Biology subjects

Lu, W.

Publications and source records attributed to Lu, W..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Tissue Factor Expression in Penile Squamous Cell Carcinoma: A Marker of HPV-Independent Disease

In a series of 33-patients, we evaluated tissue factor (TF) expression in penile squamous cell carcinoma (PSCC). A tissue microarray (TMA) was constructed with 3 cores per patient tumor (99 total cores). Anti-TF antibody staining was performed by immunohistochemistry and H-scores for membrane and cytoplasm staining were assessed (range 0-300). Percentage of cores and patient tumors staining positive for TF ([≥]10% of tumor cells with at least 1+ intensity in cytoplasm and/or membrane) and H-scores were described and compared with HPV and p16 status. Association of TF expression with tumor grade, presence of metastatic disease, lymphovascular invasion (LVI), perineural invasion (PNI), aberrant p53 expression, recurrence free survival (RFS), and cancer specific survival (CSS) were assessed. Nectin-4 and TROP2 staining and their association with clinical/pathological data was determined in a similar manner. TF staining was more prominent in HPV-negative tumors in both the membrane (H-score 69.6 vs 18.8; p=0.003) and cytoplasm (H-score 59.2 vs. 17.7, p=0.007). Cytoplasmic (H-score 61.7 vs 11.7, p=<0.001) and membrane TF staining (H-score 71.7 vs 15.0, p=<0.001) favored p16 negative tumors. The p53 status was more likely to be aberrant in the higher TF staining samples (cytoplasm H-score 61.7 vs 18.3, p=0.012; membrane H-score 67.5 vs 20.3, p=0.006). We observed an association with TROP2 staining and positive p16 status (membrane H-score 120.3 vs. 85, p=0.052; cytoplasmic H-score 135 vs. 107.5, p=0.041). We observed an association of TROP2 staining with positive LVI (membrane H-score 136.7 vs. 66.7, p=0.014; cytoplasmic H-score 110 vs. 93.3, p=0.04). We found no association between TF, TROP2, or nectin-4 staining with CSS or RFS; however, we suspect that this is due to our small sample size. Our results indicate that TF expression could be positive biomarker for HPV-independent, p53-aberrant PSCC, while TROP2 could be associated with HPV-associated PSCC.

pathology↗

bioRxiv2025

Sustained Lysosomal Delivery of Enhanced Cy3-Labeled Acid Nanoparticles Restores Lysosomal pH in Retinal Pigment Epithelial Cells and Astrocytes

Lysosomal pH is frequently elevated in age-dependent neurodegenerations like Age-related Macular Degeneration (AMD), Alzheimers Disease (AD), and Parkinsons Disease (PD). Tools that restore lysosomal pH to an optimal acidic range could enhance enzymatic degradation and reduce waste accumulation. Acidic nanoparticles offer a promising strategy for restoring lysosomal function, but accurate tracking of organelle delivery and long-term retention is needed to optimize dosage. To improve detection and enhance delivery, nanoparticles were synthesized from Poly(D,L-lactide-co-glycolide) (PLGA) polymers covalently linked to the fluorescent Cyanine3 amine (Cy3) probe. Nanoparticle concentration and loading times were optimized to achieve >90% delivery to lysosomes in cultured induced pluripotent stem cell-derived retinal pigment epithelial (iPS-RPE) cells. Uptake was heterogeneous, varying between adjacent cells. Once loaded into lysosomes, the nanoparticles were stably retained, with no detectable changes in concentration, distribution, or size for at least 28 days. iPS-RPE cells internalized more nanoparticles than the ARPE-19 cell line or mouse optic nerve head astrocyte cultures. Functionally, PLGA nanoparticles restored an acidic pH and cathepsin D levels in compromised lysosomes. In summary, Cy3-PLGA nanoparticles enabled improved tracking and long-term delivery to lysosomes, supporting future in vivo applications to restore lysosomal pH in aging and degenerating tissues. Graphical AbstractIncreased lysosomal pH reduces degradative enzyme efficiency and contribute to age-dependent neurodegeneration. This study describes synthesis of nanoparticles to restore an acidic lumen and degradative function. Nanoparticles were optimized for lysosomal delivery to astrocytes and iPS-derived retinal pigmented epithelial (RPE) cells. The fluorescent marker Cy3 was covalently bound to polymers for improved tracking to lysosomes. Particles were stably retained inside the lysosomal lumen for at least 28 days. Nanoparticles restored pH to compromised lysosomes to baseline levels and increased active Cathepsin D. The improved design will aid in vivo tracking and repair in models where lysosomal alkalinization contributes to pathology. Created in BioRender. Mitchell, C. (2025) https://BioRender.com/8hvj96m. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/659991v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@d6fcf1org.highwire.dtl.DTLVardef@7833aborg.highwire.dtl.DTLVardef@188593corg.highwire.dtl.DTLVardef@9aa531_HPS_FORMAT_FIGEXP M_FIG C_FIG New and NoteworthyTools that restore acidic pH in compromised lysosomes can enhance autophagy and waste degradation in degenerative disorders marked by excessive accumulation. Here, we describe the novel synthesis of lysosome-targeted nanoparticles composed of PLGA polymers covalently bound to Cy3 fluorescent dye. These Cy3-PLGA nanoparticles enabled improved tracking of lysosomal delivery and demonstrated sustained long-term retention within lysosomes, supporting their potential for future applications to restore lysosomal pH in aging and degenerating tissues.

