A Novel Method for In Situ RT-PCR Based on Capsules from Centrifuge Tubes, Ideal for Transcripts Detection in Plant Tissues.

In situ RT-PCR presents advantages over other expression analysis methods due to its rapid processing and low-cost equipment. However, this technique is not without its challenges. A protocol based on a capsule made from centrifuge tubes that offers advantages over slides is presented. This capsule protects histological sections from drying out, and its easy assembly reduces time pauses between incubations. In addition, the container size where the sample is deposited allows the addition and withdrawal of the different solutions. The capsule does not need previous sealing after each incubation, and, above all, it is a low-cost and accessible material. A guideline for tissue sectioning using a cryostat that offers advantages over other sectioning methods is also described.

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.

Chromosome analysis of 303 pregnancy losses in Mexico.

BACKGROUND: Chromosomal abnormalities are present in 50 to 60% of miscarriages and in 6 to 19% of stillbirths. Although microarrays are preferred for studying chromosomal abnormalities, many hospitals cannot offer this methodology. OBJECTIVE: To present the results of the cytogenetic analysis of 303 products of conception (POC), which included 184 miscarriages, 49 stillbirths and 17 cases of undefined age. MATERIAL AND METHODS: Karyotyping, fluorescence in situ hybridization, short tandem repeats and microarrays were used, depending on the type of loss and available sample. RESULTS: In 29 POCs we found maternal tissue and were eliminated from the analyses. Informative results were obtained in 250 (91.2 %)/274 cases; the karyotyping success rate was 80.7%; that of single nucleotide polymorphism microarrays, 94.5%; and that of fluorescence in situ hybridization and short tandem repeat, 100%. Cytogenetic abnormalities were observed in 57.6% of miscarriages and in 24.5% of stillbirths; 94% of total anomalies were numerical and 6% were submicroscopic. CONCLUSIONS: Karyotyping with simultaneous short tandem repeat study to rule out contamination of maternal cells is effective for studying miscarriages; in stillbirths, microarrays are recommended.

ANTECEDENTES: Las alteraciones cromosómicas están presentes en 50 a 60 % de los abortos espontáneos y en 6 a 19 % de los mortinatos. Aunque se prefieren los microarreglos para estudiarlos, numerosos hospitales no pueden ofrecerlos. OBJETIVO: Presentar los resultados del estudio citogenético de 303 productos de la concepción (POC), 184 se obtuvieron de abortos espontáneos, 49 fueron mortinatos y en 17 no se identificó la de edad gestacional. MATERIAL Y MÉTODOS: Se empleó cariotipo, hibridación in situ con fluorescencia, secuencias cortas repetidas en tándem y microarreglos, según el tipo de pérdida y la muestra disponible. RESULTADOS: En 29 POC se encontró tejido materno, por lo que fueron eliminados de los análisis. En 250 (91.2 %)/274 casos se obtuvieron resultados informativos; la tasa de éxito del cariotipo fue de 80.7 %; la de los microarreglos de SNP, de 94.5 %; y la de la hibridación fluorescente in situ y la repetición corta en tándem, de 100 %. Se observaron anomalías citogenéticas en 57.6 % de los abortos espontáneos y en 24.5 % de los mortinatos; 94 % de las anomalías fueron numéricas y 6 %, submicroscópicas. CONCLUSIONES: El cariotipo en conjunto con el estudio de secuencias cortas repetidas en tándem para descartar contaminación de células maternas es efectivo para estudiar abortos espontáneos; los microarreglos se recomiendan en los mortinatos.

Comparative Study of Colorimetric In Situ Hybridization and Quantitative Real-Time Polymerase Chain Reaction for Diagnosis of Infection by Leishmania infantum in Dogs in Formalin-Fixed and Paraffin-Embedded Skin.

The zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and dogs are reservoirs for this parasite. For the diagnosis of Leishmania at the species level in dogs in formalin-fixed, paraffin-embedded skin (FFPES) samples, colorimetric in situ hybridization (CISH) and quantitative real-time polymerase chain reaction (qPCR) are options, but their sensitivities are not well established. Therefore, the aim of this study was to determine the sensitivity of these two techniques in FFPES for the diagnosis of the L. infantum infection in dogs using culture as the reference standard. The FFPES of 48 dogs with cutaneous infection by L. infantum confirmed by culture and by multilocus enzyme electrophoresis were examined by CISH and qPCR using specific probes for L. infantum. The sensitivities of qPCR, CISH and their combination were, respectively, 77.0%, 58.0% and 83.3%. The sensitivities of qPCR in dogs with and without clinical signs were, respectively, 74.2% and 82.4%. The sensitivities of CISH in dogs with and without clinical signs were, respectively, 61.3% and 52.9%. The CISH and qPCR showed satisfactory sensitivities for the diagnosis of L. infantum in the FFPES of dogs, even in dogs without clinical signs, and their combination increases the sensitivity for this diagnosis.

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.

Karyotypic stasis and its implications for extensive hybridization events in corallivores species of butterflyfishes (Chaetodontidae).

The butterflyfishes (Chaetodontidae), emblematic inhabitants of coral reef environments, encompass the majority of known coralivorous species and show one of the highest hybridization rates known among vertebrates, making them an important evolutionary model. The vast knowledge about their life history and phylogenetic relationships contrasts with scarce information on their karyotype evolution. Aiming to expand the cytogenetic data of butterflyfishes and evaluate their karyotype evolution in association with evolutionary aspects, we conducted an extensive cytogenetic analysis in 20 species (Heniochus pleurotaenia and 19 Chaetodon spp.) from the Atlantic and Indo-Pacific regions, comparing the karyotype macrostructure and the arrangement of the 18S and 5S rDNA repetitive DNA classes in their chromosomes. The results demonstrate that butterflyfishes underwent a period of karyotypic stasis, as evidenced by their homoploid and structurally identical basal karyotype, which has 2n = 48 acrocentric chromosomes and is shared by 90% of species. Only C. trifascialis (2n = 48; FN = 50) and C. andamanensis (2n = 48; FN = 52) stood out because they both had karyotypes that diverged due to pericentric inversions. The microstructural arrays of 18S rDNA and 5S rDNA sequences were primarily comprised by single and independent loci on homologous chromosomes, indicating that there was little reshuffling among sets of orthologue chromosomes of species. Geographical comparisons revealed similar karyotypes between individuals of C. striatus from the Greater Caribbean and those of the coast of Brazil, corroborating previous data of gene flow through Amazon/Orinoco plume. The conservative chromosomal patterns in the butterflyfishes, likely overcome the limitations related to segregation and pairing of heterospecific complements and reinforce their contribution to the high degree of hybrid viability and introgression in Chaetodon species.

Estudio cromosómico de 303 pérdidas gestacionales en México / Chromosome analysis of 303 pregnancy losses in Mexico

Resumen Antecedentes: Las alteraciones cromosómicas están presentes en 50 a 60 % de los abortos espontáneos y en 6 a 19 % de los mortinatos. Aunque se prefieren los microarreglos para estudiarlos, numerosos hospitales no pueden ofrecerlos. Objetivo: Presentar los resultados del estudio citogenético de 303 productos de la concepción (POC), 184 se obtuvieron de abortos espontáneos, 49 fueron mortinatos y en 17 no se identificó la de edad gestacional. Material y métodos: Se empleó cariotipo, hibridación in situ con fluorescencia, secuencias cortas repetidas en tándem y microarreglos, según el tipo de pérdida y la muestra disponible. Resultados: En 29 POC se encontró tejido materno, por lo que fueron eliminados de los análisis. En 250 (91.2 %)/274 casos se obtuvieron resultados informativos; la tasa de éxito del cariotipo fue de 80.7 %; la de los microarreglos de SNP, de 94.5 %; y la de la hibridación fluorescente in situ y la repetición corta en tándem, de 100 %. Se observaron anomalías citogenéticas en 57.6 % de los abortos espontáneos y en 24.5 % de los mortinatos; 94 % de las anomalías fueron numéricas y 6 %, submicroscópicas. Conclusiones: El cariotipo en conjunto con el estudio de secuencias cortas repetidas en tándem para descartar contaminación de células maternas es efectivo para estudiar abortos espontáneos; los microarreglos se recomiendan en los mortinatos.

