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Li, Z.

Publications and source records attributed to Li, Z..

At least 415 records · Page 23Linked to original sources

TDP-43 pathology induces CD8+ T cell activation through cryptic epitope recognition

Aggregation and nuclear depletion of the RNA binding protein TDP-43 are the crucial pathological features of amyotrophic lateral sclerosis (ALS) and inclusion body myositis (IBM), two degenerative diseases of the CNS and muscle. The loss of TDP-43 nuclear function results in the aberrant inclusion of cryptic exons in mRNA transcripts, leading to the expression of de novo proteins. Clonally expanded and highly differentiated CD8+ T cells have been observed in individuals with TDP-43 proteinopathies and therapeutics modulating the T cell response have recently been found to extend survival. However, the target antigens mediating T cell activation have remained elusive. Here, we investigate whether the de novo proteins induced by aberrant cryptic splicing due to TDP-43 nuclear loss can act as neo-antigens. We detect the HDGFL2 cryptic peptide and multiple other TDP-43 cryptic exons in IBM skeletal muscle, where their presence correlates with enrichment of T cells and class I antigen presentation pathways. Furthermore, we identify epitopes deriving from HDGFL2 and IGLON5 cryptic peptides which are recognized by clonally expanded and functionally differentiated populations of CD8+ T cells in ALS and IBM Patients. Finally, we demonstrate that T cells engineered to express the identified TCRs can bind and activate in response to the cryptic peptide derived epitopes (cryptic epitopes) and are able to kill TDP-43 deficient astrocytes. This work identifies for the first time specific T cell antigens in ALS and IBM, directly linking adaptive immune response to TDP-43 pathology.

immunology↗

Chromosomal-level genome of the Pinus massoniana 'Minlin WY36'

Owing to conifers complex and large genomes, it is challenging to construct a comprehensive reference genome and conduct evolutionary and genomic research. Here, we present the chromosome-level assembly of Pinus massoniana Minlin WY36, a pioneering conifer species used for afforestation, timber production, and oleoresin extraction. The genome size was determined to be 23.41 Gb, with a contig N50 value of 1.15 Mb and a scaffold N50 value of 2.02 Gb. A total of 23.16 Gb of reads (98.93% of the assembled genome) were anchored and oriented onto 12 pseudochromosomes, with chromosome lengths from 1.47 to 2.29 Gb. A total of 32,144 protein-coding genes were predicted, of which 27,173 (84.54%) were functionally annotated. The P. massoniana Minlin WY36 genome consists of 79.41% (18.57 Gb) repetitive sequences, among which long terminal repeat sequences (LTR) were the most abundant. Through comparative genomic analysis, we found that the unique and significantly expanded genes in P. massoniana Minlin WY36 were enriched in the metabolism related to resin synthesis. This study offers valuable insights into conifer evolution and contributes resources for further investigations of conifer adaptation and developmental processes.

genomics↗

A spatial atlas of chemoradiation therapy in pancreatic cancer identifies cellular and microenvironmental determinants of persister populations

The molecular pathways involved in the response to radiation therapy in pancreatic ductal adenocarcinoma (PDAC) remain poorly understood. We aimed to elucidate the adaptive mechanisms and cellular interactions within PDAC to radiation therapy (RT). We constructed a transcriptomic landscape of the cellular subtypes and spatially resolved neighborhoods from 50 patient samples, including 16 longitudinally matched single cell RNA sequencing and 34 spatial transcriptomics specimens. To resolve shortcomings of cell-type mixtures in spatial data, we developed a novel statistical method called SpaCCI (spatially aware analysis of cell-cell interactions) to profile cell-cell interactions and ligand-receptor enrichment. This revealed CXCL12/TGF{beta}-driven persister cell niches where activated fibroblasts reprogram tumor- associated macrophages and spatially exclude stress-response CD8 T cells after RT. Persister cancer cells displayed transcriptional evidence of recalcitrance to metal-induced cell death pathways of ferroptosis and cuproptosis which were recapitulated in preclinical models. Our study reveals the selective pressures experienced by PDAC following RT that may help provide insight for future multimodal therapeutic strategies.

cancer biology↗

Bidirectional regulation of glycoprotein nonmetastatic melanoma protein B by β-glucocerebrosidase deficiency in GBA1 isogenic dopaminergic neurons from a patient with Gaucher disease and parkinsonism