cell biology↗

bioRxiv2025

Spatial metabolic gradients in the liver and small intestine

Mammalian organs are composed of cells, whose properties differ depending upon their spatial location. Gene expression in liver varies between periportal and pericentral hepatocytes1-3, and in intestine from crypts to villus tips4,5. A key element of tissue spatial organization is likely metabolic, but direct assessments of spatial metabolism remain limited. Here we map spatial metabolic gradients in murine liver and intestine. We developed an integrated experimental-computational workflow using MALDI imaging mass spectrometry, isotope tracing, and deep-learning artificial intelligence. Most measured metabolites (> 95%) showed significant spatial concentration gradients in liver lobules and intestinal villi. In the liver, tricarboxylic acid (TCA)-cycle metabolites and their labeling from both glutamine and lactate localized periportally. Energy-stress metabolites including adenosine monophosphate (AMP) also localized periportally, consistent with high periportal energy demand. In intestine, the TCA intermediates malate (tip) and citrate (crypt) showed opposite spatial patterns, which aligned with higher glutamine catabolism in tips and lactate oxidation in crypts based on isotope tracing. Finally, we mapped the fate of the obesogenic dietary sugar fructose. In the intestine, oral fructose was catabolized faster in the villus bottom than the tips. In the liver, fructose-derived carbon accumulated pericentrally as fructose-1-phosphate and triggered pericentral adenosine triphosphate (ATP) depletion. Thus, we both provide foundational knowledge regarding intestine and liver metabolic organization and identify fructose-induced focal derangements in liver metabolism.

biochemistry↗

bioRxiv2025

Mechanism of age-related accumulation of mitochondrial DNA mutations in human blood

One of the strongest signatures of aging is an accumulation of mutant mitochondrial DNA (mtDNA) heteroplasmy. Here we investigate the mechanism underlying this phenomenon by calling mtDNA sequence, abundance, and heteroplasmic variation in human blood using whole genome sequences from [~]750,000 individuals. Our analyses reveal a simple, two-step mechanism: first, individual cells randomly accumulate low levels of "cryptic" mtDNA mutations; then, when a cell clone proliferates, the cryptic mtDNA variants are carried as passenger mutations and become detectable in whole blood. Four lines of evidence support this model: (1) the mutational spectrum of age-accumulating mtDNA variants is consistent with a well-established model of mtDNA replication errors, (2) these mutations are found primarily at low levels of heteroplasmy and do not show evidence of positive selection, (3) high mtDNA mutation burden tends to co-occur in samples harboring somatic driver mutations for clonal hematopoiesis (CH), and (4) nuclear GWAS reveals that germline variants predisposing to CH (such as those near TERT, TCL1A, and SMC4) also increase mtDNA mutation burden. We propose that the high copy number and high mutation rate of mtDNA make it a particularly sensitive blood-based marker of CH. Importantly, our work helps to mechanistically unify three prominent signatures of aging: common germline variants in TERT, clonal hematopoiesis, and observed mtDNA mutation accrual.

