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Bone is a three‐dimensional (3D) highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or provide only two‐dimensional (2D) spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH‐BONE, to label osteocyte messenger RNA (mRNA) and protein in whole‐mount mouse bone. For mRNA labeling, we used hybridization chain reaction‐fluorescence in situ hybridization (HCR‐FISH) to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy‐based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone (CB). However, the relatively dim signal observed from nanobodies' staining limited detection. mRNA and protein labeling were performed in separate samples. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole‐mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH‐BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone‐related fields of research. [ABSTRACT FROM AUTHOR]

Pre-implantation genetic testing (PGT) is a crucial process for selecting embryos created through assisted reproductive technology (ART). Couples with chromosomal rearrangements, infertility, recurrent miscarriages, advanced maternal age, known single-gene disorders, a family history of genetic conditions, previously affected pregnancies, poor embryo quality, or congenital anomalies may be candidates for PGT. Preimplantation genetic testing for aneuploidies (PGT-A) enables the selection and transfer of euploid embryos, significantly enhancing implantation rates in assisted reproduction. Fluorescence in situ hybridization (FISH) is the preferred method for analyzing biopsied cells to identify these abnormalities. While FISH is a well-established method for identifying sperm aneuploidy, NGS offers a more comprehensive assessment of genetic material, potentially enhancing our understanding of male infertility. Chromosomal abnormalities, arising during meiosis, can lead to aneuploid sperm, which may hinder embryo implantation and increase miscarriage rates. This review provides a comparative analysis of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) in sperm evaluations, focusing on their implications for preimplantation genetic testing. This analysis explores the strengths and limitations of FISH and NGS, aiming to elucidate their roles in improving ART outcomes and reducing the risk of genetic disorders in offspring. Ultimately, the findings will inform best practices in sperm evaluations and preimplantation genetic testing strategies. [ABSTRACT FROM AUTHOR]

Small supernumerary marker chromosomes (sSMCs) are structurally abnormal chromosomes that cannot be clearly identified or characterized by traditional karyotyping. They can appear in a variety of forms, including rings, centric fragments, and other structural abnormalities, and are often detected during prenatal diagnosis. sSMCs occur in approximately 0.075 % of unselected prenatal cases and can be associated with a wide range of phenotypic presentations, from normal development to severe congenital malformations and syndromes. Understanding and interpreting the clinical significance of sSMCs remains challenging in genetic counseling due to their high heterogeneity and potential impact on fetal phenotype. Advances in the field of molecular cytogenetics, including techniques such as fluorescence in situ hybridization (FISH), as well as microarray comparative genomic hybridization (aCGH) and next-generation sequencing (NGS), have significantly improved the ability to accurately characterize sSMCs. These methods can determine their origin, size and genetic content, which is critical for risk assessment and decision-making during pregnancy. Thus, modern molecular cytogenetics techniques play a key role in the identification and characterization of sSMC, allowing for more accurate genetic counseling and helping to make informed pregnancy decisions. However, despite technological advances, sSMCs continue to pose a challenge for prenatal diagnosis due to their complexity and potential impact on fetal development. In this article, we aim to provide a general overview of sSMCs and of their impact on prenatal diagnosis, as well as consider the clinical significance and potential impact of sSMCs on pregnancy outcome.

Several reports of concurrent MYC , BCL2 , BCL6 , and CCND1 rearrangements in high-grade B-cell lymphoma (HGBL) have been recently described. Herein, we aimed to delineate the scope of this entity through a review of HGBL with a "quadruple-hit" genetic profile identified at our institution. We performed a retrospective review (2015-2023) at our institution of B-cell lymphoma (BCL) cases that were evaluated with concurrent MYC , BCL2 , and BCL6 break-apart and IGH::MYC and IGH::CCND1 dual-color dual-fusion fluorescence in situ hybridization studies. Of 203 cases meeting inclusion criteria, 2 (1%) with a quadruple-hit genetic profile were identified. Case 1 represented a 59-year-old female with widespread lymphadenopathy and a diagnosis of HGBL who exhibited primary refractoriness to dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R) chemotherapy. Case 2 represented a 58-year-old male with mediastinal and abdominal lymphadenopathy and a diagnosis of large BCL who died from disease after 1 cycle of DA-EPOCH-R chemotherapy. Similarly, a literature review of 7 previously reported cases of HGBL with a quadruple-hit profile also demonstrated aggressive disease behavior. Our study adds 2 new cases to the rarely encountered quadruple-hit HGBL, and a brief meta-analysis of the 9 available cases indicates aggressive disease behavior conferred by this constellation of genetic events. [ABSTRACT FROM AUTHOR]

The vertical stratification of oxygen concentration in deep reservoirs impacts nutrient cycling and ecosystem biodiversity. The Serra da Mesa reservoir, the largest in Brazil, was studied to evaluate the structure and production of the prokaryote community at five depths. Using 3H leucine incorporation and fluorescent in situ hybridization (FISH), the study focused on different depths near the dam, particularly within the euphotic zone. The water column was characterized into oxic, transitional, and hypoxic layers based on dissolved oxygen concentration. The highest densities and biomasses of prokaryotes were found at the euphotic zone's depth limit, where bacterial production was low, suggesting inactive or slow-growing bacteria. Cell size differences and filamentous bacteria presence near the surface were observed, likely due to varying predation pressures. Prokaryote community composition differed across depths. At the subsurface level, with high dissolved organic carbon, alphaproteobacteria, betaproteobacteria, and Cytophaga–Flavobacter had similar densities, but the lowest bacterial biomass was recorded. The highest dissolved oxygen concentration depth had the lowest bacterial density, dominated by alphaproteobacteria and gammaproteobacteria. The study revealed that prokaryotic community structure and production vary with depth, indicating that microbial participation in layer dynamics is differentiated, with variations in abundance and distribution linked to oxygen concentrations. [ABSTRACT FROM AUTHOR]