Variants in GBA1 are common genetic risk factors for several synucleinopathies. The increased risk has been attributed to the toxic effects of misfolded glucocerebrosidase (GCase) (gain-of-function), and the accumulation of lipid substrates due to reduced enzyme activity (loss-of-function). To delineate GBA1 pathogenicity, an iPSC line was generated from a patient with both type 1 Gaucher disease (GBA1: N370S/N370S; p.N409S/p.N409S) and Parkinson disease (PD). From this line, we created a reverted wild-type (WT) line and a GBA1 knockout (KO) line to eliminate misfolded GCase and intensify lipid accumulation. N370S/N370S and KO dopaminergic neurons (DANs) exhibited decreasing GCase levels and progressive accumulation of lipid substrates compared to WT DANs. Notably, the expression of GPNMB, whose levels correlate with PD risk, was upregulated by the mild lipid accumulation in N370S/N370S DANs but disrupted in KO DANs. These findings refine the loss-of-function mechanism by associating PD risk levels of GPNMB with lipid levels specific to GBA1 risk variants.

neuroscience↗

Biocatalytic Construction of a CEST MRI Nucleoside Probe: Synthesis and Evaluation of 5-Methyl-5,6-dihydrothymidine

Magnetic Resonance Imaging (MRI) is a cornerstone of modern clinical diagnostics, often enhanced by contrast agents. Traditionally, these agents are chemically synthesized, which can involve complex, costly, and environmentally unfriendly processes. Here, we report a novel biocatalytic approach for the efficient, safe, and eco-friendly synthesis of 5-methyl-5,6-dihydrothymidine (5-MDHT), a potent Chemical Exchange Saturation Transfer (CEST) MRI probe for imaging in vivo expression of the Herpes Simplex Virus Type-1 Thymidine Kinase (HSV1-TK) reporter gene. We demonstrate that 5-MDHT can be biosynthesized via one- or two-step enzymatic reactions using human purine nucleoside phosphorylase (hPNPase) and the SgvMVAV SAM-dependent methyltransferase. hPNPase catalyzed the base-exchange reaction with catalytic efficiencies (kcat/KM) between 138-316 s-1 M-1, while SgvMVAV methylation of 5,6-dihydrothymidine yielded 5-MDHT with a catalytic efficiency of 26 s-1 M-1. Molecular dynamics simulations supported the enzymatic binding and selectivity observed experimentally. The resulting 5-MDHT was validated using CEST-MRI, showing a distinct exchangeable imino proton signal at 5.3 ppm. These findings highlight the chemo- and regioselectivity of the biocatalysts and establish biocatalysis as a viable platform for producing clinically relevant MRI contrast agents.

biochemistry↗

A multimodal imaging approach for imaging the metabolic changes resulting from bronchopulmonary dysplasia

Lung tissue is composed of various functional units, each essential for maintaining the intricate functions of the lung. Disruptions in the molecular and cellular mechanisms in the lung can cause tissue fibrosis, inflammation, and severe breathing difficulties, which are common in conditions such as bronchopulmonary dysplasia (BPD). BPDs molecular changes are not well understood, which hinders effective diagnosis and treatment. Here, we present a new multimodal imaging workflow for detailed molecular and metabolic characterization of tissues at multiple spatial scales. We applied a combined imaging approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and ultrafast focused light-based imaging & photonics platform (U-FLIP) that included two-photon fluorescence (TPF), second harmonic generation (SHG), and stimulated Raman scattering (SRS). We also developed a hierarchical multimodal registration network (HiMReg) for the precise co-registration of each modality. This approach revealed previously unknown metabolic changes in distinct functional tissue units affected by BPD, including altered lipid distributions, reduced optical redox states, and specific collagen remodeling in bronchioles. Our findings evidenced alterations in lipid composition and metabolism of BPD-affected alveoli compared to healthy tissue, providing novel insights into disease pathophysiology. Our findings elucidate the intricate spatial and molecular complexity of BPD, building on prior research that did not provide the spatial resolution necessary to capture the nuances of metabolic alterations. This multimodal approach offers exceptional insights into disease exploration and could transform the way we study spatially heterogeneous conditions. By providing detailed maps of the metabolic shifts occurring in distinct tissue microanatomical features, the methods developed here could enable the discovery of new therapeutic avenues, making it highly attractive for the field of biomedical research.

