Telomere Length Measurement in Human Tissue Sections by Quantitative Fluorescence In Situ Hybridization (Q-FISH).

Telomeres in most somatic cells shorten with each cell division, and critically short telomeres lead to cellular dysfunction, cell cycle arrest, and senescence. Thus, telomere shortening is an important hallmark of human cellular senescence. Quantitative fluorescence in situ hybridization (Q-FISH) using formalin-fixed paraffin-embedded (FFPE) tissue sections allows the estimation of telomere lengths in individual cells in histological sections. In our Q-FISH method, fluorescently labelled peptide nucleic acid (PNA) probes are hybridized to telomeric and centromeric sequences in FFPE human tissue sections, and relative telomere lengths (telomere signal intensities relative to centromere signal intensities) are measured. This chapter describes our Q-FISH protocols for assessing relative telomere lengths in FFPE human tissue sections.

Highly divergent satellitomes of two barley species of agronomic importance, Hordeum chilense and H. vulgare.

In this paper, we have performed an in-depth study of the complete set of the satellite DNA (satDNA) families (i.e. the satellitomes) in the genome of two barley species of agronomic value in a breeding framework, H. chilense (H1 and H7 accessions) and H. vulgare (H106 accession), which can be useful tools for studying chromosome associations during meiosis. The study has led to the analysis of a total of 18 satDNA families in H. vulgare, 25 satDNA families in H. chilense (accession H1) and 27 satDNA families in H. chilense (accession H7) that constitute 46 different satDNA families forming 36 homology groups. Our study highlights different important contributions of evolutionary and applied interests. Thus, both barley species show very divergent satDNA profiles, which could be partly explained by the differential effects of domestication versus wildlife. Divergence derives from the differential amplification of different common ancestral satellites and the emergence of new satellites in H. chilense, usually from pre-existing ones but also random sequences. There are also differences between the two H. chilense accessions, which support genetically distinct groups. The fluorescence in situ hybridization (FISH) patterns of some satDNAs yield distinctive genetic markers for the identification of specific H. chilense or H. vulgare chromosomes. Some of the satellites have peculiar structures or are related to transposable elements which provide information about their origin and expansion. Among these, we discuss the existence of different (peri)centromeric satellites that supply this region with some plasticity important for centromere evolution. These peri(centromeric) satDNAs and the set of subtelomeric satDNAs (a total of 38 different families) are analyzed in the framework of breeding as the high diversity found in the subtelomeric regions might support their putative implication in chromosome recognition and pairing during meiosis, a key point in the production of addition/substitution lines and hybrids.

Detecting Cholangiocarcinoma in the Setting of Primary Sclerosing Cholangitis: Is Biliary Tract Fluorescence In Situ Hybridization Helpful?

INTRODUCTION/OBJECTIVE: Biliary brushing cytology (BB) to detect cholangiocarcinoma (CCA) is integral in the surveillance of patients with primary sclerosing cholangitis (PSC). Since reactive changes can mimic carcinoma, indeterminant results are frequent. Fluorescence in situ hybridization (FISH) using the UroVysion probe set has been advocated to enhance the detection of CCA. This study evaluates the performance of FISH for detecting CCA in patients with and without PSC. MATERIALS AND METHODS: A query of our pathology database for atypical and suspicious BB with concurrent FISH results was performed from 2014 to 2021. FISH (using UroVysion probe set containing centromere enumeration probes to chromosomes 3, 7, and 17) was positive if at least 5 cells demonstrated polysomy. Electronic medical records were reviewed to identify patients with PSC and CCA. CCA was confirmed by pathology or clinical impression. RESULTS: Of the 65 patients (103 BB) in the PSC cohort, 59 patients (94 BB) without CCA and 6 patients (9 BB) with CCA were identified. 33 non-PSC patients (41 BB) with CCA were included for comparison. Positive FISH was highest in non-PSC patients with CCA (10/41 BB, 24%). Positive FISH was seen in both PSC with (1/9 BB, 11%) and without (2/94 BB, 2%) CCA. CONCLUSIONS: FISH positivity was lower than expected and was positive in PSC patients without CCA. These results question the clinical utility of FISH for CCA surveillance in PSC patients.

