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PubMed2026

Single-Molecule Fluorescence In Situ Hybridization (smFISH) for RNA Localization Relative to a DNA Locus in Bacteria.

When studying the localization of RNA within a cell, it is important to probe RNA relative to a subcellular landmark, such as a gene locus (transcription site). In this chapter, we describe a single-molecule fluorescence in situ hybridization (smFISH) protocol for labeling target RNAs and their DNA loci in bacteria. This is a versatile protocol applicable to various genes and species, providing valuable insights into RNA localization in bacterial cells.

In Situ Hybridization, Fluorescence↗

PubMed2026

An Integrative Morphological and Genomic Analysis With a Refined Fluorescence In Situ Hybridization (FISH) Threshold and Novel Kinase Fusions in a Large Asian Cohort of Spitzoid Neoplasms.

Differentiating atypical Spitz tumors (ASTs) from true Spitz melanomas (SMs) and conventional melanomas with spitzoid features (MSFs) remains a formidable diagnostic challenge. Because current molecular epidemiological data are overwhelmingly derived from Caucasian cohorts, the genomic landscape of Asian populations remains largely unexplored. To elucidate the molecular progression landscape and refine the diagnostic criteria, we performed a comprehensive multimodal analysis-integrating histomorphology, immunohistochemistry, multiprobe fluorescence in situ hybridization (FISH), and targeted RNA/DNA-based next-generation sequencing (NGS)-on a cohort of 140 spitzoid neoplasms. This cohort, comprising 126 ASTs, 8 SMs, and 6 MSFs, represents the largest Asian cohort to date. Malignant phenotype strongly correlated with lesional asymmetry, deep atypical mitoses, a sheet-like growth pattern, diffuse preferentially expressed antigen of melanoma positivity, and significant loss of p16 expression (64.3% in SM/MSF vs 9.5% in ASTs; P < .0001). Building upon the established melanoma FISH criteria, we optimized a prognostic threshold of ≥2 FISH abnormalities specifically tailored for spitzoid neoplasms. We demonstrated that isolated single chromosomal aberrations (particularly MYB loss) are relatively stable events that are frequent in indolent ASTs, whereas our refined ≥2 threshold yielded 100% sensitivity and 92.5% specificity for predicting regional lymph node metastasis/local recurrence. Molecularly, NGS identified mutually exclusive initiating driver alterations (comprising kinase fusions and HRAS mutations) in 89.9% of true Spitz neoplasms, a remarkably high prevalence suggesting a distinct genetic background in Asian populations. We also characterized 5 entirely novel kinase fusions (ZNF24::ROS1, PCBP1::ROS1, NUMA1::RET, CBWD1::ALK, and TPR::NTRK1). Furthermore, NGS definitively segregated true Spitz neoplasms from morphological mimics (MSF), which lacked fusions and were driven by canonical genomic alterations of the conventional melanoma pathway. Integrating these genomic landscapes validated a stepwise progression model. Although isolated kinase fusions drove indolent ASTs, malignant SM invariably harbored concurrent pathogenic secondary alterations, demonstrating a profound reliance on CDKN2A/B, TP53, and CDK4 aberrations. Ultimately, we propose an integrated diagnostic algorithm combining morphological evaluation, the refined FISH threshold, and comprehensive NGS profiling, providing a precise, evidence-based framework for pathway classification and clinical management of spitzoid neoplasms.

fluorescence in situ hybridization↗

PubMed2025

Transcript-guided targeted cell enrichment for scalable single-nucleus RNA sequencing.

Large-scale single-cell atlases have revealed many aging- and disease-associated cell types, yet these populations are often underrepresented in heterogeneous tissues, limiting detailed molecular analyses. To address this, we developed EnrichSci-a scalable, microfluidics-free platform that combines hybridization chain reaction RNA fluorescence in situ hybridization (FISH) with combinatorial indexing to profile single-nucleus transcriptomes of target cell types with full gene-body coverage. Applied to oligodendrocytes in the aging mouse brain, EnrichSci uncovered aging-associated molecular dynamics across distinct oligodendrocyte subtypes, revealing both shared and subtype-specific gene expression changes. Additionally, we identified aging-associated exon-level signatures missed by conventional gene-level analyses, highlighting post-transcriptional regulation as a critical dimension of cell-state dynamics in aging. By coupling transcript-guided enrichment with a scalable sequencing workflow, EnrichSci provides a versatile approach to decode dynamic regulatory landscapes in diverse cell types from complex tissues.