physiology↗

Temporal gene regulation enables controlled expression of gas vesicles and preserves bacterial viability

Gas vesicles (GVs) are genetically encodable, air-filled protein nanostructures that have rapidly emerged as a versatile platform for biomedical imaging, cell tracking, and therapeutic delivery. However, their heterologous expression in non-native hosts such as Escherichia coli can be challenging due to the complex assembly process, which often involves around ten different proteins and can lead to proteotoxic stress and impair cell growth. Here, we report the observation of a drop in cell density occurring 8 to 16 hours after GV induction in E. coli. To address these, we developed a dual-inducer transcriptional regulation system that enables orthogonal control of GV assembly factor proteins and the shell protein over a range of stoichiometries. Sequential induction in time, in which assembly factor expression is initiated before shell protein expression, restored normal bacterial growth and prevented lysis without compromising GV production. Further analysis revealed that varying the interval between the two induction steps affected both GV yields and cellular stress. By preserving cell integrity with GV expression, our approach enhances the utility of GV-expressing bacteria in applications that demand population-wide cellular stability and facilitates their broader application in biomedical engineering and synthetic biology.

synthetic biology↗

MimicNeoAI: An integrated pipeline for identifying microbial mimicry antigens and tumor neoantigen partners

Tumor-associated microbial antigens represent promising immunotherapy targets, yet systematic identification methods remain underdeveloped. We developed MimicNeoAI, a computational pipeline integrating BiLSTM networks to identify microbial epitopes, mutation-derived neoepitopes, and their microbial mimics from sequencing data. Training on validated epitope datasets yielded 0.90 AUC with 91% accuracy on experimental validation sets. Application to colorectal cancer revealed that microbial epitopes, despite originating from a nine-fold smaller peptide pool, generated twice the immunogenic candidates (153 vs 75) compared to mutation-derived neoepitopes. These microbial epitopes exhibited exclusive tumor-specificity with no overlap in normal tissues. Single-cell TCR sequencing confirmed clonal expansion against 75% of predicted highly immunogenic epitopes, with molecular dynamics simulations demonstrating positive correlation between predicted immunogenicity and HLA-epitope-TCR binding stability. Collectively, our pipeline systematically unveils abundant, tumor-specific, and highly immunogenic microbial epitopes, providing a computational framework for developing broadly applicable cancer immunotherapies that leverage the tumor microbiome as an untapped source of therapeutic targets.

bioinformatics↗

Super-giga and tiny orchid genomes illuminate evolution of Orchidaceae

Orchidaceae (orchids) is commonly known as one of the largest families of seed plants, and grow in an extensive range of habitats worldwide. In the present study, we generated chromosome-level reference genomes for two orchids using a combination of PacBio, Illumina, and Hi-C sequencing, Cypripedium singchii has the largest genome and chromosomes among the sequenced species so far, with a genome size of 43.19 Gb (1C) with ten chromosomes, and Apostasia fujianica has the smallest known genome and chromosomes in Orchidaceae, with a genome size of 340.90 Mb (1C) with 35 chromosomes. We predicted a total of 32,412 and 21,724 protein-coding genes for C. singchii and A. fujianica, respectively. The overall BUSCO score was 85.01% for C. singchii and 91.80% in A. fujianica. Based on protein-coding sequences from 55 conserved single-copy families across 21 plant species, we constructed a high-confidence phylogenetic tree and estimated the divergence times. The high-quality genomes of super-giga and tiny orchids offer key insight for future evolutionary researches.

genomics↗

Functional rescue of a fatal ERAD mutation via alternative splicing

Endoplasmic reticulum (ER)-associated degradation (ERAD) is essential for cellular proteostasis, with the SEL1L-HRD1 protein complex targeting misfolded proteins in the ER for proteasomal degradation. Disruption of this pathway underlies a recently identified infant-onset neurodevelopmental disorder (ENDI syndrome), characterized by profound developmental delay, microcephaly, and immune deficiency. Its most severe form, ENDI with agammaglobulinemia (ENDI-A), is driven by a bi-allelic SEL1L Cys141Tyr (C141Y) mutation within the fibronectin II (FNII) domain, for which no treatment currently exists. Here, we serendipitously uncover a striking mechanism of intrinsic rescue in knock-in mouse models of the C141Y mutation: enhanced usage of an alternative splice donor site within exon 4 bypasses the mutant FNII-encoding region, restoring ERAD activity and rescuing key disease phenotypes including perinatal lethality, growth retardation, B cell deficiency, and neurodevelopmental defects. Building on this discovery, we demonstrate that antisense oligonucleotide (ASO)-mediated exon skipping in patient-derived fibroblasts generates a truncated yet functional SEL1L protein, fully rescuing ERAD function and ER proteostasis. These results establish RNA splicing modulation as a viable therapeutic strategy for ERAD deficiency and extend the clinical potential of exon-skipping therapy to diseases of protein misfolding. ONE-SENTENCE SUMMARYThis study reports the discovery of using antisense oligonucleotides (ASOs) to rescue the biallelic SEL1L C141Y variant, offering a potential therapeutic strategy.