Immunophenotypic Characteristics and Cytogenetic Analysis of Adolescent and Young Adult B-Cell Acute Lymphoblastic Leukemia: Correlations With Clinicopathological Parameters.

Introduction Acute lymphoblastic leukemia (ALL) has suboptimal survival rates for adolescents and young adults (AYA) as compared to children. Very limited studies have been conducted on AYA patients in India. This study aimed to identify the cytogenetic and immunophenotype characteristics of B-cell ALL (B-ALL) in AYA patients and determine its correlation with clinicopathological parameters in the Southern India region. Method The study was a prospective study conducted for three years, from June 2019 to May 2022, in India. Newly diagnosed 90 patients with AYA (13-40 years) ALL were recruited. A B-ALL diagnosis was made based on morphology with cytochemical stains and immunophenotype by flow cytometry (FCM). Cytogenetic analysis was also performed using karyotyping and fluorescent in situ hybridization to identify chromosomal aberrations. The cytogenetics results were correlated with immunophenotyping and clinicopathological characteristics. Variables were analyzed using the Mann-Whitney U test and Chi-square test using IBM SPSS Statistics for Windows, Version 20.0 (Released 2011; IBM Corp., Armonk, NY, USA). Results The mean age was 22.68 ± 8.06 years. It was observed that the most common structural chromosomal abnormality for AYA was t(9;22) in 14 (15%) cases, followed by 6q deletions in seven (8%) cases, t(1;19) in four (4%) cases, and t(12;17) and t(6;14) in two (2%) cases each. In addition, t(3;12), t(2;11), t(12;21), t(1;9), t(2;12), and t(X;10) were found in one (1%) case each. The most common numerical abnormality was hyperdiploidy (15; 17%), followed by hypodiploidy (10; 11%). Further, myeloid antigen expression of CD33 was the most common aberrantly expressed marker found in 20 (28%) cases, followed by CD15 in three cases (5%), CD13 in three (4%) cases, and CD11b in two (3%) cases. It was also observed that in Ph+ve cases, CD33 and CD13 were most commonly expressed in three (33%) and two (17%) cases, respectively. In contrast, in Ph-ve patients, their expressions were lesser at 17 (27%) and one (2%) cases, respectively. In addition, leukemia-associated immunophenotype pattern (LAIP) markers CD44 6 (86%) and CD123 5 (55%) were also found to be significantly associated with Ph+ve, whereas their values in the Ph-ve group were lesser at 25 (42%) and 9 (17%), respectively. Our data also showed that older age wassignificantly associated with Ph+ve with a median age of 30 years (p = 0.012). In comparison, the median age of Ph-ve was only 21 years. Conclusion Our study established that the incidence of cytogenetic abnormalities for AYA was consistent with previously reported data. This study reaffirms that Ph+ve cases have significant associations with MyAg (CD13), LAIP (CD123 and CD44), and older age for the South Indian population.

A Simple and Fast Optical Clearing Method for Whole-Mount Fluorescence In Situ Hybridization (FISH) Imaging.

We report a single-step optical clearing method that is compatible with RNA fluorescence in situ hybridization (FISH) imaging. We previously demonstrated microscopy imaging with immunohistochemistry and genetic reporters using a technique called lipid-preserving refractive index matching for prolonged imaging depth (LIMPID). Our protocol reliably produces high-resolution three-dimensional (3D) images with minimal aberrations using high magnification objectives, captures large field-of-view images of whole-mount tissues, and supports co-labeling with antibody and FISH probes. We also custom-designed FISH probes for quail embryos, demonstrating the ease of fabricating probes for use with less common animal models. Furthermore, we show high-quality 3D images using a conventional fluorescence microscope, without using more advanced depth sectioning instruments such as confocal or light-sheet microscopy. For broader adoption, we simplified and optimized 3D-LIMPID-FISH to minimize the barrier to entry, and we provide a detailed protocol to aid users with navigating the thick and thin of 3D microscopy.

