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PubMed2026

Bioinformatics and Quantitative Real-Time Polymerase Chain Reaction Analysis of SUCNR1 and GPR37L1 in Schizophrenia.

Schizophrenia is a severe, complex, and multifactorial mental disorder involving numerous genetic susceptibility elements, leading to substantial disability, morbidity, and mortality. Despite significant progress in understanding its pathophysiology and etiology, specific diagnostic biomarkers for schizophrenia remain elusive. This study aimed to identify candidate molecular markers associated with schizophrenia. An integrated bioinformatics analysis was performed on the public microarray dataset GSE54913. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the most significantly enriched GO terms were related to channel activity, including passive transmembrane transporter activity, ion channel activity, gated channel activity, and substrate-specific channel activity. The top five enriched KEGG pathways were insulin secretion, cAMP signaling pathway, nucleotide excision repair, TNF signaling pathway, and glutathione metabolism. Validation was conducted using quantitative real-time polymerase chain reaction (qRT-PCR) on an independent sample set from Wuhan Rongjun Youfu Hospital. The qRT-PCR results were largely consistent with the microarray analysis (Pearson r = 0.89, 95% CI: 0.66-0.97). Protein-protein interaction (PPI) network analysis identified two hub genes, SUCNR1 and GPR37L1, which were significantly associated with the GO term 'ion channel activity' and enriched in the KEGG pathway 'insulin secretion'. Furthermore, SUCNR1 expression showed a negative correlation with verbal memory scores (r = -0.54, P = 0.015), whereas GPR37L1 expression showed a positive correlation (r = 0.59, P = 0.0034). These findings suggest that altered SUCNR1 and GPR37L1 expression may be associated with schizophrenia and may represent candidate molecular markers for further investigation.

Humans↗

PubMed2026

Generation and Phenotypic Characterization of a CRISPR/Cas9-Engineered Cracd-Deficient Mouse Model for Post-Myocardial Infarction Remodeling Studies.

Myocardial infarction (MI) remains a major cause of morbidity and mortality worldwide. This protocol describes a method for generating and characterizing a Cracd-deficient mouse line on the C57BL/6N background using CRISPR/Cas9 technology. Zygotes were co-injected with Cas9 mRNA, a gRNA construct, and a donor template designed to generate a 3153-bp genomic deletion. The edited allele was confirmed by PCR genotyping and Sanger sequencing. To assess the functional role of CRACD in post-MI remodeling, Cracd-deficient and wild-type (WT, C57BL/6N) mice underwent MI induced by permanent ligation of the left anterior descending coronary artery. On postoperative day 7, cardiac function and left ventricular wall motion were assessed using transthoracic echocardiography and speckle-tracking strain imaging, followed by histopathological evaluation with H&E and Masson's trichrome staining. Representative results showed that Cracd-deficient mice exhibited reduced ventricular dilation and preserved systolic function compared to WT controls. This protocol provides a reliable experimental platform for mechanistic studies of CRACD in cardiac pathophysiology.

Animals↗

PubMed2026

A Network Pharmacology and Molecular Docking Study of TongBi Formula for Osteoarthritis.

This study applied network pharmacology combined with molecular docking to predict the potential therapeutic targets and molecular mechanisms of TongBi Formula (TBF) in osteoarthritis (OA). Active components and corresponding targets of TBF were retrieved from the traditional Chinese medicine Systems Pharmacology Database and Analysis Platform, while OA-related targets were collected from Online Mendelian Inheritance in Man, GeneCards, DrugBank, and Therapeutic Target Database. A network visualization and analysis software was used to construct compound-target and protein-protein interaction (PPI) networks. Gene Ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery platform. Molecular docking analysis was conducted using a molecular docking software to evaluate the predicted binding affinity between key active compounds and core target proteins. A total of 47 overlapping targets between TBF and OA were identified. PPI network analysis highlighted JUN, RELA, IL6, MAPK1, and IL10 as potential hub targets. Enrichment analysis suggested that TBF may regulate inflammation, lipid metabolism, and multiple intracellular signaling pathways associated with OA progression. Molecular docking results demonstrated favorable predicted binding affinities between core active compounds and key OA-related protein targets. These findings provide a computational framework for understanding the potential mechanisms of TBF against OA and support further experimental validation.