Introduction: The mapping of the satellite DNA on chromosomes is vital to understanding the distribution and evolution of repetitions in the genome since these chromosomal studies have shown the origin, evolutionary mode, and function of repetitive sequences. This study aimed to prospect the satellitome and determine its location in the genome of two cryptic species of Hypostomus, H. aff. ancistroides and H. ancistroides, with and without XX/XY sexual chromosome system. Methods: Mitotic chromosomes and DNA extraction were obtained according to protocols. After the whole genome sequencing, the satDNAs were retrieved, amplified, and hybridized in chromosome preparations for male and female individuals. Results: We found 30 satellite families (47 variants, two superfamilies) in H. ancistroides and 38 satellite families (45 variants, four superfamilies) in H. aff. ancistroides. The sequences varied from 14 bp to 2,662 bp in H. ancistroides and from 14 bp to 2,918 bp in H. aff. ancistroides. We did not observe any tandem repeats that were exclusive to each of the libraries; however, many sequences showed very different abundances and copy numbers between the libraries. Four satDNAs did not hybridize on the chromosomes of either species. Conversely, one satDNA hybridized in both species, HxySat1-80. However, the phenotypes found varied among species, populations, and in the same individual. There was no sign of HanSat3-464 and HanSat11-335 in any individuals of H. aff. ancistroides, but markings were in the chromosomes of H. ancistroides. HxySat12-1127 and HxySat8-52, on the other hand, were only hybridized in H. aff. ancistroides, while H. ancistroides had a negative sign. No hybridization of satDNAs was found in the X and Y sex chromosomes as they were mostly composed of euchromatin. Conclusion: We distinguish H. aff. ancistroides as genetically different from H. ancistroides, recognizing that such characteristics go far beyond morphological, karyotypic, and molecular data. Our data support the differential abundance and location of satellite DNAs and confirm that many organisms, including fish, have repetitive sequences that validate the library hypothesis. All found and validated satDNAs and the characterization of the satellitomes of the two species represent important contributions to cytogenomic studies of the genus Hypostomus. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract The real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) tests are the gold standard in detecting SARS-CoV-2 virus infection. However, despite high sensitivity and specificity, they have limitations that in some cases may result in false negative results. Therefore, it is reasonable to search for additional tools that could support microbiological diagnosis of SARS-CoV-2. The aim of the study was to develop a highly specific molecular test capable of detecting and visualizing SARS-CoV-2 infection. A universal probe and a set of 18 specific oligonucleotides with a FLAP sequence attached to them on both sides were designed to visualize SARS-CoV-2 virus infection based on the fluorescence in situ hybridization method (FISH). FISH conditions using the developed kit were standardized on the Vero CCL-81 cell line infected by SARS-CoV-2 virus. The method was tested on 290 nasopharyngeal swabs (collected in a doublet) from patients with clinical symptoms of SARS-CoV-2. Each one swab from the doublet was subjected to RNA isolation and amplification by rRT-PCR. From the second swab, a microscopic preparation was performed for FISH. The use of the rRT-PCR allowed obtaining 200 positive and 90 negative results, while our FISH method allowed for 220 positive results and 70 negative results. The differences obtained using both methods were statistically significant (p = 0.008). The obtained results support the use of FISH as an additional method in microbiological diagnostics of SARS-CoV-2.

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Identifying ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. Current methods use ALK IHC, ALK FISH, or NGS, but face challenges due to low fusion frequency, possible tissue exhaustion, and test limitations. We compared RNA NGS with ALK IHC and ALK FISH in 1246 NSCLC cases, finding RNA NGS faster and equally effective. This suggests the need to replace systematic ALK IHC with RNA NGS reflex testing for more efficient assessments of ALK status. The identification of ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. The current diagnostic approach employs an algorithm using ALK immunohistochemistry (IHC) screening, followed by confirmation through ALK FISH and/or next-generation sequencing (NGS). Challenges arise due to the infrequency of ALK fusions (3–7% of aNSCLC), the suboptimal specificity of ALK IHC and ALK FISH, and the growing molecular demands placed on small tissue samples, leading to interpretative, tissue availability, and time-related issues. This study investigates the effectiveness of RNA NGS as a reflex test for identifying ALK fusions in NSCLC, with the goal of replacing ALK IHC in the systematic screening process. The evaluation included 1246 NSCLC cases using paired techniques: ALK IHC, ALK FISH, and ALK NGS. ALK IHC identified 51 positive cases (4%), while RNA NGS detected ALK alterations in 59 cases (4.8%). Of the 59 ALK-positive cases identified via NGS, 53 (89.8%) were confirmed to be positive. This included 51 cases detected via both FISH and IHC, and 2 cases detected only via FISH, as they were completely negative according to IHC. The combined reporting time for ALK IHC and ALK FISH averaged 13 days, whereas ALK IHC and RNA NGS reports were obtained in an average of 4 days. These results emphasize the advantage of replacing systematic ALK IHC screening with RNA NGS reflex testing for a more comprehensive and accurate assessment of ALK status. [ABSTRACT FROM AUTHOR]

Background: The synaptonemal complex (SC) is a protein axis formed along chromosomes during meiotic prophase to ensure proper pairing and crossing over. SC analysis has been widely used to study the chromosomes of mammals and less frequently of birds, reptiles, and fish. It is a promising method to investigate the evolution of fish genomes and chromosomes as a part of complex approach. Summary: Compared with conventional metaphase chromosomes, pachytene chromosomes are less condensed and exhibit pairing between homologous chromosomes. These features of SCs facilitate the study of the small chromosomes that are typical in fish. Moreover, it allows the study of heteromorphisms in sex chromosomes and supernumerary chromosomes. In addition, it enables the investigation of the pairing between orthologous chromosomes in hybrids, which is crucial for uncovering the causes of hybrid sterility and asexual reproduction, such as gynogenesis or hybridogenesis. However, the application of SC analysis to fish chromosomes is limited by the associated complications. First, in most fish, meiosis does not occur during every season and life stage. Second, different SC preparation methods are optimal for different fish species. Third, commercial antibodies targeting meiotic proteins have been primarily developed against mammalian antigens, and not all of them are suitable for fish chromosomes. Key Messages: In the present review, we provide an overview of the methods for preparing fish SCs and highlight important studies using SC analysis in fish. This study will be valuable for planning and designing research that applies SC analysis to fish cytogenetics and genomics. [ABSTRACT FROM AUTHOR]