genetics↗

bioRxiv2025

Characterization of hyperoxia-induced senescent lung macrophages in neonatal mice

Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, results from mechanical ventilation and hyperoxia amongst other factors. We and others have shown that neonatal hyperoxia, known to lead to a BPD-like phenotype in rodent models, causes lung cellular senescence. In our 3-day hyperoxia model, the majority of senescent cells were lung macrophages, and these peaked at postnatal day (pnd) 7. The features of these senescent macrophages are not characterized. Here, we reanalyzed scRNA-seq datasets (GSE207866) of senescent lung cells from mice exposed to hyperoxia as neonates at pnd7 (SD7), and characterized their gene express profiling and compared them with air control (AirD7) and hyperoxia-exposed group without isolation of senescent cells at pnd7 (O2D7). We first classified the cells into epithelial, endothelial, immune, and mesenchymal cells to extract immune cells. By employing the workflow to the immune clusters, mixed populations of macrophages, monocytes, and dendritic cells and pure population of macrophages, we finally identified seven clusters of macrophages. In SD7 group, 65.9% senescent cells were macrophages, and comprised M1 (64%) and alveolar (62.8%) macrophages. Clusters 0 and 1 were M1 and alveolar macrophages, which were composed of 49.2% of senescent macrophages. These two clusters highly expressed genes involved in innate immunity, inflammation, DNA repair response and phagocytosis. Metabolic switch from mitochondrial respiration to glycolysis and pentose phosphate pathway was observed in these two clusters. Conclusively, senescent macrophages are heterogenous with distinct tissue compartments and metabolic dysregulation.

cell biology↗

bioRxiv2025

The Association Between SLIT2 in Human Vitreous Humor and Plasma and Neurocognitive Test Scores

BackgroundSlit Guidance Ligand 2 (SLIT2) binds Roundabout (ROBO) guidance receptors to direct axon pathfinding and neuron migration during nervous system development. SLIT2 expression has previously been linked to dementia risk. ObjectiveTo study the association between SLIT2 expression in human vitreous humor and plasma samples and neurocognitive test scores in a cross-sectional cohort study utilizing a novel, highly-sensitive Meso Scale Discovery (MSD) assay for SLIT2 detection. MethodsSeventy-nine individuals with a mean age of 55.79 {+/-} 12.03 years underwent eye surgery with collection of vitreous humor, blood (plasma) collection, and neurocognitive assessment. Vitreous humor and plasma samples were analyzed by SLIT2 MSD electrochemiluminescence immunoassay. Associations between SLIT2 levels in vitreous humor and plasma were analyzed using GraphPad Prism. ResultsWe found up to a 7-fold higher level of SLIT2 in human vitreous humor compared to plasma. Lower vitreous SLIT2 levels were associated with a lower Montreal Cognitive Assessment (MoCA) score and Immediate Recall Verbatim (IRV) z-score, and higher plasma SLIT2 was associated with a lower MoCA score. In multivariate analysis using single and multiple predictor models, the same significant associations were found when adjusted for age, sex, race, diabetic status, diabetic retinopathy status, glaucoma status, and Apolipoprotein E (APOE) genotype. ConclusionsSLIT2 protein levels are significantly associated with MoCA score and IRV z-score in middle-aged individuals. The relationship remained significant when adjusted for demographics, co-morbidity, and APOE genotype, suggesting SLIT2 may be a sensitive biomarker for detection of mild cognitive impairment and early dementia, and warrants further studies.