Animals↗

PubMed2025

Cytotype classification and genetic diversity of Platostoma palustre revealed by rDNA localization and chloroplast genome.

BACKGROUND: Platostoma palustre A. J. Paton is an edible medicinal plant that plays a significant role in traditional food production and medicinal applications. However, the genetic basis of P. palustre remains unclear, thereby hampering research on its genome and polyploid evolution. RESULTS: To characterize the karyotype and ploidy of P. palustre, we performed fluorescence in situ hybridization (FISH) by using 35 S and 5 S rDNA probes in P. palustre. FISH results indicated that 35 S rDNA mapped to the end of the chromosome (chromosome satellite, heterochromatic region) and that 5 S rDNA was located close to the centromere of the chromosomes. Based on the rDNA sites, we identified three distinct cytotypes of P. palustre: diploid (2n = 2x = 30, x = 15), triploid (2n = 3x = 45, x = 15), and tetraploid (2n = 4x = 60, x = 15). To further explore the genetic evolutionary relationship among these P. palustre cytotypes, we conducted Illumina sequencing and assembled the chloroplast (CP) genome. The CP genomes of P. palustre accessions maintained a conserved single circular molecule with a length of 152,534 - 152,788 bp, comprising a large single-copy region (LSC) and small single-copy region (SSC) separated by two inverted repeat regions (IRs). Phylogenetic trees were also created based on CP and nuclear molecular markers, showing that most P. palustre accessions clustered together corresponding to their collection regions. Of these, GDZC2 (2n = 2x = 30) clustered with several triploid accessions, suggesting that it may share a common ancestor with these triploid accessions. CONCLUSIONS: This is the first study to characterize the karyotype, identify three cytotypes of P. palustre using FISH, and provide molecular evidence for an evolutionary relationship among different P. palustre accessions. These findings will be useful for further genomic studies and polyploid evolution of P. palustre.

Genome, Chloroplast↗

PubMed2025

Simultaneous Visualization of Protein and Genomic Regions in Plant Nuclei.

Immunohistostaining (IHS) is a widely used technique in diagnostic and research laboratories in which specific antibodies are used to detect and visualize a protein of interest in cells or tissues. Similarly, with specific oligonucleotide probes, the fluorescence in situ hybridization (FISH) method allows one to visualize genomic regions and to analyze its localization in the nuclear space. Here, we describe a combined FISH-IHS technique that enables researchers to determine the localization of protein and genomic loci in plant nuclei simultaneously. This method can be applied to extracted nuclei and sections of paraffin-embedded tissues. It provides a valuable tool to improve our understanding of nuclear dynamics by revealing the spatial relationship between specific genomic loci and target proteins.

In Situ Hybridization, Fluorescence↗

PubMed2025

Optical Genome Mapping Is a Powerful Diagnostic Tool in Non-Hodgkin Lymphoma.