Introduction: The tribes Cophomantini, Scinaxini, and Dendropsophini are anurans that belong to Hylidae, with wide distribution in tropical and subtropical regions around the world. The taxonomy and systematics of this family remain in a state of ongoing revision. Previous cytogenetic analyses of genera Boana, Bokermannohyla, Ololygon, Scinax, and Dendropsophus described some karyotypic characters such as conventional staining, C-banding and NORs, and FISH with specific probes. Methods: This study describes for the first time the karyotypes of four species: Bokermannohyla ibitipoca, Ololygon luizotavioi, Dendropsophus bipunctatus, and Dendropsophus ruschii. Furthermore, we map CA(15) and CAT(10) microsatellite sites for the aforementioned species and six more species from the same genera for insight into the chromosomal evolution within the subfamily Hyalinae. Results:B. ibitipoca and O. luizotavioi had 2n = 24 and karyotypic formulas 18m + 4sm + 2st and 8m + 12sm + 4st, while D. bipunctatus and D. ruschii showed 2n = 30 and karyotypic formulas 12m + 12sm + 4st + 2t and 10m + 10sm + 6st + 4t, respectively. The diploid numbers and karyotypic formulas revealed here follow the previously reported trend for Hylidae, except B. ibitipoca has a particularity of eight metacentric chromosomes, more than what is commonly found in species of this genus. The microsatellites probes CA(15) and CAT(10) had markings accumulated in blocks in the centromeric, pericentromeric, and terminal regions that were more specific for some species, as well as markings scattered along the chromosomes. We present a comprehensive review table of current data on cytogenetics of these genera. Conclusion: Our findings showed that the karyotypes of the hylids studied here majority fit the postulated conserved diploid number (2n = 24) and morphological chromosome patterns, while the mapping of the microsatellites enabled us to detect differences between species that share similar chromosomal morphologies. [ABSTRACT FROM AUTHOR]

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Human sperm parameters serve as a first step in diagnosing male infertility, but not in determining the potential for successful pregnancy during assisted reproductive technologies (ARTs) procedures. Here, we investigated the relationship between sperm head morphology at high magnification, based on strict morphologic criteria, and the nuclear architecture analyzed by fluorescence in situ hybridization (FISH). We included five men. Two of them had an elevated high-magnification morphology score of 6 points (Score 6) indicating high fertility potential, whereas three had a low score of 0 points (Score 0), indicating low fertility potential. We used FISH to study the inter-telomeric distance and the chromosomal territory area of chromosome 1 (Chr. 1). We then compared these two parameters between subjects with high and low scores. FISH data analysis showed that the inter-telomeric distance (ITD) and chromosomal territory area (CTA) of Chr. 1 were significantly higher in subjects with low scores (score 0) than high scores (score 6). Our results suggest that (i) there is a link between nuclear architecture and sperm head abnormalities, particularly vacuoles; and (ii) it is possible to select spermatozoa with normal nuclear architecture, which might indirectly explain the positive ART outcomes observed with this technique. [ABSTRACT FROM AUTHOR]

Malignancy after augmentation cystoplasty (AC) is reported up to 5.5 %. We assessed the use of urine fluorescence in situ hybridization (FISH) screening for bladder malignancy after AC. In this study, 36/98 patients under follow-up who have completed tenth year after ileal AC were included prospectively. Twenty-four (66.7 %) patients were tested with FISH initially and overall 28 (77.8 %) patients with conventional cytology (CC). Twenty-four (66.7 %) patients with FISH analysis also had cytology analysis. Blinded from the cytology results, 32 (88.9 %) patients who were consented underwent cystoscopy with random biopsy (native bladder, ileal segment, ileovesical junction). Two patients those were tested with FISH did not consented cystoscopy. This study was registred to the government registry (No: 71146310). Mean follow-up time after AC was 15.4 ± 4.8 years. 2/32 (5.6 %) patients were diagnosed with adenocarcinoma in cyctoscopic biopsy. FISH analysis of 3/24 (12.5 %) patients demonstrated abnormal findings consistent with malignancy. Two FISH malignant patients were patients who had adenocarcinoma. The third patient's biopsy was benign and the third year control cystoscopy was normal. 2/4 patients with malignant CC had adenocarcinoma and 2/4 patients had benign biopsy. The sensitivity and specificity of FISH in our series were 100 % and 95 % respectively. Whereas the sensitivity and specificity of CC was 100 % and 91.6 % respectively. Despite limited number of patients in this study, FISH showed higher specificity than CC in this series. FISH is a promising tool for malignancy screening after AC. Diagnostic Studies. II. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacterial communities are highly complex, with interaction networks dictating ecosystem function. Bacterial interactions are constrained by the spatial organization of these microbial communities, yet studying the spatial organization of microbial communities at the single-cell level has been technically challenging. Here, we use the recently developed high-phylogenetic-resolution microbiota mapping by fluorescence in situ hybridization technology to image the gut microbiota at the species and single-cell level. We simultaneously image 63 different bacterial species to spatially characterize the perturbation and recovery of the gut microbiota to ampicillin and vancomycin in the cecum and distal colon of mice. To decipher the biology in this complex imaging data, we developed an analytical framework to characterize the spatial changes of the gut microbiota to a perturbation. The three-tiered analytical approach includes image-level diversity, pairwise colocalization analysis, and hypothesis-driven neighborhood analysis. Through this workflow, we identify biogeographic and antibiotic-based differences in the spatial organization of the gut microbiota. We demonstrate that the cecal microbiota has increased micrometer-scale diversity than the colon at baseline and recovers better from perturbation. Also, we identify potential foundation and keystone species that have high baseline neighborhood richness and that are associated with recovery from antibiotics. Through this workflow, we add a spatial layer to the characterization of bacterial communities and progress toward a better understanding of bacterial interactions leading to improved microbiome modulation strategies.IMPORTANCEAntibiotics have broad off-target effects on the gut microbiome. When the microbial community is unable to recover from antibiotics, it can lead to increased susceptibility to gastrointestinal infections and increased risk of immunological and metabolic diseases. In this study, we work to better understand how the gut microbiota recovers from antibiotics by employing a recent technology to image the entire bacterial community at once. Through this approach, we characterize the spatial changes in the gut microbiota after treatment with model antibiotics in both the cecum and colon of mice. We find antibiotic- and biogeographic-dependent spatial changes between bacterial species and that many of these spatial colocalizations do not recover to baseline levels even 35 days after antibiotic administration.