Establishing of 3D-FISH on frozen section and its applying in chromosome territories analysis in Populus trichocarpa.

KEY MESSAGE: Fluorescence in situ hybridization with frozen sections of root tips showed difference of chromosome territories distribution between autosome and sex-chromosome homologous pairs in Populus trichocarpa. The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biologic processes. Three-dimensional fluorescence in situ hybridization (3D-FISH) is a powerful tool for analyzing CTs, but its application in plants is limited. In this study, we established a 3D-FISH technique using frozen sections of Populus trichocarpa root tips, which was an improvement over the use of paraffin sections and enabled us to acquire good FISH signals. Using chromosome-specific oligo probes, we were able to analyze CTs in interphase nuclei in three dimensions. The distribution of chromosome pairs 17 and 19 in the 3D-preserved nuclei of P. trichocarpa root tip cells were analyzed and showed that the autosome pair 17 associated more often than sex chromosome 19. This research lays a foundation for further study of the spatial position of chromosomes in the nucleus and the relationship between gene expression and spatial localization of chromosomes in poplar.

High-grade B-cell lymphoma with 11q aberration in the HIV setting: a clinicopathological study of 10 cases and literature review.

High-grade B-cell lymphoma with 11q aberration (HGBL-11q) is a distinct lymphoma entity according to the 5th edition of the WHO classification of hematolymphoid tumors. It lacks MYC translocation but carries proximal gains and/or telomeric losses of chromosome 11q. This rare type of B-cell lymphoma is less frequently reported in people living with HIV (PLWH), and its exact frequency remains unclear. Our goal was to retrospectively analyze its frequency in a cohort of aggressive B-cell lymphomas in PLWH, including Burkitt lymphoma (BL, n = 35), diffuse large B-cell lymphoma (DLBCL, n = 48), high-grade B-cell lymphoma, not otherwise specified (HGBL-NOS, n = 13), which was diagnosed as AIDS-related lymphoma (ARL) at our institution. In total, 10/96 (10.4%) cases harbored the typical 11q aberration pattern, predominantly those that had been classified as BL (6/35, 17.1%), DLBCL (2/48, 4.2%), and HGBL, NOS (2/13, 15.4%). We also evaluated 7 cases of AIDS-related HGBL-11q (AR-HGBL-11q) reported in the literature. The median age of our cohort was 35 years, and all the patients were male. Most cases (70%) had a history of HIV infection for over 1 year, and all were involved in lymph nodes (100%), frequently involved extranodal sites (60%), and Ann Arbor stage III/IV. In histomorphology, the cases exhibited diverse cytological features, reminiscent of BL (6 cases), DLBCL (2 cases), and HGBL (2 cases). A comparison of the combined cohort of 17 AR-HGBL-11q cases with 11 ARL cases that lacked both MYC rearrangement and 11q aberration at our institution showed that HGBL-11q cases were characterized by strikingly coarse apoptotic debris (P < 0.001), background rich in eosinophils (P = 0.002), higher expression of the germinal centre marker LMO2 (P = 0.080), lower expression of MUM1 (P = 0.004), BCL2 (P = 0.007), and LEF1 (P = 0.080), and lower positivity for EBER in situ hybridisation (P = 0.027). Notably, one case in our series was EBV-positive, a finding not previously reported in the literature. Furthermore, comparing the prognosis between these two groups, AR-HGBL-11q showed a relatively favorable prognosis (P = 0.15), although the difference was not statistically significant. We analyzed this rare lymphoma entity in the HIV setting and highlighted the importance of integrating histomorphological and immunophenotypic features in its diagnosis and classification.

Satellitome Analysis of Adalia bipunctata (Coleoptera): Revealing Centromeric Turnover and Potential Chromosome Rearrangements in a Comparative Interspecific Study.