Non-Hodgkin lymphoma (NHL) is a diverse and heterogeneous group of hematological malignancies. These lymphomas arise from the clonal proliferation of either B/T or natural killer lymphocytes, and their correct classification relies partly on identifying characteristic structural variants and copy number alterations. Current standard-of-care technologies for detecting these genomic features, chromosome banding analysis (CBA) and fluorescent in situ hybridization (FISH), are labor intensive and have specific limitations. CBA has low resolution and relies on viable cell culture, whereas the targeted approach of FISH does not provide the whole genome view required for comprehensive disease characterization. This highlights the need for higher-resolution nontargeted genomic methods. Previous studies have evaluated optical genome mapping (OGM) as a whole genome alternative for cytogenomic characterization in NHL diagnostics but were restricted in number and to cases with peripheral blood and/or bone marrow invasion. Here, we selected a comprehensive cohort of 110 NHL cases (79 B-NHL and 31 T-NHL/natural killer-NHL) derived from different types of tissue biopsies, all with established histopathological diagnoses. Seventy-eight samples were genomically well characterized at diagnosis by CBA and FISH. The remaining 32 cases were included because of previous CBA failure, although FISH data were available for 20 cases. OGM provided informative results in 94% of the cohort, with a high concordance rate of 97.6% compared with CBA/FISH in detecting clinically relevant aberrations. The 2 variants that were missed were both present at the detection threshold of OGM. In contrast, OGM successfully resolved 26 samples with previous CBA failure and detected 3 additional disease-defining events, resulting in diagnostic reclassification of 1 patient. Finally, OGM identified novel recurrent aberrations that warrant further investigation into their pathogenetic implications. To conclude, OGM robustly detects clinically relevant structural variants and copy number alterations and presents a promising alternative to CBA and FISH in routine diagnostic evaluation of NHL.

Humans↗

PubMed2026

Cytogenetic Diversity of Variant Philadelphia Translocations in Chronic Myeloid Leukemia.

INTRODUCTION: Chronic myeloid leukemia (CML) is a disease characterized by Philadelphia (Ph) translocations. These translocations can be classical or variant. The structural features and diagnostic implications of variant Philadelphia translocations remain incompletely defined, and they display considerable cytogenetic heterogeneity. METHODS: In this retrospective study, variant Ph translocations identified by conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) were systematically classified among 639 patients diagnosed with CML. A total of 35 patients with variant Ph translocations were included in the analysis. Molecular follow-up data, when available, were assessed using RT-qPCR analyses in a subset of patients. RESULTS: Chromosome analysis revealed 2 simple and 33 complex variant Ph translocations. FISH analysis, performed in 20 patients, identified deletions involving BCR, ABL1, or both in a limited number of cases. Additional chromosomal abnormalities and secondary translocations accompanied variant Ph translocations in four patients. The partner chromosomes involved in variant Ph translocations showed marked diversity, involving multiple chromosomal loci. CONCLUSION: Variant Philadelphia chromosome translocations in CML exhibit substantial cytogenetic diversity, reflecting the complexity of their underlying genomic architecture. The rarity and heterogeneity of these rearrangements complicate their classification and interpretation in routine diagnostic practice. Descriptive reporting of variant Ph translocations may contribute to a better understanding of their diagnostic complexity and support more accurate cytogenetic interpretation in CML.

Humans↗

PubMed2026

Testicular aggressive B-cell lymphoma with plasmablastic morphology harboring concurrent IGH::MYC and IGH::BCL2 rearrangements.

BACKGROUND: Aggressive B-cell lymphomas with plasmablastic morphology are uncommon neoplasms that may exhibit overlapping morphologic, immunophenotypic, and genetic features of plasmablastic lymphoma (PBL) and double-hit lymphoma (DHL). Concurrent IGH::MYC and IGH::BCL2 rearrangements are rarely encountered in this setting, particularly in the testis. Here, we describe an unusual case presenting significant diagnostic challenges at the interface between PBL and DHL. CASE PRESENTATION: We report a 66-year-old, immunocompetent man presenting with a 5 cm left testicular mass. Histologic examination revealed diffuse proliferation of large atypical lymphoid cells with plasmablastic morphology. Immunohistochemically, the tumor expressed CD138, CD38, and MUM1, with focal BCL2, c-MYC protein, and CD79a positivity, while CD20, CD19, PAX5, CD10, BCL6, and ALK were negative. EBV-encoded RNA in situ hybridization was negative. Fluorescence in situ hybridization identified IGH::MYC [t(8;14)] rearrangement in 45% and IGH::BCL2 [t(14;18)] rearrangement in 21% of analyzed nuclei. Next-generation sequencing additionally revealed BRAF V600E mutation, CDKN2A deletion, and human leukocyte antigen class I genomic alterations. CONCLUSION: This case highlights a rare testicular aggressive B-cell lymphoma with plasmablastic morphology and concurrent IGH::MYC and IGH::BCL2 rearrangements, representing a diagnostically challenging neoplasm at the interface between PBL and DHL. Our findings underscore the value of integrated morphologic, immunophenotypic, cytogenetic, and molecular analyses in evaluating aggressive B-cell lymphomas with plasmablastic features arising in immune-privileged sites.