Objectives: To investigate the role of oral microbiome in promoting oral squamous cell carcinoma (OSCC) development. Materials and Methods: We investigated the salivary microbiome of 108 controls and 70 OSCC cases by16S rRNA gene sequencing and detected the fluorescence signal of OSCC‐related pathological bacteria by fluorescence in situ hybridization assay (FISH). The invasion and migration assays were used to show the differences of invasive and migrative abilities between control and experimental groups. Quantitative real‐time PCR and Western blotting were used to verify the epithelial‐to‐mesenchymal transition (EMT). Results: In our study, the overall microbiome abundance and composition were richer in the 108 controls than in the 70 OSCC cases. We demonstrated that Streptococcus, Capnocytophaga, Peptostreptococcus, and Lactobacillus were highly abundant in the saliva of OSCC patients by 16S rDNA sequencing and FISH. Moreover, we found that Capnocytophaga gingivalis (C. gingivalis) was highly presented in OSCC tissues by FISH. We focused on C. gingivalis and found that its supernatant induced OSCC cells to undergo EMT, causing the cells to acquire a mesenchymal phenotype associated with highly invasive and metastatic properties. Conclusion: Taken together, these results indicated that C. gingivalis might invade OSCC tissues and played an important role in OSCC by promoting OSCC invasion and metastasis by inducing EMT. Hence, the role of C. gingivalis in cancer progression revealed a new direction for the research of OSCC. [ABSTRACT FROM AUTHOR]

Objectives Myeloid/lymphoid neoplasms with FGFR1 rearrangement are a rare group of neoplasms that share features of eosinophilia and lineage promiscuity. First, we described a challenging case of acute leukemia with lineage switch and cytogenetically cryptic FGFR1. Second, we aimed to systemically review this phenomenon in published literature. Methods A 68-year-old man with a history of chemotherapy exposure presented with acute leukemia of myeloid lineage without eosinophilia or 8p11 abnormalities on karyotyping. Over a refractory and relapsing course, the blast phenotype shifted to B lymphoid. Results Fluorescence in situ hybridization identified a cytogenetically cryptic FGFR1 rearrangement, likely a paracentric inversion. We identified 26 published cases of FGFR1 -rearranged acute leukemia with ambiguous, mixed, or switching lineage. Although there was variability in the partner gene, anatomical location of different phenotypes, and timing of lineage switch, the prognosis was consistently poor in the absence of novel therapy. Conclusions Ours is the only reported case of FGFR1 -rearranged neoplasms with a disease sequence of acute myeloid leukemia transforming to B-cell acute lymphoblastic leukemia and 1 of only 3 reported cases with cytogenetically cryptic FGFR1 rearrangement. Fluorescence in situ hybridization testing for FGFR1 rearrangement should be a standard investigation in leukemia of mixed or switching lineage. [ABSTRACT FROM AUTHOR]

Objectives To evaluate the diagnostic efficacy of a DNA methylation test, compare that test with cytology alone, fluorescence in situ hybridization (FISH) alone, and cytology plus FISH, and explore reasons that may influence the accuracy of liquid biopsy. Methods We included 37 patients and 12 negative control individuals between April 2019 and May 2022. All patients had undergone radical nephroureterectomy, nephrectomy, diagnostic ureteroscopy, or tissue biopsy. Urine samples were collected for DNA methylation testing, cytology, and FISH. Test performance was calculated, and receiver operating characteristic curves were drawn for comparison. Results Median patient age was 66 years, and κ = 0.576 (P <.001) for the DNA methylation test and tissue pathology. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the DNA methylation test were 76%, 100%, 100%, and 74%, respectively, compared with 31%, 100%, 100%, and 50%, respectively, for cytology. The sensitivity, specificity, PPV, and NPV of cytology plus FISH were 66%, 100%, 100%, and 67%, respectively. The area under the curve (AUC) of the DNA methylation test was 0.879 (P <.001), and the AUC of cytology plus FISH was 0.828 (P <.001). Conclusions The test performance of DNA methylation was satisfactory. The DNA methylation test for the detection of upper tract urothelial carcinoma demonstrated better sensitivity than did cytology alone or cytology with FISH, but the accuracy of the combined tests was still acceptable. Further prospective studies with larger samples are needed to confirm the clinical value of this promising method. [ABSTRACT FROM AUTHOR]