Molecular Docking Simulation↗

PubMed2026

Genetic Evidence Links Sex Hormone-binding Globulin to Total Body Bone Mineral Density at Age 45-60 Years: A Two-sample Mendelian Randomization Study.

The menopausal transition and early postmenopause represent important periods for women's skeletal health, but the genetic relevance of metabolic, behavioral, and hormone-related factors to bone mineral density during midlife remains incompletely understood. This study used publicly available genome-wide association study summary statistics to examine associations between body mass index, 25-hydroxyvitamin D, sex hormone-binding globulin, high-density lipoprotein cholesterol, smoking initiation, and alcohol intake frequency and total body bone mineral density at ages 45-60 years. Exposure genome-wide association study summary statistics were derived from large European-ancestry populations and were not restricted to midlife women, whereas the outcome genome-wide association study captured an age-stratified total body bone mineral density phenotype at age 45-60 years. This age range overlaps with the menopausal transition and early postmenopause in women. Univariable, reverse, and multivariable Mendelian randomization analyses were performed, with inverse-variance weighting as the primary method and complementary sensitivity analyses used to assess heterogeneity, pleiotropy, and result stability. Genetically predicted higher sex hormone-binding globulin was associated with lower total body bone mineral density (β = -0.111, 95% CI: -0.170 to -0.051; P = 0.0003). Reverse Mendelian randomization did not support reverse causation from bone mineral density to sex hormone-binding globulin. Multivariable analyses suggested that this association persisted after adjustment for selected metabolic biomarkers. The other examined exposures did not show consistent evidence of association. These findings provide genetic evidence linking sex hormone-binding globulin to total-body bone mineral density at ages 45-60 years. Further prospective and predictive studies are needed to evaluate its clinical relevance beyond established bone health assessment tools.

Humans↗

PubMed2026

Trans-Mitochondrial Cybrid Generation from mtDNA Patient Platelets: An Efficient Protocol Optimizing Colony Selection and Functional Validation.

Trans-mitochondrial cybrid cell line generation represents the gold-standard method for determining pathogenicity by enabling biochemical analyses of a specific mitochondrial DNA (mtDNA) variant of interest at high and low percentages (heteroplasmy levels) within an otherwise identical mtDNA and nuclear genome background. Historically, the cybrid generation process has been tedious and poorly efficient. Here, we describe a highly efficient and effective protocol for generating trans-mitochondrial cybrid cell lines by fusing human platelets with a standard osteosarcoma 143B cell line to provide an isogenic nuclear background depleted of mtDNA (Rho0 cells). Cell isolates capture a given mtDNA genome of interest to establish stable cell lines harboring different degrees of heteroplasmy, or to compare divergent effects of distinct mitochondrial haplogroups. Because cybrids from mitochondrial patients may be more difficult to establish with standard protocols, this current methodology focuses on isolating mtDNA variants where the electron transport chain activity is affected. We here demonstrate that colony selection techniques reduce time and improve the yield of generating high-level heteroplasmy mtDNA mutant cybrid lines. A case study is provided of cybrid generation for a variant of unknown significance in MT-ND1, m.3985G>A (p.E227K). We analyze the efficiency of the cybrid generation process using this protocol and run functional studies performed by high-resolution respirometry. High-level heteroplasmy MT-ND1 m.3985G>A cybrid mutants generated by this protocol are shown to have impaired complex I-dependent mitochondrial respiration relative to wild-type control, demonstrating m.3985G>A is likely pathogenic.

Humans↗

PubMed2026

Multistage Genetic, Transcriptomic, and Single-Cell Evidence Prioritizes MAP1LC3A among Ferroptosis-Related Genes in Glioblastoma.

