Fluorescence In Situ Hybridization Research Articles

Cytogenomic evaluation of regenerated Aralia elata using PLOP-FISH and flow cytometry

Aralia elata is closely related to Panax ginseng and contains high levels of saponins and other medicinal compounds. Successful A. elata micropropagation is commercially significant; however, the genomic stability of tissue culture-derived regenerants is unclear. In this study, callus-derived regenerated A. elata plants were obtained, and their cytogenomic constitutions were assessed. Using RepeatExplorer, pre-labeled oligonucleotide probes (PLOPs) were developed with newly mined tandem repeats from < 1× NGS whole-genome short reads, fluorescence in situ hybridization (FISH) was performed using six repeat probes, including three universal PLOPs, and genomic DNA content was estimated using flow cytometry. Regenerated A. elata plants (50) exhibited consistent ploidy, repeat distribution, and genome sizes compared with those exhibited by the mother plant. Six repeat probes were detected using FISH. Tandem repeat AeTR49 was identified as an excellent cytogenetic marker for homologous chromosomes, and AeTR161 and AeTR178 were localized in the centromeric and telomeric sections, respectively. Genomic DNA content (2C) was estimated at 2.46 ± 0.04 pg in the mother plant and 2.41 ± 0.05 pg in regenerated plants, with no significant variations in genome size or chromosome length. These results demonstrate that cytogenomics can be used to effectively evaluate chromosome-level genomic stability in regenerated A. elata plants.

Genetic Diversity Analysis and Polyploid Induction Identification of Idesia polycarpa

Idesia polycarpa from Sichuan is a valuable germplasm with high economic potential, but it faces variety scarcity. To address this, this study collected 16 varieties (lines), identifying IpHT1 as a promising parent due to its high oil content (38.5%) and red fruits. Polyploid induction via adding 0.50% colchicine to Murashige and Skoog (MS) medium yielded 520 IpHT1 mutagenized seedlings. Subsequently, flow cytometry (FCM) was performed on 401 morphologically variant seedlings which had been initially screened, resulting in the identification of 15 suspected triploids, 35 suspected tetraploids, and 3 chimeras. Furthermore, fluorescence in situ hybridization (FISH) analysis found that the probe (AG3T3)3 had terminal signals at both ends of each chromosome, allowing for the counting of 42 chromosomes in diploids and 84 in tetraploids. The probe 5S rDNA showed 2, 3, and 4 hybridization signals in the interphase nuclei of diploid, triploid, and tetraploid cells, respectively, but the probe (GAA)6 failed to produce any signal on I. polycarpa chromosomes. Ultimately, 18 polyploids were selected, including 7 triploids and 11 tetraploids. Triploids and tetraploids showed significant leaf morphological and physiological differences from diploids. Consequently, this study successfully established a polyploid breeding system for I. polycarpa, thereby enhancing its genetic diversity and breeding potential.

Symbiotic bacteria play crucial roles in a herbivorous mite host suitability.

The tomato russet mite (TRM), Aculops lycopersici, is a strictly herbivorous and economically significant pest that infests Solanaceae plants, but its host suitability varies, showing high performance on tomatoes. Although symbiotic bacteria have been suggested to play crucial roles in the host adaptation of herbivores, their effects on TRM remain unclear. In this study, using next generation high-throughput sequencing of the bacterial 16S rRNA data, we identified the bacterial diversity and community composition of TRM feeding on tomato, eggplant, and chili. Our results show no significant difference in the bacterial community composition of TRM across three host plants. However, the relative density of Escherichia coli (TRM_Escherichia) showed 9.36-fold higher on tomato than on eggplant and chili. These results align with the observed TRM performance among three host plants. When TRM_Escherichia was reduced using antibiotics, the treated TRM decreased the population density on tomato. However, when we transferred TRM from eggplant to tomato, the population density of TRM increased, coinciding with an increase of the TRM_Escherichia density. These results indicate that TRM_Escherichia may affect the host suitability of TRM. Our fluorescence in situ hybridization (FISH) results further showed that TRM_Escherichia is primarily distributed in the salivary glands. Metagenomic data results suggest that TRM_Escherichia functions in food digestion and energy metabolism. We provided the first comprehensive analysis of TRM bacterial communities. Our findings demonstrate that the symbiotic bacterium TRM_Escherichia may play crucial roles in the suitability of TRM feeding on different Solanaceae hosts. © 2024 Society of Chemical Industry.

