76555 research outputs found
Sort by
STAMP: Single-cell transcriptomics analysis and multimodal profiling through imaging.
No full textSingle-cell RNA sequencing has revolutionized our understanding of cellular diversity but remains constrained by scalability, high costs, and the destruction of cells during analysis. To overcome these challenges, we developed STAMP (single-cell transcriptomics analysis and multimodal profiling), a highly scalable approach for the profiling of single cells. By leveraging transcriptomics and proteomics imaging platforms, STAMP eliminates sequencing costs, enabling cost-efficient single-cell genomics of millions of cells. Immobilizing (stamping) cells in suspension onto imaging slides, STAMP supports multimodal (RNA, protein, and H&E) profiling, while retaining cellular structure and morphology. We demonstrate STAMP\u27s versatility by profiling peripheral blood mononuclear cells, cell lines, and stem cells. We highlight the capability of STAMP to identify ultra-rare cell populations, simulate clinical applications, and show its utility for large-scale perturbation studies. In total, we present data for 10,962,092 high-quality cells/nuclei and 6,030,429,954 transcripts. STAMP makes high-resolution cellular profiling more accessible, scalable, and affordable
Response of cultured primary gingival and periodontal ligament cells to angiotensin II and IL1β challenges.
No full textAngiotensin II (Ang II) releases inflammatory mediators from several cell types. The objective of this study was to investigate the potential of Ang II to induce mRNA expression of inflammatory mediators in primary cultured fibroblast-like cells isolated from gingival and periodontal ligament tissues. A synergistic effect of co-treatment with Ang II and Interleukin-1β (IL1β) on the mRNA expression of inflammatory mediators was explored. Immunophenotyping of STRO-1, Ang II type 1 receptor (AT1R), and Ang II type 2 receptor (AT2R) was performed using flow cytometry. Cell cultures were challenged with Ang II (1 µM) for 3, 6, and 24 h with or without co-treatment with IL1β (0.1 ng/mL) for 24 h. mRNA expression of inflammatory mediators was determined using qPCR. We present, for the first time, precise quantification of AT1R and AT2R in human gingival and periodontal fibroblast-like cell types; the percentage of positive immunostaining compared to the total cell population varied from 3.35% to 5.29% for AT1R and 2.97% to 4.57% for AT2R. Ang II slightly upregulated IL6 and CCL2/MCP1 mRNA expression in gingival cells and IL8 and PTGS2/COX2 in periodontal ligament cells. IL1β upregulated IL8, IL6, CCL2/MCP1, PTGS2/COX2, and IL1β mRNA in both cell types. Co-treatment with Ang II and IL1β did not show a synergistic effect. Ang II showed a low potential to induce mRNA of inflammatory mediators, most likely owing to the low percentage of Ang II receptors in such cells and no synergistic effect with the co-treatment with IL1β
Evaluation of hippocampal DLGAP2 overexpression on cognition, synaptic function, and dendritic spine structure in a translationally relevant AD mouse model.
No full textINTRODUCTION: Developing effective therapeutics for Alzheimer\u27s disease (AD) requires a better understanding of the molecular drivers of the disease. Our previous work nominated DLGAP2 as a modifier of age-related cognitive decline and risk for AD. We tested the hypothesis that overexpression of DLGAP2 in the hippocampus would protect against cognitive and synaptic deficits in a susceptible F1 5XFAD model.
METHODS: DLGAP2 was overexpressed in the hippocampus of F1 hybrid 5XFAD and non-transgenic littermates using a viral approach. Cognitive function, electrophysiological properties, and dendritic spine morphology were assessed at 6 and 14 months of age.
RESULTS: DLGAP2 overexpression impaired synaptic plasticity and exacerbated AD-related memory deficits but had minimal effect on spine structure or intrinsic neuronal properties.
DISCUSSION: We highlight the complex role of DLGAP2 in AD pathology. Targeted interventions involving postsynaptic proteins must consider potential adverse effects on synaptic integrity and cognitive performance, particularly in the context of AD.
HIGHLIGHTS: DLGAP2 overexpression accelerates AD-related impairment of contextual fear acquisition and memory. DLGAP2 overexpression impairs synaptic plasticity prior to AD-related memory impairment, but not intrinsic excitability. Effect of DLGAP2 overexpression on thin spine density was blunted in AD mice from in vivo dendritic spine results that were replicated in cultured rodent neurons
Advancing biological understanding of cellular senescence with computational multiomics.
