Biology subjects

Huang, E.

Publications and source records attributed to Huang, E..

At least 19 recordsLinked to original sources

bioRxiv2026

LNP Delivery of Prime Editors Enables Durable Correction of PAH R408W in Phenylketonuria Models

Phenylketonuria (PKU) is a monogenic metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, the most common of which is c.1222C>T (p.R408W). Current treatment options, including dietary restriction and enzyme substitution, can be effective but are often challenging for patients to maintain and do not address the underlying genetic defect. Gene editing represents a compelling one-time treatment approach for PKU. Herein, we demonstrate mRNA:LNP-mediated delivery of prime editors that enable therapeutically-relevant levels of correction of the R408W mutation in both in vitro and in vivo model systems. Further, we found that co-delivery of the DNA repair modulator RAD18 and engineering of the prime editor protein, enabled 2-fold and 3-fold increases in prime editing relative to PE3 respectively, resulting in ~50% correction efficiency in a humanized PKU mouse model. Collectively, this work identifies novel potential approaches to enhancing prime editing and establishes LNP-delivered prime editing as a mutation-specific, non-viral therapeutic strategy for PKU, as well as providing a framework for extending prime editing to other inherited liver diseases.

pharmacology and toxicology↗

bioRxiv2026

RNA-guided contrastive learning enhances patient-level prediction from histology

Predicting molecular receptor status, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), directly from routine hematoxylin and eosin (H&E)-stained whole-slide images (WSIs) could reduce reliance on costly immunohistochemistry, but image-only models have no access to the transcriptomic programs that define these subtypes. Here we show that coupling a whole-slide vision transformer (GigaPath) with bulk RNA-sequencing profiles through a training-time-only contrastive objective improves receptor-status classification from WSIs alone, without requiring any RNA-seq data at inference. Rather than training a dedicated bulk-transcriptomics encoder, we purpose the gene-sentence encoding and pretrained text encoder from BioBERT1 and apply them to bulk RNA-seq profiles from The Cancer Genome Atlas breast cancer cohort (TCGA-BRCA). Under five-fold cross-validation, this RNA-guided pre-training consistently improved AUROC for ER, PR, and HER2 relative to a contrastive-pre-training-free baseline and reproducibly introduced receptor-relevant structure into the learned slide representation. However, evaluation on independent external cohorts revealed a striking, receptor-dependent divergence: gains for ER and PR, both governed by broad, multi-gene luminal transcriptional programs, generalized robustly, whereas the apparent AUROC improvement for HER2, driven by focal ERBB2 amplification rather than a coordinated transcriptional signature, masked a severe loss of classification sensitivity at the operating threshold. These findings establish training-time transcriptomic supervision as a practical route to enhancing histology-only molecular subtyping, while showing that gene-selection strategy, and not model architecture alone, determines which molecular alterations such supervision can teach a vision model to recognize.

pathology↗

bioRxiv2026

The strength of a gene's first 5' splice site is a key determinant of its expression

The control of gene expression is central to cellular physiology and to various biotechnological applications. In higher eukaryotes, synthesis of most messenger RNAs involves removal of introns during splicing. Intron-containing genes are often more highly expressed than intronless versions, despite generating the same mRNA transcripts, suggesting that the splicing of transcripts from a gene positively impacts its expression. Termed intron-mediated enhancement (IME), this phenomenon has been observed in animals, plants, and fungi, with enhancement ranging from 2- to 100-fold and the strongest effects observed for promoter-proximal introns1-3. Here, we assayed thousands of natural human introns, covering all distinct human 5' splice site (5SS) motifs and thousands of 3' splice sites (3SS), for their ability to enhance (or repress) expression of a reporter gene. The results confirmed previous observations that intronic UUUU motifs augment the magnitude of IME, and revealed two fundamental features of IME: (i) only introns spliced by the major spliceosome elicit IME, with minor introns often repressing expression; and (ii) the magnitude of IME increases with the strength of the 5SS motif, but is independent of 3SS strength. Our findings imply that genetic variants that alter the strength of the first 5SS of genes may alter their expression by modulating IME without necessarily impacting splicing. We confirm this expectation using human population genetic data and further show that disease-relevant perturbations of U1 snRNP components that enhance recognition of subsets of first 5SS up-regulate expression of the associated genes. These findings demonstrate that recognition of the first 5SS is vitally important to the expression of human genes.

genetics↗

bioRxiv2026

Specialized Shh-sensing cell with unique cilia and basal body in the forebrain ventricular epithelium