Humans↗

PubMed2025

TRIM63 Overexpression in FISH-Negative MiTF Family Altered Renal Cell Carcinoma (MiTF RCC).

TFE3 and TFEB break-apart fluorescent in situ hybridization (FISH) assays are the "gold standard" for diagnostic confirmation of microphthalmia-associated transcription factor (MiTF) family-altered renal cell carcinoma (MiTF RCC), which includes TFE3-rearranged RCC and TFEB-altered RCC. However, FISH assays, for multiple reasons, may lead to equivocal or false-negative results, especially in cryptic fusions resulting from intrachromosomal inversions involving 5' partner genes, such as non-POU domain-containing octamer-binding protein (NONO); GRIPI-associated protein 1 (GRIPAP1); RNA-binding motif protein, X chromosome (RBMX); and RNA-binding motif protein 10 (RBM10). When FISH results are negative in cases with strong morphological suspicion of the listed tumor entities, pathologists may recommend targeted RT-PCR or panel-based RNA fusion sequencing for diagnostic confirmation. Our recent RNA in situ hybridization (RNA ISH)-based study demonstrated RNA expression of the tripartite motif containing 63 (TRIM63) to be highly enriched in TFE3-rearranged RCC and TFEB-altered RCC, including 2 FISH false-negative RCC cases harboring RBM10::TFE3 fusion. Based on these observations, we hypothesized that TRIM63 positivity could aid in diagnosing cases that are negative by conventional FISH assay but remain morphologically suspicious, representing an unmet clinical need in this area. We collected 20 RCC cases with morphological suspicion (with equivocal/indeterminate immunohistochemistry panel) of MiTF RCC, which were TRIM63 positive, negative/equivocal for TFE3/TFEB gene rearrangement by FISH, and underwent next-generation sequencing (NGS). On NGS correlation, 14 of 20 (70%) FISH-negative TRIM63-positive tumors harbored an MiTF gene rearrangement. In the remaining 6 cases, we were unable to fully ascertain the MiTF rearrangement status due to the inherent limitation of the NGS panel utilized. The cases with MiTF gene rearrangement include TFE3 rearrangement in 60% (12/20) and TFEB low-level copy gains (with an additional missense mutation in 1 case) in 10% (2/20) of samples. RBM10:TFE3 fusion was seen in 67% (8/12) of TFE3-rearranged RCC in this cohort. TRIM63 RNA ISH assay could aid in identifying cases that harbor TFE3 or TFEB rearrangement associated with false-negative or equivocal TFE3/TFEB FISH results, especially those involving gene fusions with a paracentric Xp11 inversion. Overall, employment of TRIM63 RNA ISH coupled with TFE3/TFEB FISH assays and follow-up genomic interrogation enhanced diagnostic accuracy for patients with MiTF RCC.

Carcinoma, Renal Cell↗

PubMed2025

Repetitive DNAs and differentiation of the ZZ/ZW sex chromosome system in the combtail fish Belontia hasselti (Perciformes: Osphronemidae).