Abstract Background: Chromosomal abnormalities are present in 50 to 60 % of miscarriages and in 6 to 19 % of stillbirths. Although microarrays are preferred for studying chromosomal abnormalities, many hospitals cannot offer this methodology. Objective: To present the results of the cytogenetic analysis of 303 products of conception (POC), which included 184 miscarriages, 49 stillbirths and 17 cases of undefined age. Material and methods: Karyotyping, fluorescence in situ hybridization, short tandem repeats and microarrays were used, depending on the type of loss and available sample. Results: In 29 POCs we found maternal tissue and were eliminated from the analyses. Informative results were obtained in 250 (91.2 %)/274 cases; the karyotyping success rate was 80.7 %; that of single nucleotide polymorphism microarrays, 94.5 %; and that of fluorescence in situ hybridization and short tandem repeat, 100 %. Cytogenetic abnormalities were observed in 57.6 % of miscarriages and in 24.5 % of stillbirths; 94 % of total anomalies were numerical and 6 % were submicroscopic. Conclusions: Karyotyping with simultaneous short tandem repeat study to rule out contamination of maternal cells is effective for studying miscarriages; in stillbirths, microarrays are recommended.

Fluorescent In Situ Hybridization for the Detection of Intracellular Bacteria in Companion Animals.

FISH techniques have been applied for the visualization and identification of intracellular bacteria in companion animal species. Most frequently, these techniques have focused on the identification of adhesive-invasive Escherichia coli in gastrointestinal disease, although various other organisms have been identified in inflammatory or neoplastic gastrointestinal disease. Previous studies have investigated a potential role of Helicobacter spp. in inflammatory gastrointestinal and hepatic conditions. Other studies evaluating the role of infectious organisms in hepatopathies have received some attention with mixed results. FISH techniques using both eubacterial and species-specific probes have been applied in inflammatory cardiovascular, urinary, and cutaneous diseases to screen for intracellular bacteria. This review summarizes the results of these studies.

Ranavirus (Frog Virus 3) Infection in Free-Living Three-Toed Box Turtles (Terrapene mexicana triunguis) in Missouri, USA.

Frog virus 3 (FV3) and related ranaviruses are emerging infectious disease threats to ectothermic vertebrate species globally. Although the impact of these viruses on amphibian health is relatively well studied, less is understood about their effects on reptile health. We report two cases of FV3 infection, 11 mo apart, in three-toed box turtles (Terrapene mexicana triunguis) from a wildlife rehabilitation center. Case 1 had upper respiratory signs upon intake but had no clinical signs at the time of euthanasia 1 mo later. Case 2 presented for vehicular trauma, had ulcerative pharyngitis and glossitis, and died overnight. In case 1, we detected FV3 nucleic acid with qPCR in oral swabs, kidney, liver, spleen, and tongue. In case 2, we detected FV3 in an oral swab, an oral plaque, heart, kidney, lung, liver, spleen, and tongue. We also detected FV3 nucleic acid with in situ hybridization for case 2. For both cases, FV3 was isolated in cell culture and identified with DNA sequencing. Histopathologic examination of postmortem tissue from case 1 was unremarkable, whereas acute hemorrhagic pneumonia and splenic necrosis were noted in case 2. The difference in clinical signs between the two cases may have been due to differences in the temporal course of FV3 disease at the time of necropsy. Failure to detect this infection previously in Missouri reptiles may be due to lack of surveillance, although cases may also represent a novel spillover to box turtles in Missouri. Our findings reiterate previous suggestions that the range of FV3 infection may be greater than previously documented and that infection may occur in host species yet to be tested.