Ependymal (E1) cells, with their tufts of [~]50 motile cilia, line the walls of the brain ventricles and help propel the cerebrospinal fluid (CSF). The CSF is rich in signaling molecules, but the cellular targets that detect these signals and their function remain unknown. Here, we describe a distinct population of ependymal cells (E2) in the forebrain of mice and humans, the majority having only 1 or 2 cilia. These cilia were motile, but unlike E1 cells cilia, their pattern of motility and high expression of Arl13b and Inpp5e suggest a sensory function. E2 cells were characterized by an enormous, donut-like basal body that contained an increased number and size of subdistal appendages. In mice, E2 cells were mostly born in the embryo, but completed their differentiation in juveniles and young adults; they were found at higher densities in regions of high CSF flow and neurogenesis. E2 cilia contained the G protein-coupled receptor Smoothened, which accumulated in their cilia upon exposure to Sonic Hedgehog (Shh). Together, these findings identify E2 cells as a novel CSF-sensing ependymal cell type and provide a cellular target for the CSF signaling.

cell biology↗

bioRxiv2026

Container-less Acoustic Levitation Expands Plasma Extracellular Vesicle Proteome Coverage by Mitigating Size-Dependent Peptide Loss

Proteomic profiling of plasma-derived extracellular vesicles (EVs) is limited in part by adsorptive loss of peptides and proteins to container walls during sample preparation. Here we apply an automated, environment-controlled acoustic levitation platform (Levcell) to the tryptic digestion of small EVs (sEVs) isolated from pooled breast cancer patient plasma, and compare it directly with digestion in low-bind microcentrifuge tubes. Across three parallel technical replicates per method, container-less digestion identified 309 {+/-} 22 protein groups versus 261 {+/-} 6 for tubes (+18.4%; Welch t-test p = 0.053), with equivalent or better quantitative reproducibility (median CV 12.0% vs 14.4%). The gain was strongly asymmetric: 66 protein groups were recovered only under levitation while 10 were recovered only in tubes (exact McNemar p = 3 x 10-11). Peptides recovered exclusively by levitation were longer and heavier than those exclusive to tubes (median 14 vs 12 residues, 1611 vs 1358 Da; p < 2 x 10-6; Cliffs {delta} {approx} 0.19-0.20), whereas the total peptide pools were indistinguishable and mean missed-cleavage rates were equivalent (0.276 vs 0.263, p = 0.41), excluding differential digestion efficiency as an explanation. No systematic difference in hydropathy, isoelectric point or hydrophobic residue frequency was detected. The levitation-rescued sub-proteome was enriched for ribosomal, proteasomal, chaperonin and RNA-binding complexes -- canonical sEV luminal cargo (MYC targets 16/19, odds ratio 31.7, q = 5.6 x 10-8) -- and covered 33 of the 100 ExoCarta reference markers versus 22 for tubes, gaining 14 markers while losing three (a single ezrin/moesin/radixin protein group; McNemar p = 9.8 x 10-4). We also report two findings that temper the approach: levitated samples carried an approximately 3.7-fold higher keratin burden, consistent with airborne contamination in an open chamber, and no individual marker showed a significant abundance difference after correction for multiple testing. Container-less processing therefore offers a reproducible gain in sEV proteome coverage attributable to reduced size-dependent peptide loss, provided that contamination control is addressed.

biochemistry↗

bioRxiv2026

Spatial Topology Reveals Biologically Distinct Recurrent Motifs in Colorectal Cancer

Most spatial transcriptomic analyses of solid tumors focus on individual cell states or single-sample spatial domains rather than on recurrent multicellular tissue architectures shared across patients, and typically depend on predefined cell-type annotations or compartment definitions. We developed STORM (Spatial Topology analysis of Recurrent Motifs), an unsupervised graph-attention variational autoencoder that learns recurrent spatial motifs directly from cell-level graph structure and molecular profiles without cell-type labels, manual annotation, or predefined compartments. We applied to 32 Xenium sections from 16 patients with paired early-onset (EOCRC) and average-onset (AOCRC) colorectal cancer, STORM identified 10 recurrent motifs that self-organized into tumor-parenchymal, stromal, and immune macro-compartments. Among these, the Desmoplastic Fibrotic Barrier (DFB) motif, a CAF- and ECM-rich boundary architecture, was associated with restricted CD8+ T-cell geodesic access to tumor cores independently of CD8+ abundance, as demonstrated by abundance-normalized neighborhood enrichment statistics and within-sample mixed-effects models. EOCRC selectively amplified this barrier-exclusion architecture, exhibiting tighter tumor parenchyma, denser DFB shells, and a DFB-specific ECM activation program that yielded an age-specific prognostic signature in TCGA-COAD. Translation of motif macro-classes to H&E images via a Vision Transformer classifier produced an image-derived DFB-barrier composite that predicted overall survival in advanced-stage TCGA colorectal cancer. STORM provides an annotation-free framework for discovering recurrent spatial motifs and identifies a fibroblast barrier architecture whose topological association with immune exclusion is independent of effector abundance, amplified in early-onset disease, and translatable to a deployable pathology-based prognostic biomarker.