cell biology↗

Inducible genome-wide mutagenesis for improvement of pDNA production by E. coli

Plasmid DNA (pDNA) is a cost-driving reagent for the production of gene therapies and DNA vaccines. Improving pDNA production in the most common production host (E. coli) has faced obstacles arising from the complex network of genes responsible for pDNA synthesis, with the specific enzyme(s) limiting pDNA yield remaining unidentified. To address this challenge, we employed an inducible genome-wide mutagenesis strategy, combined with fluorescent screening, to isolate E. coli NEB 5 strains with enhanced pDNA production. Following selection, we successfully isolated an E. coli strain (M3) with elevated plasmid copy numbers (PCNs) across multiple origin types. Specifically, we observed a 5.93-fold increase in PCN for the GFP reporter plasmid, a 1.93-fold increase for the gWiz DNA vaccine plasmid, and an 8.7-fold increase for the pAAV-CAGG-eGFP plasmid, all of which contain pUC origins. In addition, plasmids with p15A and pSC101 origins showed 1.44-fold and 1.68-fold increases in PCN, respectively. Whole-genome sequencing of the adapted strain M3 identified 85 mutations, including one in recG, which encodes an ATP-dependent DNA helicase. Replacement of the mutant recG with its wild-type counterpart in the mutant strain resulted in a 63% reduction in PCN, but the recG mutation alone was insufficient to increase PCN in the wild-type strain. These findings suggest that the recG mutation plays a synergistic role with other genomic mutations to drive PCN increases. Taken together, this study presents the development of a pDNA hyperaccumulating E. coli strain with promising applications in industrial and therapeutic pDNA production, while also offering important insights into key genes involved in pDNA production.

synthetic biology↗

Structural Basis for Cas9-Directed Spacer Acquisition in Type II-A CRISPR-Cas Systems

CRISPR-Cas systems confer prokaryotic adaptive immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-EM structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo-state structure (Cas92-Csn28-Cas18-Cas24) adopts a resting conformation, with Cas9 locked in a nuclease-inactive state and Cas1-Cas2 sterically blocked from prespacer loading. Upon DNA engagement, Cas9 undergoes a conformational transition, forming a prespacer catching complex that threads the DNA through Csn2s central channel. This architecture enables Cas9 to interrogate the PAM sequence while sliding along the DNA, with Cas9 and Csn2 jointly define a 30-bp DNA segment which matches the prespacer length. Subsequent dissociation of Cas9 triggers a structural reconfiguration of the Csn2-Cas1-Cas2 assembly. The PAM-proximal DNA becomes accessible, and Cas1-Cas2 relocates to bind to the exposed DNA, enabling further prespacer processing and directional integration. These findings reveal how Cas9 collaborates with Csn2 and Cas1-Cas2 to couple PAM recognition with prespacer selection, resolving the dynamic structural transitions that ensure fidelity during type II-A CRISPR adaptation.

molecular biology↗

Multimodal Evidence for Hippocampal Engagement and Modulation by Functional Connectivity-Guided Parietal TMS

Hippocampal activity supports memory and many other brain functions. Transcranial magnetic stimulation (TMS) guided by hippocampal functional connectivity (FC) shows promise in improving memory, but direct neural evidence of its capacity to engage and modulate hippocampal activity is lacking. Here we combined TMS with intracranial electroencephalography (iEEG) in 8 neurosurgical patients and with functional magnetic resonance imaging (fMRI) in 79 neurologically healthy participants. We identified that (1) single-pulse TMS to individualized parietal cortex guided by hippocampal-FC preferentially evoked distinct temporal and spectral activity patterns in the hippocampus, (2) variability in TMS-evoked hippocampal responses related to individual differences in parietal-hippocampus FC strength, and (3) repetitive TMS to hippocampal-FC-guided parietal cortex selectively suppressed hippocampal theta oscillations. These findings provide multimodal causal neural evidence and important mechanistic insights supporting the development of personalized neuromodulation strategies aimed at improving hippocampus-dependent functions.