neuroscience↗

bioRxiv2025

Influence of Macrocyclization Strategies on DNA-Encoded Cyclic Peptide Libraries

Discovery of cyclic peptide hits using DNA-encoded libraries (DELs) has recently been extensively researched, with significant efforts directed toward developing DEL-compatible macrocyclization methods. To investigate how different cyclic linkers influence DEL selection outcomes, we constructed eight distinct sub-libraries and screened them against two protein targets, MDM2 and GIT1, resulting in two representative yet contrasting scenarios. Validation studies for MDM2 revealed that structural similarity patterns observed across multiple sub-libraries could enhance confidence in the authenticity of identified hits. Notably, cmp-10 demonstrated potent inhibitory activity, exhibiting an inhibition constant of 11 nM. In contrast, selections against GIT1 produced discrete enrichment patterns. From these outcomes, two exemplary compounds were selected and validated through both on-DNA and off-DNA modes. cmp-17 was confirmed to bind specifically to the desired binding pocket, displaying a dissociation constant of 1.22 M. Furthermore, ITC experiments using mutant GIT1 proteins (GIT1L271A/L279A and GIT1L271L/L279L) provided additional insights into the mechanism by which cmp-17 disrupts the interaction between GIT1 and {beta}-Pix.

biochemistry↗

bioRxiv2025

Closing The Loop: A Dynamic Neural Network Model Integrating Decision making and Metacognition

Decision making under uncertainty entails selecting optimal actions from noisy evidence. Currently influential models, such as drift-diffusion and attractor frameworks, posit decisions as bottom-up stochastic evidence accumulation from sensory inputs but ignore critical interactions between decision, commitment, and metacognition. While these models explain basic choice behaviors and accompanying confidence, they fail to reconcile many empirical findings, including motor-area encoding of decision variables and time-dependent urgency signals. We present a closed-loop neural network model unifying three modules: decision module accumulating evidence, a motor module implementing action thresholds, and a metacognition module regulating deliberation through dual feedback pathways to suppress noise-driven errors and accelerate decision commitment under time constraints, respectively. This architecture can account for crucial characteristics of decision-making and metacognition that were empirically observed. By integrating decision, motor, and metacognitive dynamics, our model provides a biologically grounded framework for optimal decision making, offering testable predictions for neural and behavioral studies.

neuroscience↗

bioRxiv2025

ZEB2 signaling is essential for ureteral smooth muscle cell differentiation and maintenance

Mowat-Wilson Syndrome (MWS) is a multiple congenital anomaly syndrome caused by mutations in the ZEB2, which plays a critical role in cell fate determination and differentiation during development. Congenital anomalies of the kidney and urinary tract (CAKUT) have been reported in MWS patients. However, the role of ZEB2 in urinary tract development and the cellular and molecular mechanism underlining the CAKUT phenotypes in MWS remains unknown. We performed ZEB2 protein expression analysis in the developing mouse ureter. We generated Zeb2 ureteral mesenchyme-specific conditional knockout mice by crossing Zeb2 floxed mice with Tbx18Cre mice (Zeb2 cKO) and analyzed the urinary tract phenotypes in Zeb2 cKO mice and wild-type littermate controls by gross and histological examination. Ureteral cellular and molecular phenotypes were studied using TAGLN, ACTA2, FOXD1, POSTN, CDH1, TBX18, and SOX9 ureteral cell-specific markers. We found that ZEB2 is expressed in TBX18+ ureteral mesenchymal cells during mouse ureter development. Deletion of Zeb2 in developing ureteral mesenchymal cells causes hydroureter and hydronephrosis phenotypes, leading to obstructive uropathy, kidney failure, and early mortality. Cellular and molecular marker analyses showed that the TAGLN+ACTA2+ ureteral smooth muscle cells (SMCs) layer is not formed in Zeb2 cKO mice at E15.5, but the FOXD1+ and POSTN+ tunica adventitia cells layer is significantly expanded compared to wild-type controls. CDH1+ urothelium cells are reduced considerably in the Zeb2 cKO ureters at E15.5. Mechanistically, we found that Zeb2 cKO mice have significantly decreased TBX18 expression but an increased SOX9 expression in the developing ureter at E14.5 and E15.5 compared to wild-type littermate controls. Our results show that ZEB2 is essential for ureter development by maintaining ureteral mesenchymal cell differentiation into normal ureteral SMCs. Our study also shed new light on the pathological mechanism underlying the developmental abnormalities of the urinary tract phenotypes in MWS patients. Author SummaryHydroureter and hydronephrosis are common congenital anomalies with high incidence (1:100 to 1:500) that can lead to obstructive uropathy and renal failure in the pediatric population. Mowat-Wilson Syndrome (MWS), caused by heterozygous mutations in the ZEB2 gene, is a genetic disease with multiple congenital developmental defects, including hydroureter and hydronephrosis. However, the molecular function of ZEB2 in ureter development and pathogenesis of hydroureter and hydronephrosis remains unknown. Here, we show that ZEB2 is expressed in the developing ureteral mesenchymal cells, and deletion of ZEB2 in ureteral mesenchymal cells leads to ureteral smooth muscle cell loss, which is replaced by tunica adventitia cells causing hydroureter and hydronephrosis phenotype. Hence, our work not only demonstrates the critical role of ZEB2 in ureter development but also provides molecular mechanisms of urinary tract anomalies in MWS patients.