Background: Chronic lymphocytic leukemia (CLL) is a B lymphocyte malignancy that affect mainly adults, its clinical course is variable. 13q deletion is a common cytogenetic abnormality found in CLL patients and in general, it is associated with a good prognosis and a benign clinical course. However, it was noticed that there are clinical heterogeneity among CLL patients harboring 13q deletion which may be attributed to type, size, or rate of 13q deletion. So, it was important to clarify the source of this heterogeneity. Methods: Seventy one CLL patients were examined by conventional cytogenetic and fluorescent in situ hybridization (FISH) for 13q14 deletion. Results: Deletion of 13q14 was detected in 45/71 CLL patients (63.4%) and those had significantly lower absolute lymphocytic count and hemoglobin concentration, 53.3% of positive patients were in Binet stage A, and 31.1% of them required chemotherapy. In 38 patients 13q14 deletion was found as an isolated abnormality and those had significantly lower WBCs count, absolute lymphocyte count and LDH than those with accompanied cytogentic abnormalities (non-isolated). The deletion was found at a rate (≥80%) in (8/ 38) patients with isolated deletion type and those had a significantly shorter time to treatment. The 13q14 deletion was biallelic in only 3 patients out of 38 with isolated type and all of them had a diffuse bone marrow infiltration pattern. Conclusions: 13q14 deletion is a common cytogenetic abnormality in CLL patients and has a variable impact on the disease condition according to specific features of deletion such as isolated or nonisolated, deletion rate, and type of deletion being monoallelic or biallelic. [ABSTRACT FROM AUTHOR]

Objectives Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting. Methods The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed. Results Fluorescence-activated cell sorting–FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P <.001). It improved the detection of IGH::CCND1 (P <.001), IGH::MAF (P <.001), IGH::MAFB (P <.001), other IGH rearrangements (P <.001), and gains/amplifications of 1q (P <.001), whereas the detection rates of IGH::FGFR3 fusions (P =.3), loss of 17p (P =.3), and other abnormalities, including hyperdiploidy (P =.5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P <.001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs. Conclusions Fluorescence-activated cell sorting–FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH. [ABSTRACT FROM AUTHOR]

Simple Summary: Bacterial infections have traditionally been identified by culture; however, with advances in the understanding of the role of bacteria in disease, it is recognized that intracellular bacteria can be visualized within cells. Fluorescent in situ hybridization (FISH) is a technique used to not only identify intracellular organisms but also allows researchers to see them, in situ. In this review, we discuss the use of FISH for the identification of intracellular bacteria in companion animals. Most studies have focused on the identification of Escherichia coli, particularly within the gastrointestinal tract. Additionally, bacterial associations with inflammatory diseases affecting the liver, kidney, skin, and cardiovascular systems have been investigated with mixed results. 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. [ABSTRACT FROM AUTHOR]

The vertical stratification of oxygen concentration in deep reservoirs impacts nutrient cycling and ecosystem biodiversity. The Serra da Mesa reservoir, the largest in Brazil, was studied to evaluate the structure and production of the prokaryote community at five depths. Using 3H leucine incorporation and fluorescent in situ hybridization (FISH), the study focused on different depths near the dam, particularly within the euphotic zone. The water column was characterized into oxic, transitional, and hypoxic layers based on dissolved oxygen concentration. The highest densities and biomasses of prokaryotes were found at the euphotic zone’s depth limit, where bacterial production was low, suggesting inactive or slow-growing bacteria. Cell size differences and filamentous bacteria presence near the surface were observed, likely due to varying predation pressures. Prokaryote community composition differed across depths. At the subsurface level, with high dissolved organic carbon, alphaproteobacteria, betaproteobacteria, and Cytophaga–Flavobacter had similar densities, but the lowest bacterial biomass was recorded. The highest dissolved oxygen concentration depth had the lowest bacterial density, dominated by alphaproteobacteria and gammaproteobacteria. The study revealed that prokaryotic community structure and production vary with depth, indicating that microbial participation in layer dynamics is differentiated, with variations in abundance and distribution linked to oxygen concentrations.

Abstract 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.

Chromosome aberrations are an example of the mutation process that happens at the chromosome level of DNA packaging. The occurrence of inversions and complex translocations has not been studied well enough due to the complexity of their detection using the conventional methods of differential staining. It is well-known that ionizing radiation is one of the factors that increases the frequency of chromosomal rearrangements. The objective of this pilot research was to study the frequency of inversions involving telomere regions of the chromosomes and complex translocations in T-cells of 25 chronically exposed residents of the Southern Urals. Inversions were studied in the group of 12 exposed individuals (one man, 11 women, exposure doses ranging from 0.001 to 2.9 Gy) using the Q-FISH method. Complex translocations were analyzed in the group of 13 people (nine women, four men, exposure dose ranging from 0.5 to 3.1 Gy) with the help of M-FISH method. The groups did not overlap. As a result of this analysis, it has been revealed that complex translocations occur six times less frequently than simple ones (р < 0.001). Chromatid variants of inversions predominated as compared to chromosomal ones (9 : 0.17 per 100 cells, p < 0.001). No dependence of the studied parameters on the exposure dose to red bone marrow was noted. [ABSTRACT FROM AUTHOR]

Objectives Fluorescence in situ hybridization (FISH) assays for the detection of chromosomal rearrangements involving TFE3 and TFEB are considered the gold standard for the diagnosis of MiTF family altered renal cell carcinoma (MiTF-RCC). We reviewed 801 clinical TFE3 / TFEB FISH assays performed at our tertiary-level institution between 2014 and 2023 on kidney tumors suspicious at the morphologic or biomarker level for MiTF aberrations. Methods We summarized and analyzed clinical information, TFE3 / TFEB FISH results, and available biomarker staining results in a cohort of 453 consecutive kidney tumor cases suspicious for MiTF-RCC. Results In total, 61 of 434 (14%) kidney tumors were confirmed for TFE3 translocation; 10 of 367 cases (2.7%) were confirmed for TFEB translocation. Since TFEB amplification interpretation was implemented in our service line, 20 of 306 cases (6.5%) were diagnosed with TFEB amplification. Importantly, TFE3 and TFEB rearrangements were never co-detected within the same kidney tumor. Patients with TFEB amplification were significantly older (P <.001) than patients with TFE3 or TFEB translocation. Kidney tumors with TFEB amplification were seen to be at least 3 times as common as those with TFEB translocation. Conclusions Clinical TFE3 / TFEB FISH assays successfully identified and confirmed rare MiTF-RCC with TFE3 and TFEB rearrangements. Although morphologic and biomarker features associated with a kidney tumor may be suggestive of MiTF-RCC, clinical TFE3 / TFEB FISH assays are crucial for a confirmation and definitive subclassification of patients with MiTF-RCC. [ABSTRACT FROM AUTHOR]