neuroscience↗

Analysis of Wheat Spike Morphological Traits Using 2D Imaging

The morphological structure of wheat spikes plays a central role in wheat yield. Wheat spike morphology, closely associated with crop yield, has attracted considerable attention in the fields of genetics and breeding. However, traditional measurement methods can only measure simple traits, and precise phenotypes remain difficult to obtain, constraining the study and improvement of complex spike-related traits. This study utilized deep learning technologies to develop a pipeline, called SpikePheno, for the acquisition of precise wheat spike phenotypes. Our pipeline demonstrated high accuracy in spike segmentation, achieving a mean Intersection over Union (mIoU) of 0.948. Additionally, our method accurately identified spikelet counts, achieving an R2 of 0.9923. Using experimental data of 221 wheat cultivars from various regions of China grown in Zhao County, Hebei Province, our pipeline extracted 45 different phenotypes and studied their correlations with thousand grain weight (TGW) and spike yield. Our findings indicate that precise measurement of spike area, spikelet area, and other phenotypic traits enables a clearer understanding of the correlation between spike morphology and wheat yield. Through hierarchical clustering based on spike morphology, we categorized wheat spikes into six classes and identified phenotypic differences between these classes and their impact on TGW and yield. Furthermore, this study revealed phenotypic differences between wheat cultivars from different geographical regions and over different decades, with an increase in large-spike cultivars over time, especially in southern China. This research may help breeders understand the relationship between wheat spike morphology and yield, providing an important basis for future wheat breeding efforts.

plant biology↗

Effects of diffusion MRI spatial resolution on human brain short-range association fiber reconstruction and structural connectivity estimation

Short-range association fibers (SAFs) are critical for cortical communications but are often underestimated in conventional resolution diffusion magnetic resonance imaging (dMRI) since they locate within a [~]1.5mm thin layer of superficial white matter. With the emergence of high-resolution diffusion imaging techniques, this study timely evaluated the effects of image spatial resolution on SAF reconstruction using simulation data and multi-resolution (2, 1.5, and 0.96 mm iso.) empirical data acquired on the same 20 healthy subjects using the gSlider sequence and 20 widely used tractography approaches. Resolution effects were qualitatively assessed through model fitting and tractography results and quantitatively evaluated using global and regional short-range connectivity strength (SCS). It is found that lower resolution systematically reduces SCS across all methods in a spatially varying manner. Moreover, tractography methods exhibit significant differences in resolution sensitivity, with diffusion tensor imaging (DTI) based single-tissue single-fiber tractography showing greater vulnerability than constrained spherical deconvolution (CSD)-based multi-tissue multi-fiber tractography. Probabilistic tracking with anatomical constraints (ACT) and filtering (SIFT) improves robustness. Finally, up-sampling data to nominally higher resolution partially mitigates resolution-induced degradation and improves SAF reconstruction accuracy, particularly for DTI tractography. Based on these findings, higher resolution and multi-shell imaging is recommended if possible. For a given dataset, data up-sampling and DTI-based probabilistic tracking with ACT is recommended for single-shell low b-value data. CSD-based probabilistic tracking with ACT and SIFT is recommended for single-shell higher b-value data and multi-shell data. In summary, this study systematically and quantitatively evaluated resolution effects on SAF reconstruction and structural connectivity estimation and provided practical guidelines for more accurate mapping of SAFs. These advances hold promises to improve the characterization of healthy and diseased human brains in a wide range of neuroscientific and clinical applications.

neuroscience↗

Structural and temporal dynamics analysis on PANoptosis in sepsis: a bibliometric analysis