bioinformatics↗

bioRxiv2026

Spatial and Molecular Progression of Neural Progenitor Cells in the Developing Human Dentate Gyrus

The large diversity of neuronal and glial cell types in the human brain is underpinned by foundational cell populations known as neural progenitor cells (NPCs). The dentate gyrus (DG) of the hippocampus, a key structure in learning and memory, maintains a tightly organized NPC population into adulthood across many mammalian species. However, the emergence, organization and persistence of NPCs in the human hippocampus remain poorly characterized. Reports of NPCs in the juvenile, adult, and aged periods have been variable, reflecting differences in identification criteria and highlighting the need for a unified framework across development. In this study, we provide a spatial and molecular map of the developmental trajectory of NPCs in the human DG, combining multimodal transcriptomic analysis within a neuroanatomical context. At mid-gestation, we observed changes in the structural and cellular arrangement of the hippocampus, coinciding with the emergence of a multicellular NPC layer within the DG, herein named the granular-hilar progenitor zone (GHPZ). Neurogenic transcriptomic signatures in the GHPZ were diminished by early infancy, coinciding with a reduction in NPC number as they progressed toward an astrocytic program. At childhood, the GHPZ dissolved with only sparse radial NPCs remaining in the DG. Lastly, we validated WNT signaling pathway-associated genes as NPC identity markers in the developing human DG, observing a decline in their expression after infancy. Our study defines the steep decline of NPCs from gestation to the postnatal period, identifies their progression to an astrocytic nature, and sets the molecular blueprint for NPC identification in the human DG. HighlightsO_LIMultimodal mapping of neural progenitor cells from gestational to postnatal stages in the human hippocampus C_LIO_LIFormation of the granular-hilar progenitor zone within the dentate gyrus at mid-gestation C_LIO_LINeurogenic potential declines sharply from the prenatal period to childhood, with radial glia cells progressively acquiring astrocytic features C_LIO_LIDevelopmental modulation of the WNT signaling pathway accompanies radial glia cell transitions C_LI

neuroscience↗

bioRxiv2026

Diet alters epidemic size and timing in a trophically-transmitted parasite

A hosts diet can alter the course of parasite infection. This is especially true of trophic parasites, which a host acquires through feeding. While a large body of work attests to the role of diet in the spread of disease within-hosts, diet can also impact host density and encounter rate with parasites, both of which are expected to modify disease dynamics. When parasites are acquired through feeding, epidemics may be larger and more severe on high-quality diets if these diets support a higher density of hosts that feed more and thus ingest more parasites. Alternately, epidemics may be more severe on low-quality diets if malnourishment decreases hosts ability to resist disease. To differentiate these hypothesized effects of diet on disease, we quantified individual infections and epidemic dynamics for the natural microsporidian parasite Nematocida ironsii infecting its nematode host Caenorhabditis elegans. We measured feeding rate, parasite transmission, and host fitness across three bacterial diets that vary in quality and elicit distinct feeding behaviors in C. elegans. We found that low-quality diets reduced feeding rate, which corresponded to reduced acquisition of parasite spores. However, these diet-mediated differences in parasite acquisition did not directly map onto fitness consequences: hosts eating the poor-quality diet had similar reductions in fitness to those on higher quality diets. During epidemics, a combination of increased parasite acquisition and higher population growth rates resulted in higher parasite abundance for hosts on high-quality diets. Our work underscores the importance of considering both individual- and population-level impacts acting in concert to determine how diet affects the spread of infectious disease.

ecology↗

bioRxiv2026

Orally Bioavailable SARS-CoV-2 Protease Inhibitors Bearing a Hydroxymethyl Ketone Warhead

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@40222aorg.highwire.dtl.DTLVardef@831b88org.highwire.dtl.DTLVardef@184a910org.highwire.dtl.DTLVardef@779095_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