BACKGROUND: Java combtail fish Belontia hasselti (Cuvier, 1831), a member of the Osphronemidae family, inhabits lakes and rivers throughout Southeast Asia and Sri Lanka. Previous cytogenetic research revealed it possesses a diploid chromosome number of 48 chromosomes with a female-heterogametic ZZ/ZW sex chromosome system, where the W chromosome is distinguishable as the only metacentric element in the complement. Female-heterogametic sex chromosome systems seem to be otherwise surprisingly rare in the highly diverse order Perciformes and, therefore, B. hasselti provides an important comparative model to evolutionary studies in this teleost lineage. To examine the level of sex chromosome differentiation in B. hasselti and the contribution of repetitive DNAs to this process we combined bioinformatic analyses with chromosomal mapping of selected repetitive DNA classes, and comparative genomic hybridization. RESULTS: By providing the first satellitome study in Perciformes, we herein identified 13 satellite DNA monomers in B. hasselti, suggesting a very low diversity of satDNA in this fish species. Using fluorescence in situ hybridization, we revealed detectable clusters on chromosomes only for four satellite DNA monomers. Together with the two mapped microsatellite motifs, the repeats primarily accumulated on autosomes, with no distinct clusters located on the sex chromosomes. Comparative genomic hybridization showed no region with accumulated female-specific or enriched repeats on the W chromosome. Telomeric repeats terminated all chromosomes, and no additional interstitial sites were detected. CONCLUSION: These data collectively indicate a low degree of sex chromosome differentiation in B. hasselti despite their considerable heteromorphy. Possible mechanisms that may underlie this pattern are discussed.

Animals↗

PubMed2005

Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors.

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.

Animals↗

PubMed2026

Molecular divergence and genomic composition of B chromosomes in the fish Cyphocharax modestus (Characiformes, Curimatidae).

B chromosomes are supernumerary elements that evolve from standard A chromosomes and are primarily composed of repetitive DNAs, yet their origin, diversification, and molecular composition remain poorly understood in most vertebrates. We investigated two allopatric populations of Cyphocharax modestus (Curimatidae) combining classical cytogenetics, comparative genomic hybridization (CGH), and comparative satellitomics to characterize the repetitive DNA landscape of its B chromosomes. While both populations exhibited a conserved karyotype of 2n=54 biarmed chromosomes, five individuals from the Batalha River (BR) carried supernumerary chromosomes, comprising two distinct variants: a C-positive B1 and an C-negative B2. Comparative satellitome analysis between 3B-carrying and B-lacking individuals identified 116 satellite DNAs (CmoSatDNAs), with the 3B library showing higher abundances of specific sequences. Fluorescence in situ hybridization (FISH) revealed that both B variants share two centromeric satellites (CmoSat01-192 and CmoSat02-108) with the A complement, while CmoSat58-47 was exclusively to B2. Minimum spanning tree analysis of CmoSat58-47 revealed B-exclusive haplotypes alongside haplotypes shared with B-lacking individuals, suggesting a recent origin for these chromosomes. CGH experiments further confirm the sequence sharing between the A and B chromosomes, supporting an intraspecific origin, and revealing substantial genomic differentiation among B variants.

Animals↗

PubMed2026

Optical genome mapping enhanced by refined variant interpretation in pediatric acute lymphoblastic leukemia.

Reliable detection of structural variants (SVs) and copy number variations (CNVs) is crucial in the contemporary diagnostics of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, limitations of commonly used conventional and molecular cytogenetic methods may hinder the accurate genetic characterization of patients. Optical genome mapping (OGM) offers a reliable alternative by enabling high-resolution, genome-wide detection of CNVs and SVs. Chromosomal aberrations were screened using OGM in 51 children with B-ALL. The results were compared with those of karyotyping, fluorescence in situ hybridization (FISH), digital multiplex ligation-dependent probe amplification (digitalMLPA), and targeted RNA sequencing (RNA-seq). OGM data showed high congruency with karyotyping and FISH findings, detecting clinically relevant variants beyond G-banding results and unraveling a complex KMT2A fusion undetected by FISH. Gene fusions involved in complex ETV6::RUNX1 translocations, but not detected by RNA-seq, were confirmed using FISH. Normalization of OGM copy number values with DNA-index-improved concordance with FISH-derived copy numbers in near-tri/tetraploid cases. In the peripheral regions of OGM variants (fringe-zones), a novel evaluation strategy called 'FriZone' was applied, which significantly improved the concordance between OGM and digitalMLPA. In addition, a co-segregation analysis revealed strong associations between ETV6::RUNX1 fusion and deletions of ETV6, RAG2, and NR3C2. OGM uncovered complex rearrangements undetected by widely used methods in 15% of cases, improving genetic classification and risk stratification in 10% of the patients. The FriZone analysis and normalization by DNA-index provide a refined, more accurate approach to OGM variant interpretation, facilitating the efficient application of OGM in clinical diagnostics. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Humans↗

PubMed2026

Whole-transcriptome-scale isoform-resolved spatial imaging of single cells in tissues.