Glioblastoma (GBM) remains a highly aggressive malignancy, and the contribution of ferroptosis-related genes to disease susceptibility remains incompletely understood. A genetically anchored, multistage framework was applied to prioritize ferroptosis-related genes associated with GBM. Among 483 genes curated from FerrDb V2, 315 had candidate cis-expression quantitative trait loci (cis-eQTLs) in eQTLGen, 250 retained at least three independent instruments after linkage disequilibrium clumping, and 226 yielded valid inverse-variance weighted (IVW) Mendelian randomization estimates using a GBM genome-wide association study comprising 6,183 cases and 18,169 controls. Thirty-four genes met the exploratory discovery criteria of P < 0.05 and a Benjamini-Hochberg false discovery rate (BH-FDR) < 0.20, with directionally concordant Bayesian weighted Mendelian randomization (BWMR) estimates. Replication-stage Mendelian randomization using GTEx V10 whole-blood cis-eQTLs supported four genes: ATG7, RPTOR, MAP1LC3A, and CHMP6. Evaluation across three independent tumor-control transcriptomic cohorts demonstrated that MAP1LC3A was consistently downregulated in tumor tissue and showed a significant random-effects pooled estimate (log₂ fold change, -1.273; 95% confidence interval, -1.625 to -0.920; false discovery rate = 0.016), whereas the other three genes lacked comparable cross-cohort statistical support. Single-cell virtual knockout analysis was subsequently performed in a patient-balanced subset of 2,400 malignant cells selected from 4,916 eligible cells across 20 adult IDH-wild-type GBM tumors. Across five independently seeded runs, 3, 15, 4, and 7 robust downstream genes were identified for ATG7, RPTOR, MAP1LC3A, and CHMP6, respectively. The resulting consensus sets comprised 17 unique genes, with RND3 shared across all four targets. Gene Ontology analysis indicated enrichment of cell-adhesion and cell-surface processes, whereas no KEGG or Reactome pathways remained significant after multiple-testing correction. Collectively, these findings prioritize MAP1LC3A for future experimental investigation while distinguishing genetic association, tumor-expression concordance, and computational perturbation from definitive evidence of causality or mechanism.

Humans↗

PubMed2026

Establishment of a STAT6 Reporter Assay for Screening Environmental Toxicants Affecting Allergic Airway Inflammation.

Air pollution-associated allergic airway inflammation is an increasing public health concern. Interleukin‑4 (IL‑4) and interleukin‑13 (IL‑13), which activate the Signal Transducer and Activator of Transcription 6 (STAT6) pathway, a central mediator of allergic airway inflammation, may modulate the respiratory toxicities of pollutants. The present study describes the generation and validation of a stable STAT6 luciferase reporter assay in human airway epithelial cells for evaluating environmental toxicants that modulate STAT6 signaling. Human bronchial epithelial 16HBE14o- cells were transduced with a STAT6-responsive luciferase reporter using a lentiviral vector, followed by optimization of puromycin selection and multiplicity of infection, and monoclonal isolation by limiting dilution. A stable clone with strong and reproducible induction across serial passages was selected. Reporter responsiveness was validated by IL-4/IL-13 stimulation, and STAT6 dependence was confirmed using selective STAT6, STAT5, and STAT3 inhibitors. Assay performance was quantified by Z'-factor analysis, which indicated reproducible signal separation. Furthermore, the assay was applied to individual air-pollution constituents, and benzo[b]fluoranthene and particulate matter significantly increased STAT6 reporter activity. This method provides a scalable approach for measuring STAT6 activity in airway epithelial cells and for prioritizing environmental toxicants that modulate allergic airway signaling.

STAT6 Transcription Factor↗

PubMed2026

A Standardized Nursing-Led Protocol Integrated Pain, Sleep, Medication Adherence, and Symptom Management in Postherpetic Neuralgia.