Measurable residual disease study through three different methods can anticipate relapse and guide early interventions in childhood acute lymphoblastic leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common cancer among children. Measurable residual disease (MRD, previously named minimal residual disease) study can guide therapy adjustments or preemptive interventions that might avoid hematological relapse. METHODS: Clinical decision making and patient outcome were evaluated in 80 real-life childhood ALL patients, according to the results observed in 544 bone marrow samples analyzed with three MRD methods: multiparametric flow cytometry (MFC), fluorescent in-situ hybridization (FISH) on B or T-purified lymphocytes and patient-specific nested reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Estimated 5 year overall survival and event-free survival were 94% and 84.1%, respectively. A total of 12 relapses in 7 patients were associated with positive MRD detection with at least one of the three methods: MFC (p < 0.00001), FISH (p < 0.00001) and RT-PCR (p = 0.013). MRD assessment allowed the anticipation of relapse and adapted early interventions with different approaches including chemotherapy intensification, blinatumomab, HSCT and targeted therapy to halt relapse in five patients, although two of them relapsed afterwards. CONCLUSION: MFC, FISH and RT-PCR are complementary methods for MRD monitoring in pediatric ALL. Although, our data clearly show that MDR positive detection is associated with relapse, continuation of standard treatment, intensification or other early interventions were able to halt relapse in patients with different risks and genetic background. More sensitive and specific methods are warranted to enhance this approach. However, whether early treatment of MRD can improve overall survival in patients with childhood ALL needs to be evaluated in adequately controlled clinical trials.

Human Papillomavirus-Associated Oral Epithelial Dysplasia: Report of 5 Illustrative Cases from Latin America.

BACKGROUND: Human papillomavirus-associated oral epithelial dysplasia (HPV-OED) is a distinct oral epithelial disorder characterized by viral cytopathic changes caused by transcriptionally active high-risk HPV. The aim of the present study was to report 5 additional cases from Latin America. METHODS: Clinical data from five patients with HPV-OED were obtained from the archives of three oral pathology services from Brazil and Chile. All cases were submitted to morphological, p16 expression and in situ hybridization (ISH) for HPV analyses. RESULTS: Four patients were male and one patient was female, with a mean age of 55.4 years. Four patients were HIV seropositive and two were smokers. Three cases affected the buccal mucosa and commissure, one of which had an additional plaque in the soft palate, and one case each occurred on the floor of mouth and lower labial mucosa. Most cases presented as well-demarcated white plaques with a verrucous surface. One case presented multiple lesions ranging from normal to white-colored slightly elevated plaques with a cobblestone surface. Peripheral mucosal pigmentation was observed in two cases. All five cases presented with the characteristic microscopic features of HPV-OED, including severe dysplasia with numerous karyorrhectic and apoptotic cells, full-thickness "block positivity" for p16 and high Ki-67 index (> 90%) sharply demarcated from the adjacent non-dysplastic epithelium. Wide-spectrum DNA ISH-HPV was positive in 4 cases. All patients were treated with conservative surgical excision with no signs of recurrence after a mean of 39-month follow-up. CONCLUSION: This represents the first series of HPV-OED from Latin America; most cases presented as well-demarcated papillary white plaques affecting the buccal mucosa and commissure of HIV-positive middle-aged men, two of them exhibiting peripheral pigmentation caused by reactive melanocytes. The typical microscopic findings of HPV-OED were observed in all cases, which also showed strong p16 positivity in a continuous band through the full thickness of the epithelium and high Ki67.

Differential Spreading of Microsatellites in Holocentric Chromosomes of Chagas Disease Vectors: Genomic and Evolutionary Implications.