developmental biology↗

bioRxiv2025

Therapeutic assessment of a novel mitochondrial complex I inhibitor in in vitro and in vivo models of Alzheimer's disease

Despite recent approval of monoclonal antibodies that reduce amyloid (A{beta}) accumulation, the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimers disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target, where its weak inhibition activates neuroprotective signaling, benefiting AD mouse models with A{beta} and p-Tau pathologies. Rational design and structure-activity relationship studies yielded novel mtCI inhibitors profiled in a drug discovery funnel designed to address their safety, selectivity, and efficacy. The new lead compound C458 is highly protective against A{beta} toxicity, has favorable pharmacokinetics, and has minimal off-target effects. C458 exhibited excellent brain penetrance, activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted via functional tests, metabolic assessment, in vivo 31P- NMR spectroscopy, blood cytokine panels, ex vivo electrophysiology, and Western blotting. Chronic oral administration improved long-term potentiation, reduced oxidative stress and inflammation, and enhanced mitochondrial biogenesis, antioxidant signaling, and cellular energetics. These studies provide further evidence that the restoration of mitochondrial function and brain energetics in response to mild energetic stress represents a promising disease- modifying strategy for AD.

neuroscience↗

bioRxiv2025

Convergent Agonist and Heat Activation of Nociceptor TRPM3

Detecting noxious heat is vital for survival, triggering pain responses that protect against harm1,2. The TRPM3 channel is a key nociceptor for sensing noxious heat and a promising therapeutic target for pain treatment and neurological disorders such as epilepsy3-11. Here, we functionally and structurally characterized TRPM3 in response to diverse stimuli: the synthetic superagonist CIM0216 Ref12, the anticonvulsant antagonist primidone13,14, and heat1,10,15. Our findings reveal that TRPM3 is intrinsically dynamic, with its intracellular domain (ICD) sampling both resting and activated states, though strongly favoring the resting state without stimulation. CIM0216 binds to the S1-S4 domain, inducing conformational changes in the ICD and shifting the equilibrium toward activation. Remarkably, heat induces similar ICD rearrangements, revealing a converged activation mechanism driven by chemical compounds and temperature. This mechanism is supported by functional data showing that mutations facilitating the ICD movement markedly increase the sensitivity of TRPM3 to both chemical and thermal signals. These findings establish a critical role of the ICD in temperature sensing in TRPM3, a mechanism likely conserved across the TRPM family. Finally, we show that primidone binds to the same site as CIM0216 but acts as an antagonist. This study provides a framework for understanding the thermal sensing mechanisms of temperature-sensitive ion channels and offers a structural foundation for developing TRPM3-target therapeutics for pain and neurological disorders.

biophysics↗

bioRxiv2024

Same-Slide Spatial Multi-Omics Integration Reveals Tumor Virus-Linked Spatial Reorganization of the Tumor Microenvironment