Postherpetic neuralgia (PHN) is a persistent neuropathic pain condition after herpes zoster that frequently coexists with sleep disturbance, medication-related problems, and fluctuating symptoms. This study evaluated whether a standardized nursing-led protocol could improve multidimensional short-term outcomes beyond usual care. In this prospective, parallel-group randomized controlled trial, 128 adults with PHN were allocated 1:1 to usual care or usual care plus an eight-week protocol integrating structured pain assessment, sleep monitoring, medication-adherence support, and rule-based digital symptom monitoring. The primary outcome was the between-group difference in change in Numeric Rating Scale (NRS) pain score from baseline to Week 8. Secondary outcomes included Pittsburgh Sleep Quality Index (PSQI), MMAS-8 medication adherence, symptom burden, pain-related nocturnal awakenings, breakthrough pain, rescue analgesic use, adverse events, rule-based alerts, and nursing satisfaction. Week-8 data were available for 116 participants (57 usual care; 59 protocol). Mean NRS scores decreased from 7.19 ± 1.08 to 4.82 ± 1.53 in the usual-care group and from 7.28 ± 1.05 to 3.24 ± 1.28 in the protocol group. An NRS reduction of at least 2 points occurred in 49.1% and 76.3% of participants, respectively. The protocol group also showed larger improvements in PSQI, MMAS-8, symptom burden, and nocturnal awakenings, with fewer breakthrough-pain episodes and less rescue-analgesic use. These findings support further evaluation of the standardized nursing-led protocol in preregistered multicenter trials with intention-to-treat analyses and longer follow-up.

Humans↗

PubMed2026

S100P as a Shared Biomarker in Inflammatory Bowel Disease, Colorectal Cancer, and Pancreatic Adenocarcinoma: An Integrated Transcriptomic Analysis.

Inflammatory bowel disease (IBD) is associated with an increased risk of colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD), yet the molecular features shared among these diseases remain incompletely understood. This study aimed to identify common genes and biological pathways associated with IBD, CRC, and PAAD through integrated transcriptomic analysis and experimental validation. Gene expression datasets for IBD, CRC, and PAAD were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases. Weighted gene co-expression network analysis and differential expression analysis were performed to identify disease-associated and shared genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (analyses were used to explore enriched biological functions and pathways. Immune cell infiltration was evaluated using Cell-type Identification by Estimating Relative Subsets of RNA Transcripts. Receiver operating characteristic analysis was performed to assess the diagnostic performance of common genes. Single-cell RNA sequencing analysis was conducted to examine the cellular distribution of S100P. In addition, the effects of S100P downregulation were evaluated in lipopolysaccharide (LPS)-stimulated colonic epithelial cells. A total of 162 disease-associated genes and four common genes were identified. Functional enrichment analyses indicated significant enrichment of immune- and inflammation-related pathways, including the interleukin-17 signaling pathway. Immune infiltration analysis revealed similar trends in several immune cell populations across IBD, CRC, and PAAD. Single-cell analysis showed elevated S100P expression in epithelial cells from all three diseases. Downregulation of S100P restored the proliferative capacity of LPS-stimulated colonic epithelial cells and reduced inflammatory cytokine expression. Integrated transcriptomic analysis identified S100P as a biomarker associated with IBD, CRC, and PAAD and highlighted shared immune-related features across these diseases.

Humans↗

PubMed2026

Generation of Fibrin-Based Three-Dimensional Engineered Vascular Tissues from Human Aortic Smooth Muscle Cells for Proteomic Analysis.

Vascular smooth muscle cells (SMCs) reside within the medial layer of blood vessels, where they interact with an extracellular matrix (ECM) composed of collagen, elastin, and proteoglycans to maintain vascular structure and function. Aberrant ECM remodeling contributes to multiple vascular diseases; however, conventional two-dimensional culture systems do not adequately recapitulate the three-dimensional (3D) cellular and matrix environment required to study SMC-ECM interactions and matrix remodeling. This protocol describes the generation of engineered vascular tissues (EVTs) from primary human aortic SMCs cultured within fibrin-based 3D hydrogels. Following casting between flexible polydimethylsiloxane posts, EVTs undergo cellular alignment, contraction, and deposit de novo ECM, providing a physiologically relevant platform for studying vascular matrix biology. The protocol details tissue fabrication, culture, harvesting, and downstream analysis of newly deposited ECM by immunofluorescence staining. In addition, a workflow is presented for qualitative and quantitative characterization of EVT-derived proteins using Western blotting and mass spectrometry-based proteomics. Sequential protein extraction enables assessment of soluble and ECM-enriched protein fractions, facilitating in-depth evaluation of ECM composition. This platform provides a reproducible approach for investigating ECM production and remodeling by human SMCs in a 3D environment.

Humans↗

PubMed2026

SHMT2: a Metabolic and Immune Biomarker of Aggressive Lung Adenocarcinoma.