This study focused on analyzing the distribution of microsatellites in holocentric chromosomes of the Triatominae subfamily, insect vectors of Chagas disease. We employed a non-denaturing FISH technique to determine the chromosomal distribution of sixteen microsatellites across twenty-five triatomine species, involving five genera from the two principal tribes: Triatomini and Rhodniini. Three main hybridization patterns were identified: strong signals in specific chromosomal regions, dispersed signals dependent on microsatellite abundance and the absence of signals in certain chromosomal regions or entire chromosomes. Significant variations in hybridization patterns were observed between Rhodniini and Triatomini species. Rhodniini species displayed weak and scattered hybridization signals, indicating a low abundance of microsatellites in their genomes. In contrast, Triatomini species exhibited diverse and abundant hybridization patterns, suggesting that microsatellites are a significant repetitive component in their genomes. One particularly interesting finding was the high abundance of GATA repeats, and to a lesser extent AG repeats, in the Y chromosome of all analyzed Triatomini species. In contrast, the Y chromosome of Rhodniini species did not show enrichment in GATA and AG repeats. This suggests that the richness of GATA repeats on the Y chromosome likely represents an ancestral trait specific to the Triatomini tribe. Furthermore, this information can be used to elucidate the evolutionary relationships between Triatomini and other groups of reduviids, contributing to the understanding of the subfamily's origin. Overall, this study provides a comprehensive understanding of the composition and distribution of microsatellites within Triatominae genomes, shedding light on their significance in the evolutionary processes of these species.

The spread of satellite DNAs in euchromatin and insights into the multiple sex chromosome evolution in Hemiptera revealed by repeatome analysis of the bug Oxycarenus hyalinipennis.

Satellite DNAs (satDNAs) are highly repeated tandem sequences primarily located in heterochromatin, although their occurrence in euchromatin has been reported. Here, our aim was to advance the understanding of satDNA and multiple sex chromosome evolution in heteropterans. We combined cytogenetic and genomic approaches to study, for the first time, the satDNA composition of the genome in an Oxycarenidae bug, Oxycarenus hyalinipennis. The species exhibits a male karyotype of 2n = 19 (14A + 2 m + X1 X2 Y), with a highly differentiated Y chromosome, as demonstrated by C-banding and comparative genomic hybridization, revealing an enrichment of repeats from the male genome. Additionally, comparative analysis between males and females revealed that the 26 identified satDNA families are significantly biased towards male genome, accumulating in discrete regions in the Y chromosome. Exceptionally, the OhyaSat04-125 family was found to be distributed virtually throughout the entire extension of the Y chromosome. This suggests an important role of satDNA in Y chromosome differentiation, in comparison of other repeats, which collectively shows similar abundance between sexes, about 50%. Furthermore, chromosomal mapping of all satDNA families revealed an unexpected high spread in euchromatic regions, covering the entire extension, irrespective of their abundance. Only discrete regions of heterochromatin on the Y chromosome and of the m-chromosomes (peculiar chromosomes commonly observed in heteropterans) were enriched with satDNAs. The putative causes of the intense enrichment of satDNAs in euchromatin are discussed, including the possible existence of burst cycles similar to transposable elements and as a result of holocentricity. These data challenge the classical notion that euchromatin is not enriched with satDNAs.

Supernumerary B Chromosomes of Tetragonisca fiebrigi Share Repeat Content with Standard Chromosome Set of both T. fiebrigi and Tetragonisca angustula (Apidae: Meliponini).

The stingless bees Tetragonisca angustula and Tetragonisca fiebrigi are widely distributed in Brazil, and both are commonly known as "jataí." Our goal was to investigate the possible origin of the B chromosomes in T. fiebrigi, a cytotaxonomic trait that differentiates T. fiebrigi from T. angustula. We analyzed diploid chromosome number (2n), B chromosome incidence, patterns of constitutive heterochromatin, and in situ localization of different repetitive DNA probes in T. angustula and T. fiebrigi. Both species displayed 2n = 34, with similar karyotype structures. One to three B chromosomes were observed in T. fiebrigi only. Constitutive heterochromatin was distributed on one arm of all chromosomes in both species, and T. fiebrigi B chromosomes were mainly heterochromatic with one euchromatic extremity. The (GA)15 and (CAA)10 microsatellite probes marked the euchromatic arms of all chromosomes in both species without marking the B chromosomes. The 18S ribosomal DNA (rDNA) probe marked 10 chromosomes in T. angustula and 6 A chromosomes in T. fiebrigi with an additional marking on 1B in individuals with 3B. The Tan-Bsp68I repetitive DNA probe marked the heterochromatic portion of all T. fiebrigi A and B chromosomes. This probe also marked the heterochromatic portion of all T. angustula chromosomes; therefore, both alternative hypotheses to the B chromosome origin are possible: (i) from the A chromosome complement of T. fiebrigi (intraspecific origin); or (ii) a by-product of genome reshuffling following the hybridization between T. fiebrigi and T. angustula (interspecific origin).