A Brain-wide Neuronal Spiking and Behavior Dataset for Working Memory-Specific Activation and Reactivation in Mice

AbstractHow do millisecond-scale neural activities mediate multi-second behaviors such as working memory? This dataset provides brain-wide, high-spatiotemporal-resolution single-unit spiking activity recorded from 40 head-fixed mice performing an olfactory delayed paired-association (ODPA) task, encompassing 33,028 neurons across 62 anatomically defined brain regions. Utilizing this dataset enables the extraction of millisecond-scale within- and cross-region spike couplings enriched among memory-content-selective neurons. These couplings may be used to identify higher-order, hierarchically structured activity motifs. Because these multi-neuron patterns are captured both during active task performance and spontaneous inter-trial intervals, this dataset provides a resource for studying replay-related activity and for testing hypotheses about circuit-level processes associated with perceptual working memory. The dataset supports broad investigations into cross-regional spike organization, functional coupling, and temporal coding in working memory circuits.

neuroscience↗

bioRxiv2026

Blood-brain barrier dysfunction predicts cognitive trajectory after ischemic stroke

Ischemic stroke doubles the risk of dementia.1-4 Stroke severity and location affect cognition early,5,6 but late dementia risk is not related to infarct characteristics, nor is it reduced by preventing additional strokes,3,6,7 and its mechanism is unknown. We identified a plasma proteomic signature of chronic stroke that was consistent with blood-brain barrier (BBB) dysfunction, including a 58% decrease in plasma levels of platelet-derived growth factor B and downregulation of its pathway compared to healthy controls. During 2 years of follow-up, the stroke-specific proteome was accentuated in stroke survivors who subsequently declined in the processing speed/executive function cognitive domain. To test BBB function, we performed dynamic contrast-enhanced MRI 6-9 months after stroke in an additional cohort and found 1.7-fold higher whole brain BBB leakage compared to controls. Finally, we compared autopsy tissue from people with infarcts and dementia at death to those with infarcts and no dementia. Those who died with dementia had dramatic loss of vascular mural cell coverage compared to those without dementia (median 0.7% vs. 27%). Thus, our proteomic, functional, and structural data implicate chronic BBB dysfunction in cognitive decline late after stroke, revealing potential proteomic and imaging biomarkers and, importantly, a novel target for intervention.

neuroscience↗

bioRxiv2026

Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

Frontotemporal dementia (FTD) is a major cause of early-onset neurodegeneration characterized by progressive behavioral, emotional, and cognitive decline. Progranulin haploinsufficiency, a leading genetic cause of familial FTD, disrupts lysosomal function, lipid metabolism, autophagy, and neuroimmune signaling across multiple cell types. Increasing evidence indicates that microglia are particularly sensitive to progranulin loss, exhibiting elevated complement activation that contributes to TDP-43 proteinopathy and neuronal dysfunction. Here, we investigate the biological role of restoring progranulin exclusively within microglia by transplanting human induced pluripotent stem cell-derived microglia (iMG) into progranulin (Grn)-deficient mice. We find that wild-type, but not Grn-deficient, human iMG restore brain-wide progranulin levels, normalize microglial transcriptional states, and ameliorate pathological, functional, and behavioral phenotypes associated with progranulin loss. Because microglia are the only source of progranulin in this system, these findings demonstrate that microglial progranulin is sufficient to restore key aspects of cellular, circuit, and behavioral homeostasis in a progranulin-deficient FTD model. More broadly, this work highlights a central, microglia-intrinsic role for progranulin in maintaining brain function and provides a framework for dissecting microglia-specific mechanisms across FTD and related neurodegenerative disorders. One Sentence SummaryOur study demonstrates that xenotransplantation of wild-type human iPSC-derived microglia into progranulin-deficient mice mitigates core neuropathological, network-level, and behavioral features of Frontotemporal Dementia.

neuroscience↗

bioRxiv2025

Cell-Type-Resolved Isoform Atlas of Human Tissues Reveals Age and Alzheimer's Disease-Associated Splicing Changes

Alternative splicing is a key mechanism for transcriptomic diversity, but how isoforms map to specific cell types in bulk tissues remains unclear. We present Sciege, a multimodal method that integrates bulk short-read RNA-seq with single-cell and long-read data to estimate cell type-specific isoform distributions. Through simulations, we demonstrate that Sciege accurately estimates isoform abundances and identifies differentially abundant transcripts through statistical tests. Applied to seven tissues in GTEx and brain tissue in ROSMAP datasets, Sciege generates a first-to-date multi-tissue isoform atlas and reveals isoform changes linked to cell types, aging, and Alzheimers disease. Validation with external cohorts and experimental data confirms our findings. Notably, we identify upregulation of the MAPT-010 isoform in AD inhibitory neurons, consistent with known methylation signatures. Our approach demonstrates the value of integrating RNA-seq data to study cell type-specific splicing and provides a foundation for further genetic and functional studies of alternative splicing across biological contexts.