PANoptosis, as a new type of programmed cell death, is characterized by pyroptosis, apoptosis and necroptosis, and is a key mechanism causing a variety of inflammatory diseases. Despite the growing number of studies indicating the crucial role of PANoptosis in sepsis, there has been no bibliometric analysis of the research hotspots and trends in this field. Therefore, this study aims to explore the history, research hotspots and emerging trends of PANoptosis in sepsis related research in the past 20 years from the perspective of structure and temporal dynamics. The articles related to PANoptosis in sepsis were retrieved from the Web of Science Core Collection (WoSCC) database from 2000 to 2024. CiteSpace and HistCite were used to analyse the historical features, the evolution of active topics, and emerging trends about PANoptosis in sepsis. 6165 original articles and reviews on PANoptosis in sepsis were included in the bibliometric analysis. In the last 20 years, the number of published documents is increasing year by year and reaches a peak in 2022. At the same time, many activation themes have emerged at different times, as evidenced by a total of 96 categories, 865 keywords and 629 reference bursts. Keyword clustering anchored eight emerging research subfields, namely 0# oxidative stress, 1#pyroptosis, 2#sepsis-associated encephalopathy, 3#acute kidney injury, 4#immunosuppression, 5#necroptosis, 7#lung injury and 8#extracellular vesicles. The keyword alluvial map shows that the most persistent research concepts in this field are phosphatase. And the emerging keywords are toll_like_receptors, regulatory T cells, recognition, etc. In a timeline visualization based on the time span of the citations, we find five relevant emerging topics, namely 1# immunosuppression, 2# sepsis-induced cardiomyopathy, 3# pyroptosis, 4# acute kidney injury, and 13# COVID-19. Our study provides a comprehensive bibliometric analysis and summary about the current status and trends on PANoptosis in sepsis, which will aid researchers in conducting further scientific research in this field.

bioinformatics↗

Detection of astrocyte epigenetic memory in in vitro systems, experimental autoimmune encephalomyelitis and multiple sclerosis samples

We recently described astrocyte pro-inflammatory epigenetic memory based on multiple complementary in vivo and in vitro studies, and the analysis of multiple sclerosis samples. Based on bioinformatic analyses, ODea and Liddelow argued that the astrocyte epigenetic memory we described is the result of contamination with immune cells, particularly myeloid cells. We rebut ODea and Liddelow arguments as follows: (1) We show substantial purity of astrocytes analyzed in in vivo and in vitro systems; (2) We recapitulate astrocyte memory responses using five independent pure astrocyte in vitro systems, and show its dependency on the histone acetyl transferase p300; and (3) Using the Liddelow lab bioinformatic pipeline to implement purity and cell-quality criteria, we detect astrocyte epigenetic memory in five independent scRNA-seq experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) astrocyte datasets. These additional analyses and studies provide further support for the existence of astrocyte pro-inflammatory epigenetic memory.

neuroscience↗

BifurcatoR: A Framework for Revealing Clinically Actionable Signal in Variance Masquerading as Noise

BackgroundDisease heterogeneity is a persistent challenge in medicine, complicating both research and treatment. Standard analytical pipelines often assume patient populations are homogeneous, overlooking variance patterns that may signal biologically distinct subgroups. Variance heterogeneity (VH)--including skewness, outliers, and multimodal distributions--offers a powerful but underused lens for detecting latent etiological structures relevant to prognosis and therapeutic response. MethodsA major barrier to VH analysis is the fragmented landscape of available methods, many of which rely on normality assumptions that biological data frequently violate. In addition, existing tools often require programming expertise, and clear guidance on study design considerations--such as sample size and method selection--is lacking. To address these issues, we developed BifurcatoR, an open-source software platform that simplifies the detection, modeling, and interpretation of VH. BifurcatoR integrates simulation-based method evaluation, study design recommendations, and a user-friendly web interface to support VH analysis across a range of data distributions. We benchmarked VH methods through simulation and applied BifurcatoR to two clinical datasets: acute myeloid leukemia (AML) and obesity. ResultsSimulation studies revealed that VH method performance is highly context-specific, varying with distribution shape, mean-variance coupling, and underlying subgroup structure. In AML, BifurcatoR identified two molecularly distinct subgroups with different treatment responses, including an EVI1-high group with significantly poorer prognosis (p < 0.005) among KMT2A-rearranged cases. In a separate study, VH analysis uncovered immunophenotypic subgroups in obesity based on gene-level discordance across monozygotic twin pairs, highlighting latent variation in adipose immune cell composition. ConclusionsVH is not "noise", biological variation without clinical relevance. Instead, VH is a structured signal that can reveal latent and clinically meaningful subtypes. BifurcatoR offers a practical, accessible framework for incorporating VH into biomedical research, with implications for biomarker discovery, patient stratification, and precision medicine.

bioinformatics↗