Hybrid taxa from the genus Pelophylax can propagate themselves in a modified way of sexual reproduction called hybridogenesis ensuring the formation of clonal gametes containing the genome of only one parental (host) species. Pelophylax grafi from South-Western Europe is a hybrid composed of P. ridibundus and P. perezi genomes and it lives with a host species P. perezi (P-G system). Yet it is unknown, whether non-Mendelian inheritance is fully maintained in such populations. In this study, we characterize P. perezi and P. grafi somatic karyotypes by using comparative genomic hybridization, genomic in situ hybridization, fluorescent in situ hybridization, and actinomycin D-DAPI. Here, we show the homeology of P. perezi and P. grafi somatic karyotypes to other Pelophylax taxa with 2 n = 26 and equal contribution of ridibundus and perezi chromosomes in P. grafi which supports F1 hybrid genome constitution as well as a hemiclonal genome inheritance. We show that ridibundus chromosomes have larger regions of interstitial (TTAGGG)n repeats flanking the nucleolus organizing region on chromosome no. 10 and a high quantity of AT pairs in the centromeric regions. In P. perezi , we found species-specific sequences in metaphase chromosomes and marker structures in lampbrush chromosomes. Pericentromeric RrS1 repeat sequence was present in perezi and ridibundus chromosomes, but the blocks were stronger in ridibundus. Various cytogenetic techniques applied to the P-G system provide genome discrimination between ridibundus and perezi chromosomal sets. They could be used in studies of germ-line cells to explain patterns of clonal gametogenesis in P. grafi and broaden the knowledge about reproductive strategies in hybrid animals. [ABSTRACT FROM AUTHOR]

Abstract—DNA in situ hybridization (ISH) is a valuable technique in molecular cytogenetics for precisely localizing specific DNA sequences on chromosomes. To perform ISH, DNA probes are essential, which can be either commercially available or custom-designed for specific research purposes (homemade probes). However, a drawback of homemade probes is their reduced hybridization signal intensity when they are small. Therefore, it is crucial to develop approaches that optimize the noise/signal ratio when using these DNA probes, which is a current focus in molecular cytogenetics. Tyramide signal amplification (TSA) is a technique that addresses this issue by enabling the visualization of small DNA sequences directly on chromosomes. The TSA system relies on the formation of a covalent bond between electron-rich fragments of sample proteins and tyramide molecules that are linked to a hapten (in chromogenic ISH) or a fluorophore (in fluorescent ISH). This process involves the conversion of tyramide molecules into free radical intermediates by horseradish peroxidase (HRP), followed by the deposition of precipitated molecules in close proximity. As a result, the low-intensity signal is amplified, enhancing the detection sensitivity. TSA serves as an excellent complement to DNA hybridization in situ due to its high sensitivity, allowing the detection of small genomic imbalances. Therefore, it has the potential to be a valuable tool in diagnosing chromosomal rearrangements in clinical practice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: The garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis. Methods: Raw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species. Results: Seven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays. Discussion: These results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA. [ABSTRACT FROM AUTHOR]

Genome or genomic dominance (GD) is a phenomenon observed in hybrids when one parental genome becomes dominant over the other. It is manifested by the replacement of chromatin of the submissive genome by that of the dominant genome and by biased gene expression. Nucleolar dominance (ND) - the functional expression of only one parental set of ribosomal genes in hybrids - is another example of an intragenomic competitive process which, however, concerns ribosomal DNA only. Although GD and ND are relatively well understood, the nature and extent of their potential interdependence is mostly unknown. Here, we ask whether hybrids showing GD also exhibit ND and, if so, whether the dominant genome is the same. To test this, we used hybrids between Festuca and Lolium grasses (Festulolium), and between two Festuca species in which GD has been observed (with Lolium as the dominant genome in Festulolium and F. pratensis in interspecific Festuca hybrids). Using amplicon sequencing of ITS1 and ITS2 of the 45S ribosomal DNA (rDNA) cluster and molecular cytogenetics, we studied the organization and expression of rDNA in leaf tissue in five hybrid combinations, four generations and 31 genotypes [F. pratensis × L. multiflorum (F1, F2, F3, BC1), L. multiflorum × F. pratensis (F1), L. multiflorum × F. glaucescens (F2), L. perenne × F. pratensis (F1), F. glaucescens × F. pratensis (F1)]. We have found that instant ND occurs in Festulolium, where expression of Lolium-type rDNA reached nearly 100% in all F1 hybrids and was maintained through subsequent generations. Therefore, ND and GD in Festulolium are manifested by the same dominant genome (Lolium). We also confirmed the concordance between GD and ND in an interspecific cross between two Festuca species. [ABSTRACT FROM AUTHOR]

The results of karyological and cytogenetic studies of populations of conifer species from the families Pinaceae and Cupressaceae growing under extreme conditions have been summarized. A chromosomal variability has been revealed in coniferous plants growing at the southern and northern borders of their natural habitats, at ecological limits of distribution, in anthropogenically disturbed ecosystems, and under introduction conditions. A high frequency of changes in the number of chromosomes (mixoploidy, aneuploidy, and polyploidy) and their morphology has been observed. Different types of chromosomal anomalies (ring and polycentric chromosomes, chromosome fragments, and multiple chromosomal abnormalities), chromosome agglutination, mitotic and meiotic abnormalities, and the appearance of B chromosomes have been revealed. Using classical methods and fluorescent in situ hybridization (FISH), a polymorphism of nucleolar-forming regions of chromosomes has been studied and features of the 5S and 45S ribosomal DNA loci have been revealed. [ABSTRACT FROM AUTHOR]