Serine/glycine-one-carbon (SGOC) metabolism is frequently altered in lung adenocarcinoma (LUAD), but its relationship to tumor behavior and predicted immunotherapy responsiveness remains incompletely defined. Metabolomic profiling of 23 paired LUAD and adjacent normal lung tissues was performed using internal extractive electrospray ionization mass spectrometry. Transcriptomic and clinical data from The Cancer Genome Atlas LUAD cohort (TCGA-LUAD) were analyzed to assess SHMT2 expression, prognosis, differentially expressed genes, and immune-related features. Predicted response to immune checkpoint blockade was evaluated using Tumor Immune Dysfunction and Exclusion (TIDE) and The Cancer Immunome Atlas (TCIA), and drug sensitivity was inferred using oncoPredict. Single-cell RNA-seq data were used to examine the cellular distribution of SHMT2. Experimental validation included quantitative reverse-transcription PCR (RT-qPCR), western blotting, Human Protein Atlas (HPA) immunohistochemistry, and short hairpin RNA (shRNA)-mediated SHMT2 knockdown followed by proliferation, wound-healing and colony formation assays. Metabolomic analysis identified glycine, serine, and threonine metabolism as a prominently altered pathway in LUAD. SHMT2 was upregulated in LUAD and associated with worse overall survival and adverse clinicopathological features. SHMT2-high tumors displayed enrichment of cell-cycle and SGOC-related transcriptional programs, lower immune and stromal scores, and reduced predicted responsiveness to immunotherapy. Single-cell analysis showed relative enrichment of SHMT2 expression in B cell populations. In vitro, SHMT2 was overexpressed in LUAD cells, and its knockdown suppressed proliferation, migration, and clonogenic growth. Collectively, SHMT2 is associated with SGOC metabolic reprogramming, aggressive tumor phenotypes, and an immune-disadvantaged state in LUAD, supporting its potential relevance as a biomarker; therapeutic targeting requires additional pharmacologic and in vivo validation.

Humans↗

PubMed2026

Pan-cancer Bioinformatics Analysis Combined with Colon Cancer Experimental Validation: A Study on TMED3 as a Diagnostic and Prognostic Biomarker.

Transmembrane Emp24 Protein Transport Domain 3 (TMED3), a member of the p24 protein family, has been implicated in tumor proliferation, invasion, and migration. This study aimed to evaluate the expression patterns, prognostic significance, immune associations, and potential biological functions of TMED3 across multiple cancer types using pan-cancer bioinformatics analysis combined with immunohistochemical (IHC) validation in colon cancer. Multiomics datasets from The Cancer Genome Atlas, Genotype-Tissue Expression, UALCAN, Human Protein Atlas, and cBioPortal databases were analyzed to investigate TMED3 expression and genetic alterations in pan-cancer. Immunohistochemistry was performed to evaluate TMED3 protein expression in colon cancer tissues. Kaplan-Meier survival analysis and Cox regression analysis were used to assess the prognostic value of TMED3. Spearman correlation analysis was conducted to evaluate the associations of TMED3 with tumor mutational burden, microsatellite instability (MSI), immune cell infiltration, and immune checkpoints. Gene Set Enrichment Analysis was performed to investigate potential biological pathways associated with TMED3 in colon cancer. TMED3 expression was elevated in most tumor types and was associated with unfavorable overall survival and disease-specific survival in adrenocortical carcinoma, colon adenocarcinoma, and uveal melanoma. The greatest frequency of TMED3 genetic alterations was identified in mesothelioma, with amplification representing the predominant alteration type. In addition, TMED3 expression showed significant correlations with tumor mutational burden and microsatellite instability in kidney renal clear cell carcinoma, stomach adenocarcinoma, and uterine corpus endometrial carcinoma. TMED3 expression was also associated with immune infiltration and immune checkpoint expression in several tumors. IHC analysis demonstrated increased TMED3 expression in colon cancer tissues compared with normal colon tissues and showed an association with T stage. Functional enrichment analysis identified pathways related to ribosome, antigen processing and presentation, oxidative phosphorylation, and pentose phosphate. These findings indicate that TMED3 may represent a promising biomarker for the diagnosis and prognostic evaluation of colon cancer as well as other tumor types.