Cell and tissue functions arise from complex interactions among numerous genes, and a systematic understanding of these functions requires isoform-resolved transcriptomic analysis of single cells with high spatial resolution. Here, we introduce an in situ RNA amplification method and its integration with multiplexed error-robust fluorescence in situ hybridization (MERFISH) to detect short RNA sequences and enable whole-transcriptome-scale, isoform-resolved spatial transcriptomics of individual cells in intact tissues. Using this approach, we imaged ∼33,000 distinct RNAs-including ∼23,000 genes and ∼10,000 isoforms-in the mouse brain. Our data enabled systematic analyses of region- and cell-type-specific gene programs and ligand-receptor-based cell-cell communications. These data further revealed rich spatial diversity and cell-type specificity in isoform usage across numerous genes, as well as brain structures particularly rich in isoform specificity. We anticipate broad application of this method for characterizing the molecular and cellular basis of tissue functions, unlocking previously inaccessible discoveries in cell and organismal biology.

Animals↗

PubMed2025

Spatial transcriptomics-aided localization for single-cell transcriptomics with STALocator.

Single-cell RNA-sequencing (scRNA-seq) techniques can measure gene expression at single-cell resolution but lack spatial information. Spatial transcriptomics (ST) techniques simultaneously provide gene expression data and spatial information. However, the data quality of the spatial resolution or gene coverage is still much lower than the quality of the single-cell transcriptomics data. To this end, we develop a ST-Aided Locator for single-cell transcriptomics (STALocator) to localize single cells to corresponding ST data. Applications on simulated data showed that STALocator performed better than other localization methods. When applied to the human brain and squamous cell carcinoma data, STALocator could robustly reconstruct the relative spatial organization of critical cell populations. Moreover, STALocator could enhance gene expression patterns for Slide-seqV2 data and predict genome-wide gene expression data for fluorescence in situ hybridization (FISH) and Xenium data, leading to the identification of more spatially variable genes and more biologically relevant Gene Ontology (GO) terms compared with the raw data. A record of this paper's transparent peer review process is included in the supplemental information.

Single-Cell Analysis↗

PubMed2025

Drought recovery in plants triggers a cell-state-specific immune activation.

All organisms experience stress as an inevitable part of life, from single-celled microorganisms to complex multicellular beings. The ability to recover from stress is a fundamental trait that determines the overall resilience of an organism, yet stress recovery is understudied. To investigate how plants recover from drought, we examine a fine-scale time series of RNA sequencing starting 15 min after rehydration following moderate drought. We reveal that drought recovery is a rapid process involving the activation of thousands of recovery-specific genes. To capture these rapid recovery responses in different Arabidopsis thaliana (A. thaliana) leaf cell types, we perform a single-nucleus transcriptome analysis at the onset of drought recovery, identifying a cell type-specific transcriptional state developing independently across cell types. To further validate the cell-type specific transcriptional changes observed during drought recovery, we employ spatial transcriptomics using multiplexed error-robust fluorescence in situ hybridization (MERFISH), revealing anatomical localization of recovery-induced gene expression programs across Arabidopsis leaf tissues. Furthermore, we reveal a recovery-induced activation of the immune system that occurs autonomously, and which enhances pathogen resistance in vivo in A. thaliana, wild tomato (Solanum pennellii) and domesticated tomato (Solanum lycopersicum cv. M82). Since rehydration promotes microbial proliferation and thereby increases the risk of infection, the activation of drought recovery-induced immunity may be crucial for plant survival in natural environments. These findings indicate that drought recovery coincides with a preventive defense response, unraveling the complex regulatory mechanisms that facilitate stress recovery in different plant cell types.