Annotation of transposable elements in the transcriptome of the Neotropical brown stink bug Euschistus heros and its chromosomal distribution.

Transposable elements (TEs) are DNA sequences capable of moving within the genome. Their distribution is very dynamic among organisms, and despite advances, there are still gaps in the understanding of the diversity and evolution of TEs in many insect species. In the case of Euschistus heros, considered the main stink bug in the soybean crop in Brazil, little is known about the participation of these elements. Therefore, the objective of the current work was to identify the different groups of transposable elements present in the E. heros transcriptome, evidencing their chromosomal distribution. Through RNA-Seq and de novo assembly, 60,009 transcripts were obtained, which were annotated locally via Blastn against specific databases. Of the 367 transcripts identified as TEs, 202 belong to Class II, with emphasis on the TIR order. Among Class I elements or retrotransposons, most were characterized as LINE. Phylogenetic analyses were performed with the protein domains, evidencing differences between Tc1-mariner sequences, which may be related to possible horizontal transfer events. The transposable elements that stood out in the transcriptome were selected for fluorescent in situ hybridization. DNA transposon probes hAT, Helitron, and Tc1-mariner showed mostly scattered signals, with the presence of some blocks. Retrotransposon probes Copia, Gypsy, Jockey, and RTE showed a more pulverized hybridization pattern, with the presence of small interstitial and/or terminal blocks. Studies like this one, integrating functional genomics and molecular cytogenetic tools, are essential to expanding knowledge about transcriptionally active mobile elements, and their behavior in the chromosomes.

A novel approach for direct detection of the IGH::CRLF2 gene fusion by fluorescent in situ hybridization.

BACKGROUND: High expression of the Cytokine Receptor-Like Factor 2 (CRLF2) gene has been observed in patients with acute lymphoblastic leukemia BCR-ABL1-like subtype. Currently, there is no commercial system available for the direct detection of the IGH::CRLF2 fusion by fluorescent in situ hybridization (FISH), as there are for many other leukemia-related gene fusions. In an effort to verify the IGH::CRLF2 fusion, some researchers prepare home-grown FISH probes from bacterial artificial chromosome clones flanking the IGH and CRLF2 genes, which is the best alternative to confirm the fusion, however difficult to reproduce in most cytogenetic laboratories. RESULTS: For the direct observation of the IGH::CRLF2 gene fusion we designed a methodological approach requiring the two commercially available IGH and CRLF2 break-apart probes. CONCLUSIONS: Our methodological approach allows direct visualization of the IGH::CRLF2 gene fusion and has the potential to be used for identification of other gene fusions.

Xp22.33p22.13 Duplication in a Male Patient Carrying a Recombinant X Chromosome Derived from an Inherited Intrachromosomal Insertion.

Intrachromosomal insertions are complex structural rearrangements that are challenging to interpret using classical cytogenetic methods. We report a male patient carrying a recombinant X chromosome derived from a maternally inherited intrachromosomal insertion. The patient exhibited developmental delay, intellectual disability, behavioral disorder, and dysmorphic facial features. To accurately identify the rearrangements in the abnormal X chromosome, additional cytogenetic studies were conducted, including fluorescence in situ hybridization (FISH), multicolor-banding FISH, and array comparative genomic hybridization. The results showed a recombinant X chromosome, resulting in a 13.05 Mb interstitial duplication of segment Xp22.33-Xp22.13, which was inserted at cytoband Xq26.1. The duplicated region encompasses 99 genes, some of which are associated with the patient's clinical manifestations. We propose that the combined effects of the Xp-duplicated genes may contribute to the patient's phenotype.

Abundance of distal repetitive DNA sequences in Capsicum L. (Solanaceae) chromosomes.