The advent of spatial transcriptomics and spatial proteomics have enabled profound insights into tissue organization to provide systems-level understanding of diseases. Both technologies currently remain largely independent, and emerging same slide spatial multi-omics approaches are generally limited in plex, spatial resolution, and analytical approaches. We introduce IN-situ DEtailed Phenotyping To High-resolution transcriptomics (IN-DEPTH), a streamlined and resource-effective approach compatible with various spatial platforms. This iterative approach first entails single-cell spatial proteomics and rapid analysis to guide subsequent spatial transcriptomics capture on the same slide without loss in RNA signal. To enable multi-modal insights not possible with current approaches, we introduce k-bandlimited Spectral Graph Cross-Correlation (SGCC) for integrative spatial multi-omics analysis. Application of IN-DEPTH and SGCC on lymphoid tissues demonstrated precise single-cell phenotyping and cell-type specific transcriptome capture, and accurately resolved the local and global transcriptome changes associated with the cellular organization of germinal centers. We then implemented IN-DEPTH and SGCC to dissect the tumor microenvironment (TME) of Epstein-Barr Virus (EBV)-positive and EBV-negative diffuse large B-cell lymphoma (DLBCL). Our results identified a key tumor-macrophage-CD4 T-cell immunomodulatory axis differently regulated between EBV-positive and EBV-negative DLBCL, and its central role in coordinating immune dysfunction and suppression. IN-DEPTH enables scalable, resource-efficient, and comprehensive spatial multi-omics dissection of tissues to advance clinically relevant discoveries.

systems biology↗

bioRxiv2024

The biophysical mechanism of mitochondrial pearling

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, "beads-on-a-string" morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of "pearls" connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation, neuronal firing, and replicative senescence. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling generated by ionic flux, and ii) external tension-driven pearling generated by the cytoskeleton. By applying live-cell STED and FIB-SEM imaging we document the structural reorganization of inner cristae membranes during mitochondrial pearling and the role of the MICOS complex in regulating the frequency of pearling events. We then establish numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux producing internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.

biophysics↗

bioRxiv2024

Developmentally regulated actin-microtubule crosstalk in Drosophila oogenesis

Actin-microtubules (MT) interactions are essential, but how those mechanisms are orchestrated in complex developing systems is poorly understood. Here we show that actin-MT crosstalk regulates actin cable assembly and the assembly and organization of MTs in Drosophila nurse cells during oogenesis. We found that a stable MT meshwork develops concurrently with actin cable initiation and requires acetylation for its maintenance. These {gamma}-tubulin nucleated MTs are cortically tethered via Patronin and Shortstop, extend into the cytoplasm, and coalign with the elongating actin cables. The MT network promotes the cortical enrichment of the actin assembly factors Diaphanous and Enabled and loss of MTs dramatically reduced actin cable density and growth rate. We further demonstrated that actin filament assembly promotes cortical tethering of MTs and that loss of the actin filament bundler Villin/Quail results in fewer, shorter, and more highly coaligned MTs. Together, our data reveal multiple modes of coordinated actin-MT crosstalk producing actin cable-MT network synergy that is instrumental for oogenesis. SummaryThis study demonstrates actin-microtubule crosstalk in Drosophila oogenesis, revealing coregulation between actin filament assembly/bundling and microtubule nucleation/organization, highlighting the coordinated regulation of the cytoskeleton required during development.

cell biology↗

bioRxiv2024

Comparison of imaging-based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed, paraffin-embedded tumor samples

Imaging-based spatial transcriptomics (ST) is evolving rapidly as a pivotal technology in studying the biology of tumors and their associated microenvironments. However, the strengths of the commercially available ST platforms in studying spatial biology have not been systematically evaluated using rigorously controlled experiments. In this study, we used serial 5-m sections of formalin-fixed, paraffin-embedded surgically resected lung adenocarcinoma and pleural mesothelioma tumor samples in tissue microarrays to compare the performance of the single cell ST platforms CosMx, MERFISH, and Xenium (uni/multi-modal) platforms in reference to bulk RNA sequencing, multiplex immunofluorescence, GeoMx Digital Spatial Profiler, and hematoxylin and eosin staining data for the same samples. In addition to objective assessment of automatic cell segmentation and phenotyping, we performed pixel-resolution manual evaluation of phenotyping to carry out pathologically meaningful comparison between ST platforms. Our study detailed the intricate differences between the ST platforms, revealed the importance of parameters such as tissue age and probe design in determining the data quality, and suggested reliable workflows for accurate spatial profiling and molecular discovery.