No full textCellular senescence is a complex biological process that plays a pathophysiological role in aging and age-related diseases. The biological understanding of senescence at the cellular and tissue levels remains incomplete due to the lack of specific biomarkers as well as the relative rarity of senescent cells, their phenotypic heterogeneity and dynamic features. This Review provides a comprehensive overview of multiomic approaches for the characterization and biological understanding of cellular senescence. The technical capability and challenges of each approach are discussed, and practical guidelines are provided for selecting tools for identifying, characterizing and spatially mapping senescent cells. The importance of computational analyses in multiomics research, including senescent cell identification, signature detection and interactions of senescent cells with microenvironments, is highlighted. Moreover, tissue-specific case studies and experimental design considerations for individual organs are presented. Finally, future directions and the potential impact of multiomic approaches on the biological understanding of cellular senescence are discussed
Consistent performance of large language models in rare disease diagnosis across ten languages and 4917 cases.
No full textBACKGROUND: Large language models (LLMs) are increasingly used medicine for diverse applications including differential diagnostic support. The training data used to create LLMs such as the Generative Pretrained Transformer (GPT) predominantly consist of English-language texts, but LLMs could be used across the globe to support diagnostics if language barriers could be overcome. Initial pilot studies on the utility of LLMs for differential diagnosis in languages other than English have shown promise, but a large-scale assessment on the relative performance of these models in a variety of European and non-European languages on a comprehensive corpus of challenging rare-disease cases is lacking.
METHODS: We created 4917 clinical vignettes using structured data captured with Human Phenotype Ontology (HPO) terms with the Global Alliance for Genomics and Health (GA4GH) Phenopacket Schema. These clinical vignettes span a total of 360 distinct genetic diseases with 2525 associated phenotypic features. We used translations of the Human Phenotype Ontology together with language-specific templates to generate prompts in English, Chinese, Czech, Dutch, French, German, Italian, Japanese, Spanish, and Turkish. We applied GPT-4o, version gpt-4o-2024-08-06, and the medically fine-tuned Meditron3-70B to the task of delivering a ranked differential diagnosis using a zero-shot prompt. An ontology-based approach with the Mondo disease ontology was used to map synonyms and to map disease subtypes to clinical diagnoses in order to automate evaluation of LLM responses.
FINDINGS: For English, GPT-4o placed the correct diagnosis at the first rank 19.9% and within the top-3 ranks 27.0% of the time. In comparison, for the nine non-English languages tested here the correct diagnosis was placed at rank 1 between 16.9% and 20.6%, within top-3 between 25.4% and 28.6% of cases. The Meditron3 model placed the correct diagnosis within the first 3 ranks for 20.9% of cases in English and between 19.9% and 24.0% for the other nine languages.
INTERPRETATION: The differential diagnostic performance of LLMs across a comprehensive corpus of rare-disease cases was largely consistent across the ten languages tested. This suggests that the utility of LLMs in clinical settings may extend to non-English clinical settings.
FUNDING: NHGRI 5U24HG011449, 5RM1HG010860, R01HD103805 and R24OD011883. P.N.R. was supported by a Professorship of the Alexander von Humboldt Foundation; P.L. was supported by a National Grant (PMP21/00063 ONTOPREC-ISCIII, Fondos FEDER). C.M., J.R. and J.H.C. were supported in part by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy (Contract No. DE-AC0205CH11231)
Olaparib in Patients With Solid Tumors With
No full textPURPOSE: The Targeted Agent and Profiling Utilization Registry Study is a phase II basket trial evaluating the antitumor activity of targeted agents in patients with advanced cancer and genomic alterations. Results of five cohorts of patients with
METHODS: Eligible patients had advanced tumors, measurable disease (RECIST), Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and no standard treatment options. The primary end point was disease control (DC), defined as complete or partial response or stable disease (SD) of at least 16-weeks duration. For histology-specific cohorts, Simon two-stage design is based on a null DC rate of 15% versus 35% (power = 0.85; α = .10). Cohorts that were closed before achieving the planned stage II sample size were analyzed using a one-sided exact binomial test. For the HP cohort, the hypothesized null DC rate of 15% was rejected if the lower limit of a one-sided 90% CI was \u3e15%. Secondary end points were objective response, progression-free survival, overall survival, duration of response or SD, and safety.
RESULTS: Patients with BC (n = 28), BTC (n = 19), LC (n = 25), UC (n = 15), or other advanced cancers (n = 32) with
CONCLUSION: Olaparib met prespecified criteria to declare a signal of activity in patients with various advance
Extrachromosomal DNA associates with nuclear condensates and reorganizes chromatin structures to enhance oncogenic transcription.