Chili peppers (Solanaceae family) have great commercial value. They are commercialized in natura and used as spices and for ornamental and medicinal purposes. Although three whole genomes have been published, limited information about satellite DNA sequences, their composition, and genomic distribution has been provided. Here, we exploited the noncoding repetitive fraction, represented by satellite sequences, that tends to accumulate in blocks along chromosomes, especially near the chromosome ends of peppers. Two satellite DNA sequences were identified (CDR-1 and CDR-2), characterized and mapped in silico in three Capsicum genomes (C. annuum, C. chinense, and C. baccatum) using data from the published high-coverage sequencing and repeats finding bioinformatic tools. Localization using FISH in the chromosomes of these species and in two others (C. frutescens and C. chacoense), totaling five species, showed signals adjacent to the rDNA sites. A sequence comparison with existing Solanaceae repeats showed that CDR-1 and CDR-2 have different origins but without homology to rDNA sequences. Satellites occupied subterminal chromosomal regions, sometimes collocated with or adjacent to 35S rDNA sequences. Our results expand knowledge about the diversity of subterminal regions of Capsicum chromosomes, showing different amounts and distributions within and between karyotypes. In addition, these sequences may be useful for future phylogenetic studies.

Carcinoma tipo-linfoepitelioma pulmonar con expresión de virus de Epstein-Barr y PD-L1 / Pulmonary lymphoepithelioma-like carcinoma with ex pression of Epstein-Barr virus and PD-L1

Resumen El carcinoma tipo-linfoepitelioma pulmonar es una variante rara de carcinoma de células no pequeñas de pulmón, representa aproximadamente 0.7% de todos los casos. Está usualmente asociado con la infección por el virus de Epstein-Barr y es más prevalente en el Sureste de Asia; sin embargo, es extremadamente raro en Améri ca Latina. Informamos el caso de un hombre de 65 años de edad con un carcinoma tipo-linfoepitelioma pulmo nar, que se presentó con tos, disnea y pérdida de peso. La TAC de tórax mostró nódulo mal definido localizado en el pulmón derecho. Se realizó biopsia transtorácica de la lesión, y el estudio microscópico reveló células gran des poligonales dispuestas en mantos, infiltrados por abundantes linfocitos y células plasmáticas, alrededor del intersticio. Las células neoplásicas fueron positivas para citoqueratina 5/6 y p63, y negativas para Napsina A y el factor de transcripción tiroideo 1 (TTF-1). La expre sión de PD-L1 fue positivo (aproximadamente 100%) por inmunohistoquímica; así como el núcleo de las células neoplásicas mediante hibridación in situ para el RNA codificado por el virus de Epstein-Barr (EBER-ISH). El paciente recibió seis ciclos de un esquema combinado de quimioterapia basado en platino (gencitabina/cisplatino) más durvalumab. Presentó progresión de la enfermedad y finalmente murió 9 meses después del diagnóstico.

Abstract Pulmonary lymphoepithelioma-like carcinoma is a rare type of non-small cell lung cancer, it accounts for approximately 0.7% of all cases. It is usually associated with Epstein-Barr virus infection and is more prevalent in Southeast Asia; however, it is extremely rare in Latin America. We present a 65-year-old man with a primary pulmonary lymphoepithelioma-like carcinoma, who presented with cough, dyspnoea and weight loss. Com puter tomographic scan of the thorax showed a nodule localized in the right lung. A transthoracic biopsy of the lung lesion was made and the microscopic obser vation revealed large polygonal cells that proliferated in a nest pattern with infiltration by lymphocytes and plasma cells around the interstitium. The tumour cells were positive for citokeratin 5/6 and p63, and negative for Napsin A and thyroid transcription factor 1 (TTF-1). PD-L1 expression was positive (approximately 100%) in the immunohistochemical study, and the nuclei of the tumour cells were positive for EBV-encoded small RNA in-situ hybridization (EBER-ISH). The patient underwent six cycles of platinum-based combination (gencitabine/ carboplatin) chemotherapy plus durvalumab. He pre sented progression of the disease and finally he died 9 months after diagnosis.