WITHDRAWN: Expert Consensus on the Diagnosis and Treatment of FGFR Gene-altered Solid Tumors

The fibroblast growth factor receptor (FGFR) is a crucial receptor tyrosine kinase involved in essential biological processes, including growth, development, and tissue repair. However, FGFR gene mutations, including amplification, fusion, and mutation, can disrupt epigenetics, transcriptional regulation, and tumor microenvironment interactions, leading to cancer development. Targeting these kinase mutations with small molecule drugs or antibodies has shown clinical benefits. For example, Edatinib is approved for treating locally advanced or metastatic urothelial cancer patients with FGFR2/FGFR3 mutations, and Pemitinib is approved for treating cholangiocarcinoma with FGFR2 fusion/rearrangement. Effective screening of FGFR variant patients is crucial for the clinical application of FGFR inhibitors. Various detection methods, such as polymerase chain reaction (PCR), next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC), are available, and their selection should be based on diagnostic and treatment decision-making needs. Our developed expert consensus aims to standardize the diagnosis and treatment process for FGFR gene mutations and facilitate the practical application of FGFR inhibitors in clinical practice.

Giant Cell-Rich Tumors of the Skeleton.

The accurate diagnosis of giant cell-rich tumors of bone is challenging, especially in limited tissue samples. This diverse group of neoplasms have similar and often ambiguous clinical presentations, radiologic features, and morphologic characteristics. During the last decade, the discovery of pathogenic recurrent genetic alterations has allowed the development of immunohistochemical surrogate markers and FISH assays that can help differentiate the entities of this broad group from one another. The correct diagnosis of these neoplasms is essential in the management of the affected patients.

Navigating the implementation gap for Trastuzumab's journey from health insurance to patient access: a preliminary study in a hospital in China

BackgroundTrastuzumab, a monoclonal antibody for breast cancer, faces global accessibility challenges, primarily due to high costs. This study examines how changes in medical insurance policies and price adjustments influence Trastuzumab utilization in China, focusing on implementation challenges and their impact on drug accessibility and affordability.MethodsThis retrospective study investigated Trastuzumab utilization among HER2-positive breast cancer patients at a tertiary hospital from May 2013 to December 2022 in JX, a prefecture-level city in eastern China. Patients were stratified based on their CerbB2 indicator, because those with a 2 + CerbB2 status require further FISH testing to confirm Trastuzumab eligibility. We analyzed the utilization rates of both FISH test and Trastuzumab using logistic regressions, identifying factors that influence their usage.Results766 patients were included. The utilization rate of Trastuzumab among patients with CerbB2 3+ rose from 40.40 to 77.94% in September 2017 following the expansion of insurance coverage, and further increased to 90.41% after a price reduction in 2020. The FISH test was not covered by health insurance, and it was not available in the local hospital until Trastuzumab became available in JX city. For patients with CerbB2 2+, the proportion undergoing the FISH test increased dramatically from 8.89 to 82.08% after the price reduction in 2020. The mere inclusion into basic medical insurance, regardless of insurance types, significantly increased the utilization of Trastuzumab and the FISH test. However, rural patients in JX city are still facing financial burdens with Trastuzumab's out-of-pocket cost accounting for 62.9% of their annual disposable income in 2020.ConclusionsOur findings indicate that insurance coverage and price reduction significantly increased Trastuzumab utilization. However, failure to improve the accessibility of the FISH test can pose challenges in enhancing the uptake of Trastuzumab among eligible patients. With the expiration of Trastuzumab's patent and the development of affordable biosimilars, there are now greater opportunities to enhance treatment access globally. These insights can inform policy makers of implementation guidance about providing financial support for breast cancer patients in other LMICs.