Eukaryotic genomes exhibit a dynamic interplay between single-copy sequences and repetitive DNA elements, with satellite DNA (satDNA) representing a substantial portion, mainly situated at telomeric and centromeric chromosomal regions. We utilized Illumina next-generation sequencing data from Adalia bipunctata to investigate its satellitome. Cytogenetic mapping via fluorescence in situ hybridization was performed for the most abundant satDNA families. In silico localization of satDNAs was carried out using the CHRISMAPP (Chromosome In Silico Mapping) pipeline on the high-fidelity chromosome-level assembly already available for this species, enabling a meticulous characterization and localization of multiple satDNA families. Additionally, we analyzed the conservation of the satellitome at an interspecific scale. Specifically, we employed the CHRISMAPP pipeline to map the satDNAs of A. bipunctata onto the genome of Adalia decempunctata, which has also been sequenced and assembled at the chromosome level. This analysis, along with the creation of a synteny map between the two species, suggests a rapid turnover of centromeric satDNA between these species and the potential occurrence of chromosomal rearrangements, despite the considerable conservation of their satellitomes. Specific satDNA families in the sex chromosomes of both species suggest a role in sex chromosome differentiation. Our interspecific comparative study can provide a significant advance in the understanding of the repeat genome organization and evolution in beetles.

Use of high-resolution fluorescence in situ hybridization for fast and robust detection of SARS-CoV-2 RNAs.

Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (-) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures.

The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri. In pursuing our goal to capture each organism's population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture's health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE: Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, "state-of-the-art" labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum, C. ljungdahlii, and Clostridium kluyveri. We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Diagnosing B-cell acute lymphoblastic leukemia in 2 pediatric patients with recent SARS-CoV-2 infection.

COVID-19 infection is still a mystery in terms of its long-term effect on health and its consequences on hematological disorders. Prior studies including ours have shown the abnormal changes in hematopoietic cells in COVID-19 patients. In this article, we are presenting 2 cases of pediatric B-lymphoblastic leukemia (B-ALL) with a previous history of COVID-19 infection. The first case describes a 22-month-old boy presenting with lymphadenopathy, neutropenia, and anemia with concurrent COVID-19 infection without any evidence of a hematolymphoid neoplasm as per bone marrow and lymph node biopsy. However, he presented after 2 months with bone marrow biopsy confirming B-ALL. The second case is that of a 4-year-old girl presenting with B-ALL who has had asymptomatic COVID-19 infection 5 months before this current presentation. Both the cases had complete resolution of COVID-19 infection during the time of presentation with acute leukemia. There were notably 2 rare findings along the course of the patients' illnesses. First, the unusual plasmacytosis in the marrow during active COVID-19 infection in the first patient and the second, is predilection of development of B-ALL following COVID-19. In both the cases the fluorescence in situ hybridization (FISH) studies showed pathologic alteration of the RUNX1 gene. Overall, there are no literature to support a causal association between acute B-ALL and COVID-19. The diagnosis of B-ALL in these patients after COVID-19 infection may be totally unrelated. However, if we consider Greaves proposed 2-hit model for childhood acute leukemia, that an infectious agent can precipitate development of B-ALL in a genetically susceptible individual. Alteration of the RUNX1 gene in both the patients, opens a door for further exploration of the "second-hit" hypothesis regarding an infectious agent precipitating development of B-ALL in a genetically susceptible individual.

Protein Expression, Amplification, and Mutation of HER2 Gene in Canine Primary Pulmonary Adenocarcinomas: Preliminary Results.

Recently, human epidermal growth factor receptor 2 (HER2) has emerged as a therapeutic target of interest for non-small-cell lung cancer in humans. The role of HER2 in canine pulmonary adenocarcinomas is poorly documented. To address this gap, this study employed three methodologies: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS) to investigate the protein expression, gene amplification, and mutation of HER2 in 19 canine primary pulmonary adenocarcinomas. By IHC, 3 out of 19 cases were overexpressed 3+, 6 were 2+, and 10 were negative. With FISH, 2 cases were amplified (12.5%), 3 were inadequate for the analyses, and the others were non-amplified. With NGS, seven cases were inadequate. All other cases were wild-type, except for one IHC 3+ case, which was amplified with FISH and with a specific mutation already described in human pulmonary adenocarcinoma, V659E. This mutation is probably sensitive to tyrosine kinase inhibitory drugs. These results are similar to those in human medicine and to the few data in the literature on canine lung carcinomas; the presence of 12.5% of amplified cases in dogs lays the foundation for future targeted drugs against HER2 alterations.