Humans↗

PubMed2026

Estimating Rhizobial Fitness During Legume Symbiosis: Enriching Viable Undifferentiated Bacteria from Root Nodules.

Advances in understanding the evolutionary ecology of the rhizobia-legume mutualism have been constrained by methodological limitations in efficiently measuring relative strain frequencies alongside measurements of absolute population sizes of rhizobia living in nodules. To examine strain competition in natural and agricultural ecosystems that harbor multiple strains of rhizobia, an increasing number of manipulative and observational studies have recently begun to examine dozens or hundreds of strains simultaneously. Assessing the competitive fitness of multiple strains in legume nodules requires, first, processing pools of dozens to hundreds of nodules to overcome the stochasticity of nodule formation; second, focusing on the reproductively viable rhizobial population, since this trait represents rhizobia's reproductive success in nodules and is pivotal for evolutionary interpretations. Our approach has been optimized in the Medicago truncatula-Sinorhizobium meliloti system, where rhizobia induce the formation of indeterminate nodules that harbor two subpopulations: terminally-differentiated bacteroids and undifferentiated rhizobia that retain reproductive viability. This protocol has also been used for other legumes with terminally-differentiated bacteroids, such as pea and vetch, as well as for those with non-terminally differentiated bacteroids, such as soybean and cowpeas. The protocol we present enables rapid and reproducible homogenization of pools containing hundreds of nodules using a tissue homogenizer. We also enrich for undifferentiated rhizobia using two centrifugation steps: first, a low-speed centrifugation to deplete nodule debris and large, endoreduplicated, terminally-differentiated bacteroids, followed by a high-speed centrifugation to pellet the remaining undifferentiated rhizobia. The pellet can later be used for DNA extraction, followed by whole-genome or amplicon sequencing, and then downstream analysis to estimate strain fitness. Finally, we include an optional step for a reliable, reproducible system for nodule imaging, which is especially useful for quantifying nodule abundance and studying morphological variation. .

Symbiosis↗

PubMed2026

Standardized Xenograft Models for Preclinical Cancer Research.

Xenograft models are the principal in vivo platform of preclinical oncology and the most established experimental link between cell culture and clinical investigation. From the carcinogen-exposed rabbit models of the early twentieth century through the current generation of humanized patient-derived xenograft (PDX) systems, these platforms have evolved in response to the demands of translational cancer research. This review critically examines the biological principles, methodological standards, and translational applications of the principal xenograft platforms in current use. Cell line-derived xenograft (CDX) models remain the most widely used and most cost-effective modality for preclinical efficacy testing, offering the reproducibility, scalability, and accessibility that have sustained their role across oncology drug development pipelines for decades. PDX models have emerged as the preferred platform for co-clinical trial design, predictive biomarker discovery, and personalized oncology applications, preserving the genomic landscape, intratumor heterogeneity, and histological architecture of the donor tumor across serial passages. The engraftment biology of PDX systems, including immunodeficient host strain selection, implantation site, tumor source, and passage biology, is reviewed, together with humanized and autologous humanized configurations that extend the platform to immune checkpoint inhibitors, bispecific T-cell engagers, and chimeric antigen receptor T (CAR-T) cell therapy evaluation. This review addresses preclinical-to-clinical translation as a function of immunological divergence, incomplete tumor microenvironment recapitulation, and standardization. Formal frameworks, including the PDX Model Minimal Information (PDX-MI) standard and the Minimal Information for Standardization of Humanized Mice (MISHUM), are examined alongside global biobank infrastructure and emerging AI-driven translational modeling approaches.

Animals↗

PubMed2026

Preserving the Pineal Gland in Perinatal Mouse Brain Preparations.