Copy number alterations in pediatric B-cell precursor acute lymphoblastic leukemia patients and their association with patients' outcome.

Genetic abnormalities provide diagnostic and prognostic information for pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. The aim of this study was to determine the effects of genetic CNAs and RUNX1 gene abnormalities on the outcome of pediatric BCP-ALL patients. This study included 78 de novo-BCP-ALL pediatric patients who presented to the Pediatric Oncology Department of the National Cancer Institute (NCI), Cairo University. We aimed to study the impact of copy number alteration (CNA) of 8 of the most altered genes in BCP-ALL patients, in addition to RUNX1 gene abnormalities, on patient survival and response to treatment. Multiplex ligation-dependent probe amplification (MLPA) was used to detect CNA, while RUNX1 gene alterations were detected by fluorescence in situ hybridization (FISH). CNA of the PAX5 gene was significantly associated with worse overall survival (OS) and event-free survival (EFS) (P = 0.012 and P = 0.025, respectively). An increase in the CNA of ETV6 was associated with an increase in minimal residual disease (MRD) on day 15 (P = 0.041). Although RUNX1 gene abnormalities were not a predictor of shorter OS or EFS, an interesting significant association was found between PAX5 CNA and RUNX1 gene gain and translocation (P = 0.017 and P = 0.041, respectively). PAX5 CNA is an adverse prognostic factor. ETV6 CNA is associated with high MRD on day 15.

Dynamic changes in gut microbiota and production phenotypes driven by host genetic background in large yellow croaker

The gut microbiota is becoming increasingly important in enhancing aquaculture productivity. However, there are fewer studies on the effect of host genetic background on the gut microbiota of cultured fish. In the current study, we aimed to determine whether the genetic background of large yellow croaker influences differences in gut microbial composition and growth. To address this objective, we conducted a comparative experiment involving the nearshore cage culture of a selected line (SL) of large yellow croaker, which was subjected to genetic selection for swimming performance, alongside a control line (CL). Both lines were reared under identical environmental and dietary conditions for a duration of six weeks. We employed 16S rRNA gene sequencing technology to analyze the gut microbial composition of the large yellow croaker, and utilized bioinformatics methods to assess the abundance and diversity of the gut microbiota. The findings revealed that the core gut microbiota of both lines primarily comprised Proteobacteria, Firmicutes, and Bacteroidetes. However, a significant disparity in gut microbiota abundance was observed between SL and CL following nearshore cage culture. Additionally, the survival rate of SL reached 64.8 %, significantly higher than the 37.3 % observed in CL. The feed conversion efficiency of SL reached 47.7 %, significantly higher than that of CL (28.6 %). These results underscore the influence of host genetic background on driving differences in gut microbial community composition and production phenotypes. This research offers intriguing insights into the interconnectedness of gut microbiota, fish genetics, and production phenotypes.

Clinical features and genetic analysis of 471 cases of fetal congenital heart disease