Challenges and solutions in FISH for formalin-fixed paraffin-embedded tissue: A scoping review.

Fluorescence in situ hybridization (FISH) has revolutionized molecular cytogenetic analysis since the 1980s, enabling precise localization of DNA sequences in cells and tissues. Despite its relevance, applying FISH to formalin-fixed paraffin-embedded (FFPE) tissue samples encounters significant technical challenges. This review addresses the main issues encountered in this context, such as inadequate fixation, contamination, block and slide age, inadequate pretreatment, and FISH technique. Proposed solutions include optimized pretreatment protocols, monitoring of blockage, careful selection of probes, and thorough analysis of results. Implementing good laboratory practices and quality control strategies are essential to ensure reliable results. Additionally, the use of emerging technologies such as artificial intelligence and digital pathology offers new perspectives for improving the efficiency and accuracy of FISH in FFPE samples. This review highlights the importance of a careful and personalized approach to overcome the technical challenges associated with FISH in FFPE samples, strengthening its role in research and clinical diagnosis. RESEARCH HIGHLIGHTS: Few FISH studies on FFPE: The scarcity of studies specifically addressing FISH applications in FFPE tissues highlights a critical gap in the literature. Troubleshooting FISH in FFPE tissues: Identifying and addressing common challenges in FISH techniques when applied to FFPE samples, such as signal quality and hybridization efficiency. Critical aspects of FISH technique: Discuss the main technical considerations crucial for successful FISH in FFPE tissues, including sample preparation, probe selection, and protocol optimization.

Cytogenetic analysis and visualization of genetic relationships in wild lilies.

Lilies are highly regarded for their ornamental appeal and striking flowers, which are of significant importance in horticulture. Understanding the genetic makeup of these plants is crucial for breeding and developing new cultivars. This study presents a comprehensive cytogenetic analysis of 45 S and 5 S rDNA loci in 34 wild Lilium species. To reveal the genetic relationships within the genus, advanced visualization methods, such as heatmaps and 3D network plots, were utilized. The results of this study identified both conserved and divergent genetic features, which offer insights into the evolutionary history and potential genetic compatibility of these species. Notably, the clustering of species based on rDNA locus patterns highlights the need for potential taxonomic re-evaluation and reveals candidates for cross-breeding. This integrated approach emphasizes the importance of combining cytogenetic data with traditional morphological classifications to refine our understanding of the Lilium species. Future research should expand the range of analyzed species, incorporate additional molecular markers to further elucidate genetic relationships, and support the development of resilient and diverse ornamental crops. The findings of this study provide a novel framework for genetic analysis of Lilium, offering valuable insights for both scientific understanding and practical breeding programs.

Insights from single-strain and mixed culture experiments on the effects of heatwaves on freshwater flagellates.

The increasing frequency and intensity of heatwaves driven by climate change significantly impact microbial communities in freshwater habitats, particularly eukaryotic microorganisms. Heterotrophic nanoflagellates are important bacterivorous grazers and play a crucial role in aquatic food webs, influencing the morphological and taxonomic structure of bacterial communities. This study investigates the responses of three flagellate taxa to heatwave conditions through single-strain and mixed culture experiments, highlighting the impact of both biotic and abiotic factors on functional redundancy between morphologically similar protist species under thermal stress. Our results indicate that temperature can significantly impact growth and community composition. However, density-dependent factors also had a significant impact. In sum, stabilizing effects due to functional redundancy may be pronounced as long as density-dependent factors play a minor role and can be overshadowed when flagellate abundances increase.