The pineal gland, a neuroendocrine structure that secretes melatonin, forms from an anlage at the dorsal midline in the embryonic forebrain. At embryonic day (E) 14.5 in the mouse, a pineal diverticulum is seen out-pouching from the roof of the diencephalon between the paired telencephalic vesicles. This diverticulum is in close contact with overlying cartilage, meningeal, and vascular tissue. It is easily lost when the cranial vault is opened to extract the brain at late embryonic/ early postnatal stages. Despite its important neuroendocrine role, the early development of the pineal gland and the signaling mechanisms that regulate this process remain poorly understood, partly due to the difficulty of preserving this structure intact during embryonic stages of mouse brain development. This article presents an innovative dissection procedure that preserves the pineal gland while maintaining its structural integrity and its attachment to the roof of the diencephalon during brain isolation from embryonic stages through postnatal day (P) 7. The procedure is demonstrated using an E18.5 mouse brain and is therefore suitable for downstream applications requiring preservation of tissue architecture, including in situ hybridization, immunohistochemistry, and spatial transcriptomics. The protocol may also be used to isolate the developing pineal gland, thereby improving pineal-specific representation in transcriptomic and proteomic analyses, which is otherwise challenging because of its small size.

Animals↗

PubMed2026

Role of Sanqi Baiji San in Mitigating Ethanol-Induced Gastric Epithelial Cell Injury via PI3K/AKT-related Signaling.

This work aimed to clarify the protective mechanism of Sanqi Baiji San (SQBJ) against ethanol-induced gastric epithelial cell injury and to explore its potential relevance to gastric ulcer (GU). Network pharmacology was used to screen SQBJ's active components (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform [TCMSP], Oral Bioavailability [OB] ≥ 20%, Drug-Likeness [DL] ≥ 0.1), map their targets (Universal Protein Resource [UniProt]), collect GU-related targets (GeneCards/OMIM/DrugBank), and analyze overlapping targets via Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and molecular docking. Ethanol-injured Gastric Epithelial Cell Line-1 (GES-1) cells were treated with SQBJ or the PI3K inhibitor LY294002. CCK-8 was used to determine the optimal SQBJ concentration. Network pharmacology analyses identified 126 common targets enriched within PI3K/AKT/MAPK-related cascades and suggested potential interactions between principal SQBJ constituents and PI3K/AKT-related proteins. In ethanol-challenged cells, SQBJ alleviated cell injury by reducing inflammatory mediator release and oxidative stress, as evidenced by decreased intracellular reactive oxygen species and malondialdehyde levels. SQBJ restored mitochondrial membrane potential and ATP content and reduced apoptosis-associated changes in B-cell lymphoma-2, cleaved caspase-3, and Bcl-2-associated X protein levels. SQBJ also modulated PI3K/AKT- and MAPK-associated signaling markers. These protective effects were largely weakened by LY294002, suggesting that PI3K/AKT-related signaling is involved in SQBJ-mediated cytoprotection. These findings provide an in vitro mechanistic basis for the potential application of SQBJ in GU, although further validation in animal models of ethanol-induced gastric ulcer is required.

Ethanol↗

PubMed2026

Integrated Network Pharmacology and Molecular Docking Analysis of Sishen Decoction Identifies Potential Targets and Pathways in Gout.

Gout is a disease characterized by hyperuricemia and the deposition of urate crystals in joints and soft tissues, leading to recurrent acute arthritis. Its increasing prevalence imposes substantial clinical and socioeconomic burdens. Sishen Decoction (SSD) has been used in the treatment of gout, but its potential molecular mechanisms remain unclear. This study applied an integrated network pharmacology and molecular docking approach to identify potential targets and signaling pathways associated with SSD in gout. Active compounds and corresponding targets of SSD were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), while gout-related targets were collected from the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. Overlapping targets were identified and used to construct a drug-component-target-disease network. A protein-protein interaction (PPI) network was established using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed, followed by molecular docking using the docking server analysis module. A total of 37 bioactive compounds were associated with 116 overlapping gout-related targets. The top hub targets included TP53, IL6, IL1B, TNF, AKT1, EGFR, CASP3, JUN, BCL2, and MMP9. GO analysis suggested that these targets are involved in gene expression regulation and signal transduction. KEGG enrichment analysis indicated significant associations with the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/protein kinase B (PI3K-Akt), interleukin-17 (IL-17), and tumor necrosis factor (TNF) signaling pathways. Molecular docking predicted favorable interactions between key compounds and hub targets, with all binding energies of ≤-5 kcal/mol. These computational findings provide potential mechanistic hypotheses for the action of SSD in gout and may support future experimental validation.

Molecular Docking Simulation↗

PubMed2026

DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Humans↗