BackgroundCongenital heart disease (CHD) is a heterogeneous collection of structural abnormalities of the heart or great vessels that are present at birth. These birth defects are one of the leading causes of infant mortality and morbidity worldwide. The etiology and pathogenesis of CHD are unclear and largely considered to be multifactorial in nature. Since the chromosomal profile of CHD has not been analyzed in a large sample size, we aimed to summarize the clinical features, cytogenetics findings, and pregnancy outcomes of CHD to provide a clinical reference for prenatal diagnosis.MethodsAmong 21,152 pregnant women, 471 (2.23%) showed fetal CHD on cordocentesis or amniocentesis. The number of cases showing simple CHD, simple CHD with concomitant extracardiac structural abnormalities, complex CHD, and complex CHD with concomitant extracardiac structural abnormalities was 128, 124, 89, and 130, respectively. For prenatal genetic diagnosis, karyotyping was performed with single-nucleotide polymorphism array(SNP-array)-based chromosomal microarrays, fluorescence in situ hybridization (FISH), copy number variation sequencing (CNV-seq), and BACs-on-Beads™ (BoBs) analyses. The results of ultrasonography examinations, genetic analyses, and pregnancy outcomes were recorded in detail.ResultsVentricular septal defects (VSDs) were observed in 245 (52.02%) cases of fetal CHD. Among the 471 cases of CHD, 258 (54.78%) showed other ultrasound abnormalities. The most common ultrasound abnormalities were abnormalities of the central nervous system. The 471 cases included 93 (19.75%) cases showing chromosomal abnormalities, and the incidence of these abnormalities increased with advanced maternal age or the presence of other ultrasound abnormalities. In eight cases, karyotype analysis showed normal results while SNP-array or CNV-seq results were abnormal. Among the 453 cases that were followed up, 166 (36.64%) involved pregnancy termination, 273 (60.26%) involved live births, 7 (1.55%) involved fetal death in utero, and 7 (1.55%) involved neonatal death after birth.ConclusionsFetuses with CHD showed higher rates of chromosomal abnormalities. In cases diagnosed with fetal CHD during fetal ultrasonic examination, the mothers should undergo a careful and comprehensive fetal ultrasound scan as well as prenatal genetic testing, including karyotype analysis and SNP-array or CNV-sequencing. The prognosis for simple fetal CHD is good, while the prognosis for complex fetal CHD and extracardiac anomalies is poor.

Development of Aegilops comosa and Aegilops caudata-specific molecular markers and fluorescence in situ hybridization probes based on specific-locus amplified fragment sequencing.

As tertiary gene pools of wheat, Aegilops comosa and Ae. caudata contain many excellent genes/traits and gradually become important and noteworthy wild resources for wheat improvement worldwide. However, the lack of molecular markers and cytological probes with good specificity and high sensitivity limits the development and utilization of Triticum aestivum-Ae. comosa (Ta. Aeco)/Ae. caudata (Ta. Aeca) introgression lines. Using specific-locus amplified fragment sequencing, two Ae. comosa and one Ae. caudata accessions, Chinese Spring, and three Ta. Aeco and Ta. Aeca introgression lines each were sequenced to develop new molecular markers and cytological probes. After strict sequence comparison and verification in different materials, a total of 39 molecular markers specific to three chromosomes in Ae. comosa (nine, seven, and 10 markers for 1M, 2M, and 7M, respectively) and Ae. caudata (two, six, and five markers for 3C, 4C, and 5C, respectively) and 21 fluorescence in situ hybridization (FISH) probes (one centromeric probe with signals specific to the M chromosomes, two centromeric probes with signals in all the tested genomes, and six, eight, and four FISH probes specific to the M, C, and M, C, and U chromosomes, respectively) were successfully exploited. The newly developed molecular markers and cytological probes could be used in karyotype studies, centromere evolutionary analyses of Aegilops, and had the ability to detect the fusion centromeres and small-fragment translocations in introgression lines.

Fast and accurate multi-bacterial identification using cleavable and FRET-based peptide nucleic acid probes

Fast and accurate identification of pathogenic microbes in patient samples is crucial for the timely treatment of acute infectious diseases such as sepsis. The fluorescence in situ hybridization (FISH) technique allows the rapid detection and identification of microbes based on their variation in genomic sequence without time-consuming culturing or sequencing. However, the recent explosion of microbial genomic data has made it challenging to design an appropriate set of probes for microbial mixtures. We developed a novel set of peptide nucleic acid (PNA)-based FISH probes with optimal target specificity by analyzing the variations in 16S ribosomal RNA sequence across all bacterial species. Owing to their superior penetration into bacteria and higher mismatch sensitivity, the PNA probes distinguished seven bacterial species commonly observed in bacteremia with 96–99.9% accuracy using our optimized FISH procedure. Detection based on Förster resonance energy transfer (FRET) between pairs of adjacent binding PNA probes eliminated crosstalk between species. Rapid sequential species identification was implemented, using chemically cleavable fluorophores, without compromising detection accuracy. Owing to their outstanding accuracy and enhanced speed, this set of techniques shows great potential for clinical use.