bioinformatics↗

bioRxiv2025

RIP3 and MLKL regulate Hepatic ER stress in alcohol-associated liver disease and pharmacological ER stress models: insights beyond necroptosis

Background and AimEndoplasmic reticulum (ER) stress is an important contributor to liver disease progression, including alcohol-associated liver disease (ALD). While receptor-interacting protein kinase-3 (RIP3) and mixed lineage kinase domain-like pseudokinase (MLKL) are known for their roles in necroptosis, emerging evidence highlights their non-canonical functions in metabolic regulation and cellular stress responses. However, their specific role in regulating hepatic ER stress remains unclear. This study investigates how RIP3, its kinase activity, and MLKL regulate ER stress pathways during chronic ethanol exposure and pharmacological ER stress induction. MethodsRip3-/-, Rip3K51A/K51A and Mlkl-/- mice alongside WT controls and pharmacological necroptosis inhibitors were used to study the role of RIP3 and MLKL in modulating ER stress. Chronic ethanol feeding and pharmacological agents (tunicamycin, thapsigargin) were utilized to induce ER stress in vivo and in isolated primary hepatocytes. ER stress markers were assessed by qPCR and western blot, ER expansion was evaluated by confocal microscopy, and hepatocyte viability was measured using MTS assay. ResultsChronic ethanol increased expression of ER stress markers in WT mice; this response was attenuated in Rip3-/- mice. Tunicamycin exposure increased hepatic ER stress markers in WT mice; this response was diminished in Rip3-/-, Rip3K51A/K51A and Mlkl-/-mice. In primary hepatocytes, genetic and pharmacological inhibition of RIP3 and MLKL also reduced thapsigargin-induced ER stress responses. Hepatocytes isolated from Rip3-/-, Rip3K51A/K51Aand Mlkl-/- mice exhibited enhanced cell viability under ER stress conditions compared to hepatocytes from WT mice, which was associated with ER expansion as a potential mechanism for mitigating ER stress. ConclusionThis study highlights a novel function of RIP3 and MLKL in regulating hepatic ER stress responses, expanding their known roles beyond programmed necrosis. Impact and ImplicationsThis study provides new mechanistic insight into how RIP3 and MLKL regulate hepatic ER stress responses, extending their roles beyond necroptosis. By demonstrating that genetic or pharmacological inhibition of Rip3, RIP3 kinase activity and Mlkl attenuates ER stress signaling, reduces cell death, and promotes adaptive ER remodeling, our findings identify these proteins as key modulators of hepatocyte survival under stress. These results are important for researchers and clinicians focused on alcohol-associated liver disease and other ER stress-driven liver disorders, as they highlight novel therapeutic targets. In practical terms, modulation of the RIP3- MLKL axis could inform the development of interventions aimed at enhancing ER stress resilience, with potential applications in drug development for ER stress-associated liver injury.

cell biology↗

bioRxiv2025

Preventing Microglial Reactivity Protects from Acute and Progressive Neuronal Dysfunction, Motor Impairments and Sedation following Alcohol Abuse

Alcohol abuse is the primary risk factor for alcohol use disorder (AUD), a leading cause of preventable morbidity and mortality, characterized by systemic inflammation, multi-organ damage, and neurological impairments. While direct effects of alcohol on brain function are well-established, the role of microglia in acute and chronic neurological dysfunction in AUD remains unclear. Using longitudinal in vivo imaging in mice during acute and repeated alcohol abuse, we found that microglia exhibit dynamic morphological responses that precede but parallel ethanol-induced sedation. Ethanol also induced microglia-dependent synapse elimination and reduced neuronal activity and density. Genetic disruption of microglial MyD88 reversed these ethanol-associated changes in microglial reactivity, neuronal structure, and function, while protecting against alcohol-induced intoxication and motor impairments. These findings identify microglia as cellular drivers of acute and chronic brain dysfunction following alcohol abuse, and highlight MyD88 as a critical therapeutic target for the detrimental neurological consequences of AUD.