Background and aimsCholangioscopy is useful in establishing a visual diagnosis of cholangiocarcinoma (CCA), but this is harder to achieve in primary sclerosing cholangitis (PSC) because of the stricture-forming nature of the disease. Furthermore, it can be harder to differentiate malignant from benign features of the underlying inflammation. This case series demonstrates the varied features of nonmalignant inflammatory findings in PSC.MethodsA single experienced endoscopist performed cholangioscopy for PSC cases referred for ERCP.ResultsCholangioscopy in these 5 cases without CCA demonstrated the features of acute and chronic inflammation, acute inflammatory mass, dominant stricture, acute cholangitis in a duct with features of chronic inflammation with a large pigmented stone, and fibrostenotic disease. Cholangioscopic maneuvers such as advancement across strictures after balloon dilation, targeted mucosal biopsy, and electrohydraulic lithotripsy (EHL) of impacted stones are demonstrated. The relevant radiographic and histopathologic features of the disease accompany each case description. Regarding long-term prognosis, 1 case of acute inflammatory mass and a case of worsening liver function required a liver transplant evaluation, whereas the other 3 cases remain stable.ConclusionsCholangioscopic features of benign disease in PSC are varied. Knowledge of these features is essential in differentiating between benign and malignant findings. These features, combined with biopsy and cytology evaluation, can help in tailoring management in patients with benign PSC.

Background and aimsPrompt and accurate differentiation of benign and malignant strictures in primary sclerosing cholangitis (PSC) is crucial. ERCP with brush cytology, the most common modality to achieve this, is hindered by a low diagnostic yield. Cholangioscopy can overcome this limitation by establishing a visual diagnosis based on the characteristic morphologic features of cholangiocarcinoma (CCA) and can aid in targeted biopsies of suspicious lesions. However, its role in PSC remains unclear. This case series demonstrates the performance of the latest generation of single-operator cholangioscope for this indication.MethodsA single experienced endoscopist performed cholangioscopy for PSC cases referred for ERCP.ResultsCholangioscopies of patients 1 to 3 demonstrate the features of extrahepatic duct dominant strictures (DS) and the cholangioscopic maneuvers undertaken in these cases, including advancement across the DS after balloon dilation, biopsy of the DS, and electrohydraulic lithotripsy of impacted stones. Cholangioscopies of patients 4 to 6 demonstrate the varied features of CCA ranging from focal stricture with tumor vessels, papillary frond-like projections, and features of an intraductal papillary biliary neoplasm. Also shown are the radiographic and histopathologic features of the disease.ConclusionsCholangioscopy allowed us to identify morphologic features of both malignancy and benign disease in PSC in the setting of extrahepatic duct strictures, and we were able to obtain adequate targeted tissue samples for histopathologic confirmation.

IntroductionThe garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis.MethodsRaw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species.ResultsSeven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays.DiscussionThese results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA.

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. [ABSTRACT FROM AUTHOR]

This article provides a summary of the current knowledge on the cytogenetics of four genera, which are all composed of 36 chromosomes, within the Orchidinae subtribe (Orchidaceae). Previous classical studies have revealed differences in karyomorphology among these genera, indicating genomic diversity. The current study includes an analysis of the current knowledge with an update of the karyotype of 47 species with 36 chromosomes from the genera Anacamptis, Serapias, Himantoglossum, and Ophrys. The study discusses comparisons of karyotypes among these genera that used traditional techniques as well as karyotype asymmetry relationships with various asymmetry indices. Additionally, the study reports new findings on polyploidy in Anacamptis pyramidalis and Serapias lingua, which were observed through karyotype and meiotic metaphase analyses in EMC. Moreover, the study detected B chromosomes for the first time in A. papilionacea and A. palustris. The article also describes the use of fluorescent in situ hybridization in some specimens of A. papilionacea and A. collina to locate different sites of the 18S-5.8S-25S rDNA and 5S rDNA ribosomal complexes on chromosomes. The information derived from these cytogenetic analyses was used to refine the classification of these orchids and identify evolutionary relationships among different species and genera. [ABSTRACT FROM AUTHOR]

Macroptilium (Benth.) Urb. is a neotropical legume genus from the subtribe Phaseolinae. The investigated species present a stable chromosome number (2n = 22), but differ in their karyotype formulae, suggesting the presence of chromosome rearrangements. In this work, we comparatively analysed the karyotypes of six species (Macroptilium atropurpureum, Macroptilium bracteatum, Macroptilium erythroloma, Macroptilium gracile, Macroptilium lathyroides, and Macroptilium martii) from the two main clades that form the genus. Heterochromatin distribution was investigated with chromomycin A3 (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization was used to localize the 5S and 35S ribosomal DNA (rDNA) sites. Single copy bacterial artificial chromosomes (BACs) previously mapped in the related genera Phaseolus L. and Vigna Savi were used to establish chromosome orthologies and to investigate possible rearrangements among species. CMA+/DAPI− bands were observed, mostly associated with rDNA sites. Additional weak, pericentromeric bands were observed on several chromosomes. Although karyotypes were similar, species could be differentiated mainly by the number and position of the 5S and 35S rDNA sites. BAC markers demonstrated conserved synteny of the main rDNA sites on orthologous chromosomes 6 and 10, as previously observed for Phaseolus and Vigna. The karyotypes of the six species could be differentiated, shedding light on its karyotype evolution. [ABSTRACT FROM AUTHOR]