Cytogenetic features of intergeneric amphydiploids and genome-substituted forms of wheat

Synthetic intergeneric amphydiploids and genome-substituted wheat forms are an important source for transferring agronomically valuable genes from wild species into the common wheat (Triticum aestivum L.) genome. They can be used both in academic research and for breeding purposes as an original material for developing wheatalien addition and substitution lines followed by translocation induction with the aid of irradiation or nonhomologous chromosome pairing. The chromosome sets and genome constitutions of allopolyploids are usually verified in early hybrid generations, whereas the subsequent fate of these hybrids remains unknown in most cases. Here we analyze karyotypes of five hexa- (2n = 6x = 42) and octoploid (2n = 8x = 56) amphydiploids of wheat with several species of the Aegilops, Haynaldia, and Hordeum genera, and six genome-substituted wheat–Aegilops forms, which were developed over 40 years ago and have been maintained in different gene banks. The analyses involve C-banding and fluorescence in situ hybridization (FISH) with pAs1 and pSc119.2 probes. We have found that most accessions are cytologically stable except for Avrodes (genome BBAASS, a hexaploid genome-substituted hybrid of wheat and Aegilops speltoides), which segregated with respect to chromosome composition after numerous reproductions. Chromosome analysis has not confirmed the presence of the N genome from Ae. uniaristata Vis. in the genome-substituted hybrid Avrotata. Instead, Avrotata carries the D genome. Our study shows that octoploid hybrids, namely AD 7, AD 7147 undergo more complex genome reorganizations as compared to hexaploids: the chromosome number of two presumably octoploid wheatAegilops hybrids were reduced to the hexaploid level. Genomes of both forms lost seven chromosome pairs, which represented seven homoeologous groups and derived from different parental subgenomes. Thus, each of the resulting hexaploids carries a synthetic/hybrid genome consisting of a unique combination of chromosomes belonging to different parental subgenomes.

Detailed cytogenetic analysis of three duck species (the northern pintail, mallard, and common goldeneye) and karyotype evolution in the family Anatidae (Anseriformes, Aves)

Galliformes and Anseriformes are two branches of the Galloanserae group, basal to other Neognathae. In contrast to Galliformes, Anseriformes have not been thoroughly researched by cytogenetic methods. This report is focused on representatives of Anseriformes and the evolution of their chromosome sets. Detailed cytogenetic analysis (G-banding, C-banding, and fluorescence in situ hybridization) was performed on three duck species: the northern pintail (Anas acuta, 2n = 80), the mallard (A. platyrhynchos, 2n = 80), and the common goldeneye (Bucephala clangula, 2n = 80). Using stone curlew (Burhinus oedicnemus, 2n = 42, Charadriiformes) chromosome painting probes, we created homology maps covering macrochromosomes and some microchromosomes. The results indicated a high level of syntenic group conservation among the duck genomes. The two Anas species share their macrochromosome number, whereas in B. clangula, this number is increased due to fissions of two ancestral elements. Additionally, in this species, the presence of massive heterochromatic blocks in most macroautosomes and sex chromosomes was discovered. Localization of clusters of ribosomal DNA and telomere repeats revealed that the duck karyotypes contain some microchromosomes that bear ribosomal RNA genes and/or are enriched for telomere repeats and constitutive heterochromatin. Dot plot (D-GENIES) analysis confirmed the established view about the high level of syntenic group conservation among Anatidae genomes. The new data about the three Anatidae species add knowledge about the transformation of macro- and sex chromosomes of Anseriformes during evolution.