neuroscience↗

bioRxiv2025

Mitochondria-derived dsRNA-induced stress granules promote IRF3-mediated fibrotic responses

Nucleic acid-induced activation of the anti-viral transcription factor interferon regulatory factor 3 (IRF3) promotes transforming growth factor-beta (TGF{beta})-induced fibrotic responses of hepatic stellate cells in vitro. Herein, we identified molecular mechanisms underlying TGF{beta}-induced IRF3 activation. Gene silencing of IRF3 with short interfering RNA (siRNA) in LX2 human hepatic stellate cells decreased TGF{beta}-induced fibrotic responses. An unbiased proteomic analysis of homeostatic IRF3-interacting proteins revealed striking enrichment in cytoplasmic stress granule components, including G3BP stress granule assembly factor 1 (G3BP1), G3BP2, and cell cycle associated protein 1 (CAPRIN1). TGF{beta} induced cytoplasmic accumulation of mitochondria-derived double-stranded RNA (mt-dsRNA) and assembly of IRF3-containing stress granules. Finally, blockade of G3BP1 activity or depletion of mt-dsRNA decreased TGF{beta}-induced fibrotic responses. Collectively, these findings identify a novel TGF{beta}-mt-dsRNA-stress granule axis in hepatic stellate cells that promotes IRF3-mediated fibrotic responses and potentially implicate cytoplasmic stress granules as a global regulator of fibrosis.

cell biology↗

bioRxiv2025

State Space Misspecification in Morphological Phylogenetics: A Pitfall for Models and Parsimony Alike

Phylogenetic analysis relies on two fundamental levels of biological information: genotype and phenotype. Molecular data benefit from operating within a well-defined, finite state space (e.g., nucleotide alphabets), whereas morphological data present inherent challenges due to frequently ambiguous character states and variable state counts. In this study, I use simulated data to examine how state space misspecification (SSM), defined as the mismatch between the assumed and true state space, affects phylogenetic reconstruction. Results show that SSM generally reduces topological accuracy, with the extent of its impact depending on mutation rate, state space disparity, and the proportion of affected characters. Counterintuitively, under conditions typical of empirical morphological datasets (high proportions of binary characters and elevated mutation rates), SSM can improve topological precision. This creates a paradox where an incorrect model outperforms a correct one, though at the cost of distorted branch lengths. Importantly, the effects of SSM extend beyond model-based approaches. I demonstrate, through an extension of the no common mechanism (NCM) model, that standard maximum parsimony is consistent with the assumption that characters evolved under an SSM model--a largely overlooked feature. To address this, I propose a state-space-aware weighting scheme that accounts for variation in character state space. I also discuss additional strategies for mitigating SSM, including model adjustments and reducing reliance on oversimplified binary coding. This work underscores the need to explicitly address state space uncertainty in morphological phylogenetics. As morphology remains crucial for reconstructing deep-time lineages and integrating fossils, accounting for SSM is essential to improving the reliability of evolutionary trees.

evolutionary biology↗

bioRxiv2025

A unified eIF1A+ luminal cells-centered hypoxic and "cold" tumor microenvironment promotes PCa progression among different subtypes

PCa is a malignancy with high heterogeneity arises from both tumor microenvironment (TME) and histological subtypes. To achieve superior clinical efficacy, it is imperative to identify unified progression drivers within such heterogeneity in PCa, thereby enabling the development of more consistent and effective therapeutic strategies. In this study, we applied imaging mass cytometry to stain 39 proteins on 71 tissues comprising para-cancer, low-grade acinar adenocarcinoma (LgPAC), high-grade PAC (HgPAC), intraductal carcinoma (IDC) and ductal adenocarcinoma (DAC) tissues, obtaining the spatial proteomic landscape of 345,233 single cells. We discovered a hypoxic eIF1A+ luminal epithelial (LE) cluster exhibiting both translational activation and immunosuppressive properties, which was enriched in high-risk PCa including HgPAC, IDC and DAC, and associated which tumor progression and unfavorable prognosis. Additionally, eIF1A+ LE orchestrates the formation of an immunologically "cold" TME, characterized by the low infiltration of anti-cancer immune cells including PD1- T cells CD163- macrophages. Through in vivo validation and investigator-initiated study, we further found that Homoharringtonine, an inhibitor targeting protein translation process, effectively inhibited PCa growth, alleviated hypoxic microenvironment, and enhanced immune cell infiltration. This study is the first to apply spatial proteomics to delineate consistent molecular features across histological subtypes, providing transformative insights for clinical management of PCa.

cancer biology↗