In schizophrenia, somatostatin (SST) and parvalbumin (PV) mRNA levels are lower in the dorsolateral prefrontal cortex (DLPFC), but it remains unclear whether these findings reflect lower transcript levels per neuron, fewer neurons, or both. Distinguishing among these alternatives has implications for understanding the pathogenesis of, and developing new treatments for, DLPFC dysfunction in schizophrenia. To identify SST and PV neurons in postmortem human DLPFC, the authors used fluorescent in situ hybridization to label cells expressing two transcripts not altered in schizophrenia: vesicular GABA transporter (VGAT; a marker of all GABA neurons) and SOX6 (a marker of only SST and PV neurons). In cortical layers 2 and 4, where SST and PV neurons, respectively, are differentially enriched, levels of SST and PV mRNA per neuron and the relative densities of SST-, PV-, and VGAT/SOX6–positive neurons were quantified. In individuals with schizophrenia, mRNA levels per positive neuron were markedly and significantly lower for SST in both layers (effect sizes >1.48) and for PV only in layer 4 (effect size=1.14) relative to matched unaffected individuals. In contrast, the relative densities of all SST-, PV-, or VGAT/SOX6–positive neurons were unaltered in schizophrenia. Novel multiplex fluorescent in situ hybridization techniques permit definitive distinction between cellular levels of transcripts and the presence of neurons expressing those transcripts. In schizophrenia, pronounced SST and PV mRNA deficits are attributable to lower levels of each transcript per neuron, not fewer neurons, arguing against death or abnormal migration of these neurons. Instead, these neurons appear to be functionally altered and thus amenable to therapeutic interventions. [ABSTRACT FROM AUTHOR]

Synovial sarcoma is a rare malignant mesenchymal neoplasm mostly affecting young adults, characterized by a specific translocation which results in the fusion of the SS18 gene on chromosome 18 with one of the three highly homologous SSX genes on chromosome X. Its morphological diagnosis, especially in monophasic or poorly differentiated variants, can be challenging because histological features often overlap with other malignant mesenchymal tumors. Until recently, the differential diagnosis mostly relied on the use of cytogenetic or molecular analyses to detect the specific t(X;18)(p11;q11) translocation, thus virtually restricting its correct identification to referral centers with a high histological and molecular pathology workflow. The recently commercialized highly sensitive and fusion-specific SS18-SSX antibody has significantly improved the approach to these tumors, representing a relatively cheap and easy to access tool for synovial sarcoma diagnosis. Through a retrospective analysis of 79 synovial sarcomas and histological mimickers, this study confirms the usefulness of the SS18-SSX antibody in the diagnosis of synovial sarcoma, particularly focusing on its application in the pathological response evaluation after neoadjuvant treatment as well as its time- and cost-saving advantages. Finally, we here propose a new diagnostic algorithm to apply into the routine practice. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Autism is a heterogeneous neurodevelopmental disorder that influences a child's ability to think, learn, communicate, and interact socially and possess behavioral problems. It has also been observed to be associated with several medical conditions, including certain chromosomal disorders like Down syndrome (DS). Several studies have shown autistic individuals with DS. The main objective of the present study is to screen for chromosomal abnormalities in autistic children with DS characteristics in the North Karnataka population, India. MATERIALS AND METHODS: Chromosome analysis of peripheral blood of four DS children with autistic characteristics was performed to examine the chromosomal abnormalities, which were confirmed by fluorescent in situ hybridization. RESULTS: Childhood Autism Rating Scale score was calculated initially to assess the severity of autism. All four cases were found to have autism with DS. Out of four cases, three (75%) were found to be trisomy 21 and 1 (25%) had a trisomy 21 mosaic condition. CONCLUSION: Our study confirms the chromosomal abnormality present in autistic children with DS characteristics and these findings will contribute in several ways to the diagnosis and treatment of the genetic cause of autism with other comorbidities or vice versa.

The detection of ROS1 rearrangements in metastatic non-squamous non-small cell lung carcinoma (NS-NSCLC) permits administration of efficient targeted therapy. Detection is based on a testing algorithm associated with ROS1 immunohistochemistry (IHC) screening followed by ROS1 FISH and/or next generation sequencing (NGS) to confirm positivity. However, (i) ROS1 rearrangements are rare (1–2% of NS-NSCLC), (ii) the specificity of ROS1 IHC is not optimal, and (iii) ROS1 FISH is not widely available, making this algorithm challenging to interpret time-consuming. We evaluated RNA NGS, which was used as reflex testing for ROS1 rearrangements in NS-NSCLC with the aim of replacing ROS1 IHC as a screening method. ROS1 IHC and RNA NGS were prospectively performed in 810 NS-NSCLC. Positive results were analyzed by ROS1 FISH. ROS1 IHC was positive in 36/810 (4.4%) cases that showed variable staining intensity while NGS detected ROS1 rearrangements in 16/810 (1.9%) cases. ROS1 FISH was positive in 15/810 (1.8%) of ROS1 IHC positive cases and in all positive ROS1 NGS cases. Obtaining both ROS1 IHC and ROS1 FISH reports took an average of 6 days, while obtaining ROS1 IHC and RNA NGS reports took an average of 3 days. These results showed that systematic screening for the ROS1 status using IHC must be replaced by NGS reflex testing. [ABSTRACT FROM AUTHOR]

Purpose: Although outcomes of children with acute myeloid leukemia (AML) have improved over the last decades, around one-third of patients relapse. Measurable (or minimal) residual disease (MRD) monitoring may guide therapy adjustments or pre-emptive treatments before overt hematological relapse. Methods: In this study, we review 297 bone marrow samples from 20 real-life pediatric AML patients using three MRD monitoring methods: multiparametric flow cytometry (MFC), fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). Results: Patients showed a 3-year overall survival of 73% and a 3-year event-free survival of 68%. Global relapse rate was of 25%. All relapses were preceded by the reappearance of MRD detection by: (1) MFC (p = 0.001), (2) PCR and/or FISH in patients with an identifiable chromosomal translocation (p = 0.03) and/or (3) one log increase of Wilms tumor gene 1 (WT1) expression in two consecutive samples (p = 0.02). The median times from MRD detection to relapse were 26, 111, and 140 days for MFC, specific PCR and FISH, and a one log increment of WT1, respectively. Conclusions: MFC, FISH and PCR are complementary methods that can anticipate relapse of childhood AML by weeks to several months. However, in our series, pre-emptive therapies were not able to prevent disease progression. Therefore, more sensitive MRD monitoring methods that further anticipate relapse and more effective pre-emptive therapies are needed. [ABSTRACT FROM AUTHOR]

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.