Development of an immobilized Mycobacterium tuberculosis purine nucleoside phosphorylase platform for ligand fishing and inhibition assays

Purine nucleoside phosphorylase (PNP) from Mycobacterium tuberculosis (MtPNP) plays a crucial role in purine metabolism, making it an attractive target for developing new tuberculosis treatments. In this study, we developed a ligand screening platform using MtPNP covalently immobilized on magnetic particles (MtPNP-MPs). The immobilization process achieved a high enzyme loading and preserved the enzyme catalytic activity, enabling its use in both activity and affinity-based screening assays. The activity of MtPNP-MPs was monitored by quantifying hypoxanthine released from inosine phosphorolysis, and kinetic studies revealed Michaelis-Menten behavior for inosine and inorganic phosphate substrates, with KM values comparable to those of free MtPNP. A proof-of-concept inhibitor study using the transition state analog DI4G demonstrated the platform capability for recognizing and characterizing inhibitors, yielding an IC50 value of 91.4 nM and a competitive inhibition mechanism with a Ki of 69.2 nM. Furthermore, the MtPNP-MPs exhibited high stability, retaining over 80 % of their activity after six months of storage and more than 90 % after five consecutive reaction cycles, highlighting their potential for reuse in high-throughput assays. We optimized key parameters for ligand fishing assay, including the amount of MtPNP-MPs, incubation time, and elution conditions. While higher organic solvent concentrations and longer elution times improved ligand isolation, these conditions also reduced enzyme activity. This trade-off between ligand isolation yield and enzyme reusability suggests that elution conditions should be tailored based on the ligand binding strength. Overall, this study establishes the MtPNP-MPs platform as a versatile tool for ligand identification and inhibitor characterization, with promising applications in the screening of complex libraries, such as natural products, for bioactive compounds.

Overdiagnosis of atypical lipomatous tumors/well-differentiated liposarcomas by morphological diagnosis using only HE stained specimens: a case–control study with MDM2/CDK4 immunostaining and MDM2/CDK4 fluorescence in situ hybridization

BackgroundLipomatous tumors represent the most common type of soft tissue neoplasms. Mouse double minute 2 homolog (MDM2)/cyclin-dependent kinase 4 (CDK4) immunostaining is considered effective in differentiating between benign lipomas and intermediate malignant atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDLPSs). However, these tumors have traditionally been diagnosed histopathologically using hematoxylin and eosin-stained specimens, which is referred to here as morphological diagnosis. In this study, the accuracy of morphological diagnoses that had been made before MDM2/CDK4 immunostaining became available for distinguishing between lipoma and ALT/WDLPS was examined.MethodsThe study participants were 109 patients with a morphological diagnosis of lipoma (68 patients) or ALT/WDLPS (41 patients) who had undergone surgical resection of the tumor in our hospital between 2009 and 2012. Tissue samples from all patients were used for MDM2/CDK4 immunostaining and the confirmation of MDM2/CDK4 amplification by fluorescence in situ hybridization (FISH).ResultsOf the 41 patients with a morphological diagnosis of ALT/WDLPS, only 17 were positive for MDM2 FISH. In addition, one of the 68 patients with a morphological diagnosis of lipoma showed MDM2 amplification by FISH. When the definitive diagnosis of ALT/WDLPS was made by the positive results of MDM2 FISH, the sensitivity and specificity of morphological diagnosis were 41.5% and 98.5%, respectively. The sensitivity of MDM2 and CDK4 immunostaining was 55.6% and 40.0%, respectively, and their specificity was 87.0% and 84.6%, respectively. This indicates that the diagnostic accuracy of these immunostaining assays was not particularly high. The clinical features suggesting ALT/WDLPS were: patient age (older), maximum tumor diameter (large, cut-off value of 125 mm), tumor location (lower limb), and tumor depth (deep-seated).ConclusionsMorphological diagnosis alone can accurately diagnose lipomas. However, it has a propensity to overdiagnose ALT/WDLPS. Thus, MDM2 FISH should be used more proactively, not only for lesions with obvious morphological abnormalities, but also for lipomatous tumors that are clinically suggestive of ALT/WDLPS.

Development and Testing of a Small-Scale Fish Filleting Machine for an Indian Major Carp, Rohu (Labeo rohita)

ABSTRACT This paper describes a filleting machine designed for the popular Asian freshwater species rohu (Labeo rohita). The machine comprises three key units: conveying, filleting, and water spraying and collection. The filleting unit has two parallel disc blades for precise cutting, with adjustable spacing to accommodate a variety of fish backbone thicknesses. The conveying unit employs belt drives and spring-adjusted entry for smooth transport, while the water management unit sprays and cleans the filleting blades, ensuring efficient fish movement. The machine performed efficiently when set to its optimal settings and showed significant improvements compared to manual filleting while maintaining product quality.