No full textExtrachromosomal, circular DNA (ecDNA) is a prevalent oncogenic alteration in cancer genomes, often associated with aggressive tumor behavior and poor patient outcome. While previous studies proposed a chromatin-based mobile enhancer model for ecDNA-driven oncogenesis, its precise mechanism and impact remains unclear across diverse cancer types. Our study, utilizing advanced multi-omics profiling, epigenetic editing, and imaging approaches in three cancer models, reveals that ecDNA hubs are an integrated part of nuclear condensates and exhibit cancer-type specific chromatin connectivity. Epigenetic silencing of the ecDNA-specific regulatory modules or chemically disrupting nuclear condensates breaks down ecDNA hubs, displaces MED1 co-activator binding, inhibits oncogenic transcription, and promotes cell death. These findings substantiate the trans-activator function of ecDNA and underscore a structural mechanism driving oncogenesis. This refined understanding expands our views of oncogene regulation and opens potential avenues for alternative therapeutic strategies in cancer treatment
A xeno-free protocol for rapid differentiation of human iPSC-derived microglia from the KOLF2.1J reference line.
No full textWe present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Adapted from Dräger et al. (1), our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in disease modeling, neuroinflammation research, and therapeutic screening
Chlamydia muridarum Infection Impacts Murine Models of Intestinal Inflammation and Cancer.
No full textChlamydia muridarum has reemerged as a prevalent infectious agent in research mouse colonies. Despite its prevalence and ability to persistently colonize the murine gastrointestinal tract, few studies have evaluated the potential impact of C. muridarum on experimental models of gastrointestinal disease. Studies were conducted to evaluate the impact of C. muridarum on the Citrobacter rodentium, Trichuris muris, and Il10-/- mouse models of intestinal inflammation, as well as on tumorigenesis in the ApcMin/+ mouse following administration of dextran sodium sulfate (DSS). Naïve C57BL/6J (B6), B6.129P2-Il10tm1Cgn/J (Il10-/-), and C57BL/6J-ApcMin/J (ApcMin/+) mice were infected with C. muridarum by cohousing with chronic C. muridarum-shedding BALB/cJ mice for 2 weeks; controls were cohoused with C. muridarum-free mice. After cohousing, B6 mice (n = 8 C. muridarum infected and free) were infected with C. rodentium (109 CFU orally) or T. muris (200 ova orally). Il10-/- mice (n = 8/group with and without Helicobacter hepaticus [108 CFU/mouse] and with and without C. muridarum) and ApcMin/+ mice (n = 8/group) that received 2% DSS for 7 days in drinking water after cohousing. Mice were euthanized 14 days post-C. rodentium infection, 18 days post-T. muris infection, 60 days post-H. hepaticus infection, or control with Il10-/- mice, and 28 days post-DSS administration to ApcMin/+ mice. The severity of the cecal and colonic lesions was evaluated and graded using a tiered, semiquantitative scoring system. C. muridarum infection attenuated colitis associated with C. rodentium (P = 0.03), had no effect on T. muris-associated pathology (P = 0.22), worsened colitis in Il10-/- mice in the absence of H. hepaticus (P = 0.007), and reduced chemically induced colonic tumorigenesis in ApcMin/+ mice (P = 0.004). Thus, C. muridarum colonization differentially impacts several models of intestinal inflammation and tumorigenesis, and the presence of this bacterium in mouse colonies should be considered as a variable in these experimental readouts
Extrachromosomal DNA amplifications exhibit distinct molecular characteristics and prognostic implications in gastric cancer.
No full textGastric cancer (GC) is characterized by marked molecular heterogeneity that contributes to differential patient outcomes. Focal amplification in form of extrachromosomal DNA (ecDNA) is common in multiple cancer types and is associated with poor patient outcomes, but its prevalence and clinical implications in GC remain largely unclear. In this study, we analyzed whole genome and whole transcriptome sequencing from 76 GC patients collected at a single hospital (Seoul National University Bundang Hospital) in Korea. EcDNAs were detected in 22.4 % (n = 17) of GC patients. Notably, 75.0 % (n = 12) of the patients in the \u27chromosomal instability (CIN)\u27 category carried ecDNAs which frequently co-occurred with chromothripsis. We found that ecDNAs were enriched for known cancer genes, and the presence of ecDNAs was associated with poor patient prognosis. Among the CIN cases, patients carrying ecDNAs showed gene expression patterns related to chromosomal instability, as also observed in patients having only non-ecDNA chromosomal amplicons (ChAmp) but exhibited more pronounced immune suppression. Our findings show that ecDNAs display distinct molecular characteristics in GC, including the high prevalence of cancer genes and pronounced characteristic of immune suppression, alongside clinical implications, suggesting that ecDNA is a key molecular factor in the clinical management of GC patients, particularly for the CIN subtype patients