Arabidopsis↗

PubMed2025

Microbial signals in primary and metastatic brain tumors.

Gliomas and brain metastases are associated with poor prognosis, necessitating a deeper understanding of brain tumor biology and the development of effective therapeutic strategies. Although our group and others have demonstrated microbial presence in various tumors, recent controversies regarding cancer-type-specific intratumoral microbiota emphasize the importance of rigorous, orthogonal validation. This prospective, multi-institutional study included a total of 243 samples from 221 patients, comprising 168 glioma and brain metastases samples and 75 non-cancerous or tumor-adjacent tissues. Using stringent fluorescence in situ hybridization, immunohistochemistry and high-resolution spatial imaging, we detected intracellular bacterial 16S rRNA and lipopolysaccharides in both glioma and brain metastases samples, localized to tumor, immune and stromal cells. Custom 16S and metagenomic sequencing workflows identified taxa associated with intratumoral bacterial signals in the tumor microenvironment; however, standard culture methods did not yield readily cultivable microbiota. Spatial analyses revealed significant correlations between bacterial 16S signals and antimicrobial and immunometabolic signatures at regional, neighborhood and cellular levels. Furthermore, intratumoral 16S bacterial signals showed sequence overlap with matched oral and gut microbiota, suggesting a possible connection with distant communities. Together, these findings introduce microbial elements as a component of the brain tumor microenvironment and lay the foundation for future mechanistic and translational studies.

Humans↗

PubMed2026

A Novel piRNA-Mediated Epigenetic Axis: piR-36241 Exacerbates Pulpitis by Silencing the Protective Receptor ADGRG2 in Human Dental Pulp Stem Cells.

BACKGROUND: Pulpitis, a prevalent inflammatory dental disease, primarily results from bacterial infection, yet its epigenetic regulatory mechanisms remain poorly understood. PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs known to maintain genomic stability, have not been investigated in the context of pulpitis. OBJECTIVE: This study aimed to profile piRNA expression patterns in pulpitis and to elucidate the functional role and underlying mechanism of a specific piRNA, piR-36241 and its target gene ADGRG2 in disease progression. METHODS: piRNA and mRNA sequencing were performed on pulp tissues from patients with irreversible pulpitis and healthy controls. Potential piRNA targets were predicted bioinformatically. The interaction between piR-36241 and the 3'UTR of ADGRG2, an orphan adhesion G protein-coupled receptor (GPCR), was validated using dual-luciferase reporter assay, quantitative Real-Time PCR (qRT-PCR), fluorescence in situ hybridization (FISH), Western blot (WB), hematoxylin-eosin staining (HE), immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Functional analyses were conducted in LPS-stimulated human dental pulp cells (HDPCs) through gain-of-function and rescue experiments. RESULTS: We identified a distinct piRNA expression profile in pulpitis, with 21 piRNAs differentially expressed. piR-36241 was notably upregulated. It directly targeted the 3'UTR of ADGRG2 and suppressed its expression. ADGRG2, hypothesised to exert protective and homeostatic functions, was downregulated in pulpitis tissues and LPS-induced HDPCs. Overexpression of piR-36241 inhibited ADGRG2 expression and promoted the secretion of pro-inflammatory cytokines (IL-6, IL-8). Conversely, knockdown of ADGRG2 similarly enhanced inflammatory responses. Rescue experiments demonstrated that piR-36241 primarily regulates inflammation through ADGRG2 silencing. CONCLUSION: Our study reveals a novel pathogenic axis in pulpitis whereby upregulation of piR-36241 exacerbates inflammatory progression by repressing the protective receptor ADGRG2. These findings provide the first evidence of piRNA-mediated epigenetic regulation in pulpitis and highlight the piR-36241/ADGRG2 pathway as a potential therapeutic target for preserving pulp vitality.

Humans↗