bioinformatics↗

bioRxiv2024

BindingGYM: A Large-Scale Mutational Dataset Toward Deciphering Protein-Protein Interactions

Protein-protein interactions are crucial for drug discovery and understanding biological mechanisms. Despite significant advances in predicting the structures of protein complexes, led by AlphaFold3, determining the strength of these interactions accurately remains a challenge. Traditional low-throughput experimental methods do not generate sufficient data for comprehensive benchmarking or training deep learning models. Deep mutational scanning (DMS) experiments provide rich, high-throughput data; however, they are often used incompletely, neglecting to consider the binding partners, and on a per-study basis without assessing the generalization capabilities of fine-tuned models across different assays. To address these limitations, we collected over ten million raw DMS data points and refined them to half a million high-quality points from twenty-five assays, focusing on protein-protein interactions. We intentionally excluded non-PPI DMS data pertaining to intrinsic protein properties, such as fluorescence or catalytic activity. Our dataset meticulously pairs binding energies with the sequences and structures of all interacting partners using a comprehensive pipeline, recognizing that interactions inherently involve at least two proteins. This curated dataset serves as a foundation for benchmarking and training the next generation of deep learning models focused on protein-protein interactions, thereby opening the door to a plethora of high-impact applications including understanding cellular networks and advancing drug target discovery and development.

biophysics↗

bioRxiv2024

CNV-Profile Regression: A New Approach for Copy Number Variant Association Analysis in Whole Genome Sequencing Data

Copy number variants (CNVs) are DNA gains or losses involving >50 base pairs. Assessing CNV effects on disease risk requires consideration of several factors. First, there are no natural definitions for CNV loci. Second, CNV effects can depend on dosage and length. Third, CNV effects can be more accurately estimated when all CNV events in a genomic region are analyzed together to assess their joint effects. We propose a new framework for association analysis that directly models an individuals entire CNV profile within a genomic region. This framework represents an individuals CNVs using a CNV profile curve to capture variations in CNV length and dosage and to bypass the need to predefine CNV loci. CNV effects are estimated at each genome position, making the results comparable across different studies. To jointly estimate the effects of all CNVs, we use a Lasso penalty to select CNVs associated with the trait and integrate a weighted L2-fusion penalty to encourage similar effects of adjacent CNVs when supported by the data. Simulations show that the proposed model can more effectively identify causal CNVs while maintaining false positive rates comparable to baseline methods and yield more precise effect-size estimates across different settings. When applied to CNV derived from whole genome sequencing data of the Alzheimers Disease Sequencing Project, the proposed methods identify additional CNVs associated with Alzheimers Disease (AD). These identified CNVs overlap with several known AD-risk genes and are significantly enriched by biological processes related to neuron structures and functions crucial in AD development.

bioinformatics↗

bioRxiv2024

Language model-guided anticipation and discovery of unknown metabolites

Despite decades of study, large parts of the mammalian metabolome remain unexplored. Mass spectrometry-based metabolomics routinely detects thousands of small molecule-associated peaks within human tissues and biofluids, but typically only a small fraction of these can be identified, and structure elucidation of novel metabolites remains a low-throughput endeavor. Biochemical large language models have transformed the interpretation of DNA, RNA, and protein sequences, but have not yet had a comparable impact on understanding small molecule metabolism. Here, we present an approach that leverages chemical language models to discover previously uncharacterized metabolites. We introduce DeepMet, a chemical language model that learns the latent biosynthetic logic embedded within the structures of known metabolites and exploits this understanding to anticipate the existence of as-of-yet undiscovered metabolites. Prospective chemical synthesis of metabolites predicted to exist by DeepMet directs their targeted discovery. Integrating DeepMet with tandem mass spectrometry (MS/MS) data enables automated metabolite discovery within complex tissues. We harness DeepMet to discover several dozen structurally diverse mammalian metabolites. Our work demonstrates the potential for language models to accelerate the mapping of the metabolome.

bioinformatics↗