Low-Level MDM2 Amplification by FISH: An Institutional Experience With a Diagnostic Dilemma.

Background: MD M2 (murine double minute-2) amplification via fluorescence in-situ hybridization (FISH) is the gold standard test used for confirming the diagnosis of atypical lipomatous tumor/well differentiated liposarcoma and dedifferentiated liposarcoma. It is also used as a screening test in high grade spindle cell or pleomorphic neoplasms. MDM2 FISH is considered positive for amplification when the MDM2/CEP12 ratio is greater than 2; however, a ratio between 2 and 3 is considered a "low-level" amplification and raises the possibility of a false positive result; thus, posing a diagnostic dilemma. Another molecular modality, next generation sequencing (NGS) assay, can help in such situations in confirming or excluding the M D M 2 amplification status. Confronted by a number of neoplastic specimens at our institution with "low-level" MDM2 amplification via FISH, we aimed to assess the specificity of fluorescence in situ hybridization (FISH) in such tumors by comparing their NGS assay results and determine an accurate MDM2 amplification status that further aids in diagnosis. Methods: Tumors with "low-level" MDM2 amplification via FISH, defined as MDM2/CEP12 ratio between 2 and 3, and harboring a high grade and/or pleomorphic morphology were retrospectively retrieved from our institutional archives from the last five years. The retrieved specimens were evaluated for concordant retrospective Oncomine v3 analysis. Oncomine v3 is an institutional NGS assay that covers 161 genes and assesses for DNA mutations, RNA fusions, and copy number alterations including MDM2 gene gain or amplification. The tumors with Oncomine v3 results were compared and the FISH specificity was calculated. Results: Twenty-seven high grade and/or pleomorphic tumors with "low-level" MDM2 amplification were retrieved. Eight out of twenty-seven tumors had Oncomine v3 performed on them. The tumors correlated to neoplasms from different lineage types including undifferentiated melanoma, sarcomatoid squamous cell carcinoma, leiomyosarcoma, myxofibrosarcoma, undifferentiated pleomorphic sarcoma, and high-grade poorly differentiated pleomorphic neoplasm. All 8 tumors had a low-level MD M2 amplification ratio ranging between 2.09 and 2.84. Seven out of eight had no MD M2 copy number alteration. One only (leiomyosarcoma) demonstrated MD M2 copy number gain (∼5 copies) that did not qualify as amplification due to the "6 copy number" gain cutoff. TP53, CDKN2A/B, PIKC3, and PTEN alterations were the most common genetic aberrations detected by Oncomine v3. Conclusion: We demonstrated the absence of MDM2 amplification via Oncomine in all our 8 "low-level" MDM2 FISH amplification specimens confirming the FISH results as "false positive" with a corresponding FISH specificity rate of 0%. Laboratory measures and utilizing NGS assay when needed, could be implemented when encountering such problematic "low-level" MDM2 amplification specimens to avoid misdiagnosis and misuse of targeted therapy. Future studies are needed to better characterize and investigate such findings.

Knockdown of the long noncoding RNA VSIG2-1:1 promotes the angiogenic ability of human pulmonary microvascular endothelial cells by activating the VEGF/PI3K/AKT pathway