DEK::AFF2 Fusion-Associated Squamous Cell Carcinoma: A Case Series with Literature Review on an Emerging and Challenging Entity.

PURPOSE: DEK::AFF2 fusion-associated squamous cell carcinoma (DEK::AFF2 SCC), also reported in the literature as low-grade papillary sinonasal (Schneiderian) carcinoma (LGPSC), is a rare, primarily bland-appearing, but locally aggressive neoplasm. Morphologically, these tumors can closely resemble sinonasal papilloma (SP), especially on small or limited biopsy, often leading to misdiagnosis. DEK::AFF2 SCC is devoid of the underlying mutually exclusive EGFR or KRAS driver mutations of SP, suggesting it may represent a distinct unique entity. METHODS: In this study, we conducted a retrospective search of "unusual" SP reported either as atypical, dysplastic, or suspicious for malignant transformation at our institution in the last 13 years (2010-2023), to identify potential cases of DEK::AFF2 SCC. RESULTS: Of the 201 SP cases during this time period, 30 "unusual" SP cases were identified. On morphologic review of these 30 cases, 6 were worrisome for DEK::AFF2 SCC and were selected for AFF2 immunohistochemical stain (IHC), of which 3 cases were positive. All 3 AFF2 IHC positive cases were also positive for DEK::AFF2 fusion by fluorescence in situ hybridization (FISH), thereby, confirming IHC results. CONCLUSIONS: This study highlights that AFF2 IHC can be an invaluable surrogate marker to FISH in identifying DEK::AFF2 SCC in challenging cases to avoid misdiagnosis. Detailed clinical and pathologic data were collected to gain a better understanding of this emerging challenging entity. A literature review was performed to enrich our knowledge of DEK::AFF2 SCC.

MYC translocation is a valuable marker for the development and relapse of extramedullary disease in multiple myeloma.

OBJECTIVE: To study the cytogenetic characteristics of extramedullary disease (EMD) in patients with multiple myeloma (MM) and their impact on prognosis. METHODS: Patients with newly diagnosed MM (NDMM) at Peking Union Medical College Hospital (Beijing, China) between June 2007 and December 2019 were recruited for this study. Demographic information, clinical data, fluorescence in situ hybridization (FISH) results of marrow and tissue samples, and survival outcome data were collected. RESULTS: A total of 439 patients with NDMM were divided into those without EMD (non-EMD, n = 339), those with EMD with primary paraosseous plasmacytoma (pEMD-B, n = 48), those with primary EMD with soft-tissue involvement (pEMD-S, n = 33), and those with secondary EMD (sEMD, n = 19). The incidence of EMD was 18.5% (81/439) at diagnosis and 22.8% (100/439) throughout the disease course. Comparison of FISH results showed a higher proportion of RB1 deletion (n = 20; 60.0% vs. 20.0%, p = .013) and MYC translocation (n = 12; 44.4% vs. 12.5%, p = .041) in the extramedullary tissues than in the paired bone marrow samples. At diagnosis, the percentage of MYC translocations in the sEMD group was notably higher than that in the non-EMD group (55.6% vs. 15.5%, p = .012). The median overall survival (OS) of patients with pEMD-S (32 months) and sEMD (17 months) was significantly shorter (both p = .001) than that of non-EMD patients (60 months). CONCLUSION: Soft-tissue EMD can be considered a high-risk condition, even in the era of novel agents. MYC translocation can serve as a valuable marker that correlates with extramedullary spread and relapse in patients with MM and should be considered for inclusion in routine FISH panels in clinical practice.

CAPZB mRNA is a novel biomarker for cervical high-grade squamous lesions.

