of deletions associated with microdeletion syndromes. Results: 629 cases of high genetic risk were referred to the National Center of Medical Genetics. There was a prevalence of the indication of fetal abnormalities detected by ultrasound. In 612 (97 percent) cases the diagnosis was achieved in a satisfactory form, among them 50 (8.1 percent) positive cases, with predominance of Down syndrome in 26 cases. There were corroborated 312 cases by conventional cytogenetics with 98 percent of agreement with the results obtained by fluorescent in situ hybridization. It was used the cooled amniotic fluid to corroborate cases of uncertain diagnosis obtained by cytogenetics and there were detected 3 fetuses with chromosomal mosaics, the origin of a marker chromosome and the definition of fetal sex in one case. Conclusions: With the technology by fluorescent in situ hybridization, the prenatal diagnosis achieved a safe analysis option in cases of genetic high-risk pregnancies. Due to technological limitations, the test by fluorescent in situ hybridization in amniotic cells in interphase has adapted to the conditions in order to always achieve a safe diagnosis with the less possible damage to the pregnant women, the fetus and its family(AU)

Who is where in the Plastisphere, and why does it matter?

To fully understand how plastic is affecting the ocean, we need to understand how marine life interacts directly with it. Besides their ecological relevance, microbes can affect the distribution, degradation and transfer of plastics to the rest of the marine food web. From amplicon sequencing and scanning electron microscopy, we know that a diverse array of microorganisms rapidly associate with plastic marine debris in the form of biofouling and biofilms, also known as the "Plastisphere." However, observation of multiple microbial interactions in situ, at small spatial scales in the Plastisphere, has been a challenge. In this issue of Molecular Ecology Resources, Schlundt et al. apply the combination labelling and spectral imaging - fluorescence in situ hybridization to study microbial communities on plastic marine debris. The images demonstrate the colocalization of abundant bacterial groups on plastic marine debris at a relatively high taxonomic and spatial resolution while also visualizing biofouling of eukaryotes, such as diatoms and bryozoans. This modern imaging technology provides new possibilities to address questions regarding the ecology of marine microbes on plastic marine debris and describe more specific impacts of plastic pollution in the marine food webs.