BackgroundAbnormal pulmonary vascular development poses significant clinical challenges for infants with bronchopulmonary dysplasia (BPD). Although numerous factors have been suggested to control the development of pulmonary blood vessels, the mechanisms underlying the role of long noncoding RNAs (lncRNAs) in this process remain unclear.MethodsA lncRNA array was used to measure the differential expression of lncRNAs in premature infants with and without BPD. The expression of lncRNA-VSIG2-1:1 in patients with BPD and hyperoxia-induced human pulmonary microvascular endothelial cells (HPMECs) was assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Fluorescence in situ hybridization (FISH) assay was performed to detect the subcellular localization of lncRNA-VSIG2-1:1. Pulmonary microvascular endothelial cells were stably transfected with adenoviral vectors to silence or overexpress lncRNA-VSIG2-1:1. The effects of lncRNA-VSIG2-1:1 on the proliferation, migration, and tube formation abilities of HPMECs subjected to hyperoxia were examined by performing Cell Counting Kit‐8 (CCK-8), cell migration, and tubule formation assays. RNA sequencing (RNA-seq) was performed to determine the correlation between lncRNA-VSIG2-1:1 and phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT). The protein levels of vascular endothelial growth factor (VEGF), p-PI3K, PI3K, p-AKT, and AKT were determined using western blotting.ResultsThe expression of lncRNA-VSIG2-1:1 was upregulated in patients with BPD and hyperoxia-treated HPMECs. Inhibiting lncRNA-VSIG2-1:1 expression promoted the proliferation, migration, and tube-formation abilities of HPMECs, while significantly increasing VEGF, p-PI3K, and p-AKT levels.ConclusionOur findings reveal that the suppression of lncRNA-VSIG2-1:1 expression stimulates angiogenesis in vitro by inducing the initiation of the VEGF/PI3K/AKT signaling pathway. This observation may aid the development of novel therapeutic targets for treating BPD.

Adamantinoma-like Ewing Sarcoma: A Report of Two Rare Cases in the Pancreatic Tail and Spine

Adamantinoma-like Ewing sarcoma (ALES), an extremely rare variant of Ewings sarcoma occurs primarily in the head and neck sites. ALES is characterized by basaloid cytomorphology, immunohistochemically expresses keratins, p63, p40, CD99, and harbors t (11; 22) EWSR1::FLI1 translocation on fluorescence in situ hybridization (FISH). The current case report discusses two cases of ALES, one in the pancreatic tail of a 64-year-old male and the second in the lumbar spine of an 18 yr old male. The IHC for both cases showed similar picture, confirming ALES through Fluorescence in situ hybridization (FISH), which indicated EWSR1 gene rearrangement. ALES being relatively rare and aggressive, diagnosis and the treatment poses significant challenges. Diagnosis relies on morphological, immunohistochemical, and molecular findings. The diagnostic findings helped in establishing a diagnosis of sarcoma which could have easily been misdiagnosed as a carcinoma,thus aiding appropriate selection of therapeutic strategies. Due to its rarity, further research is necessary to improve diagnostic accuracy and treatment outcomes.

Approaching the sterile womb theory in dogs and cats: A multi-technique investigation

The study investigated whether bacterial seeding occurs in utero in dogs and cats using a multi-technique approach, including 16S rRNA gene sequencing, culture, and fluorescence in situ hybridization (FISH). Healthy pregnant bitches and queens (n = 8) undergoing ovariohysterectomy between 30 and 45 days of gestation were included. Placenta and amniotic fluid samples from two foetuses per dam, along with multiple controls (uterine serosa, sampling table, and surgeon's gloves), were collected and analysed.Bacterial sequences were detected in all foetal samples, with no significant differences in intra- and inter-sample diversity (i.e., alpha and beta diversities, respectively) based on sample type or species. However, the surgeon's gloves showed species-specific differences in bacterial composition. After removing control sequences, significant differences based on foeto-maternal units emerged. Moraxella spp. was cultured from the canine placenta with the highest bacterial load, and Burkholderia cepacia was isolated from two feline placentae. FISH showed low bacterial presence in 50 % of placentae without histological signs of inflammation. No bacterial growth was observed in amniotic fluid or control samples.A multi-technique approach, including multiple controls, is essential in studies involving low-biomass samples, as the results of the present study indicated that contamination could mask real bacterial composition of foetal samples. After decontamination, differences were observed based on the foeto-maternal unit.This study confirmed the presence of bacterial DNA in placental and amniotic fluid samples during mid-term, healthy pregnancies in dogs and cats. Bacterial DNA is not equivalent to living bacteria, hence bacterial DNA fragments in the uterus probably originate from the dam but are unlikely to indicate the presence of a resident foetal microbiome.