This study aimed to evaluate the potential of capping protein (actin filament) muscle Z-line subunit ß (CAPZB) messenger ribonucleic acid (mRNA) levels as a biomarker for distinguishing low-grade squamous intraepithelial lesions of the cervix (LSIL) from high-grade squamous intraepithelial lesions of the cervix (HSIL). We collected a total of 166 cervical exfoliated cells and divided them into five groups based on histopathological results. Each sample was divided into two portions, one for fluorescence in situ hybridization (FISH) detection and the other for bisulfite sequencing polymerase chain reaction (BSP) detection. We found that FISH detection of CAPZB mRNA mean fluorescence intensity (MFI) and BSP detection of CAPZB deoxyribonucleic acid (DNA) percentage of methylation rate (PMR) performed as biomarkers for distinguishing HSIL from LSIL, with an area under the receiver operating characteristic curve (AUC), sensitivity, specificity and cut-off value of 0.893, 81.25%, 80.39% and 0.616, 0.794, 64.06%, 81.37% and 0.454, respectively. Furthermore, FISH detection of CAPZB mRNA exhibited a greater AUC (0.893) for the detection of HSIL than the CAPZB DNA methylation method (0.794), indicating the CAPZB mRNA levels can be used as a biomarker for assessing cervical lesions.

Characteristics and Cytological Analysis of Several Novel Allopolyploids and Aneuploids between Brassica oleracea and Raphanus sativus.

Polyploids are essential in plant evolution and species formation, providing a rich genetic reservoir and increasing species diversity. Complex polyploids with higher ploidy levels often have a dosage effect on the phenotype, which can be highly detrimental to gametes, making them rare. In this study, offspring plants resulting from an autoallotetraploid (RRRC) derived from the interspecific hybridization between allotetraploid Raphanobrassica (RRCC, 2n = 36) and diploid radish (RR, 2n = 18) were obtained. Fluorescence in situ hybridization (FISH) using C-genome-specific repeats as probes revealed two main genome configurations in these offspring plants: RRRCC (2n = 43, 44, 45) and RRRRCC (2n = 54, 55), showing more complex genome configurations and higher ploidy levels compared to the parental plants. These offspring plants exhibited extensive variation in phenotypic characteristics, including leaf type and flower type and color, as well as seed and pollen fertility. Analysis of chromosome behavior showed that homoeologous chromosome pairing events are widely observed at the diakinesis stage in the pollen mother cells (PMCs) of these allopolyploids, with a range of 58.73% to 78.33%. Moreover, the unreduced C subgenome at meiosis anaphase II in PMCs was observed, which provides compelling evidence for the formation of complex allopolyploid offspring. These complex allopolyploids serve as valuable genetic resources for further analysis and contribute to our understanding of the mechanisms underlying the formation of complex allopolyploids.

A large colonial choanoflagellate from Mono Lake harbors live bacteria.

As the closest living relatives of animals, choanoflagellates offer insights into the ancestry of animal cell physiology. Here, we report the isolation and characterization of a colonial choanoflagellate from Mono Lake, California. The choanoflagellate forms large spherical colonies that are an order of magnitude larger than those formed by the closely related choanoflagellate Salpingoeca rosetta. In cultures maintained in the laboratory, the lumen of the spherical colony is filled with a branched network of extracellular matrix and colonized by bacteria, including diverse Gammaproteobacteria and Alphaproteobacteria. We propose to erect Barroeca monosierra gen. nov., sp. nov. Hake, Burkhardt, Richter, and King to accommodate this extremophile choanoflagellate. The physical association between bacteria and B. monosierra in culture presents a new experimental model for investigating interactions among bacteria and eukaryotes. Future work will investigate the nature of these interactions in wild populations and the mechanisms underpinning the colonization of B. monosierra spheres by bacteria. IMPORTANCE: The diversity of organisms that live in the extreme environment of Mono Lake (California, USA) is limited. We sought to investigate whether the closest living relatives of animals, the choanoflagellates, exist in Mono Lake, a hypersaline, alkaline, arsenic-rich environment. We repeatedly isolated members of a new species of choanoflagellate, which we have named Barroeca monosierra. Characterization of B. monosierra revealed that it forms large spherical colonies containing diverse co-isolated bacteria, providing an opportunity to investigate mechanisms underlying physical associations between eukaryotes and bacteria.