Biology subjects

Du, P.

Publications and source records attributed to Du, P..

At least 19 recordsLinked to original sources

bioRxiv2026

In vivo intercellular CRISPR screens using viral proximity barcoding reveal regulators of tumor-immune interactions

Cell-cell interactions shape tumor growth, immune evasion, and therapeutic response, but systematically dissecting their genetic regulators in vivo remains challenging. Existing cell-cell interaction tracing technologies rely largely on protein labels or enzymatic reactions, limiting their information content and scalability. To overcome these limitations, we developed match-seq, an imaging-free, sequencing-based cell proximity tracing system that uses virus-like particles to transmit barcodes between neighboring cells. Barcoded mRNAs are transmitted from sender cells to nearby receiver cells, thereby establishing a spatial linkage that can be computationally reconstructed by barcode sequencing after tissue dissociation. We apply this system to an in vivo syngeneic murine tumor model, where we observe robust labeling of all immune cell lineages, and leverage barcode labeling to reconstruct cell type niches that recapitulate known tumor spatial biology. Furthermore, we couple this system with CRISPR perturbations and single-cell RNA sequencing to perform genetic screens in vivo on tumor-immune interactions at single-cell resolution. We use this method to infer cell-cell spatial relationships and uncover genetic dependencies in cancer cells that change the composition and cell state of their local microenvironments. By calculating a local immune activation signature, we are able to prioritize targets whose deletion enhances anti-tumor immunity through distinct effector cell types: loss of Tgfb1 engages CD8 T cells and macrophages, Traf7 loss elicits a CD4 T cell response, and Nectin3 loss promotes NK cell-mediated immunity, relationships that we validate by in vivo immune cell depletion. Notably, Tgfb1 and Nectin3 deletion had little effect on cancer cell fitness in the pooled screen yet suppressed tumor growth when deleted throughout the tumor, demonstrating the potential of match-seq to reveal functionally important tumor-immune interactions that would otherwise remain hidden. Together, these results establish a general framework for pooled genetic dissection of cell-cell interactions in vivo, extending CRISPR screening from cell-intrinsic phenotypes to the mechanisms by which cells shape and respond to their local microenvironments.

cancer biology↗

bioRxiv2026

J-domain proteins stimulate PKL-mediated chromatin compaction at H3K4-hypomethylated genomic loci

Chromatin accessibility varies widely across distinct genomic regions in eukaryotes, yet the mechanisms governing these differential patterns remain poorly understood. Here, we identify a subfamily of functionally redundant J-domain proteins (JDPs) that assemble into a protein complex with PICKLE (PKL), an evolutionarily conserved CHD3-type chromatin remodeler, in Arabidopsis thaliana. JDPs are required not only for maintaining PKL protein stability but also for stimulating its nucleosome remodeling and ATPase activities. A previously uncharacterized histone-binding domain (HBD) within JDPs specifically recognizes the N-terminal tail of histone H3 when the H3K4me3 modification is absent. This interaction enhances PKL-mediated nucleosome sliding and ATPase activities in vitro, and promotes PKL-dependent chromatin compaction at H3K4me3-depleted genomic loci in vivo. The PKL-JDP complex drives chromatin compaction to repress developmentally regulated genes, thereby governing key developmental phase transitions, including the embryo-to-seedling transition and flowering. Collectively, these findings uncover a distinct mechanism by which the absence of H3K4me3 is sensed to initiate regional chromatin compaction, repress developmentally regulated genes, and facilitate key developmental transitions.

plant biology↗

bioRxiv2026

Maladaptive Piezo1 Mechanotransduction Drives Smooth Muscle Aging in the Gut

Age-related gastrointestinal dysfunction is common, but the mechanisms of aging-associated smooth muscle failure remain unclear. We show that aging in mice slows whole gut and colonic transit, increases regional stiffness, and reduces smooth muscle contractility. Inducible smooth muscle cell (SMC) specific deletion of Piezo1 preserved youthful transit and force generation, whereas Piezo1 activation in young mice phenocopied aging-associated transit delay. Single cell transcriptomics, RNA velocity, stiffness-controlled cell and tissue cultures, and pharmacologic studies revealed that Piezo1 couples increased stiffness to Ca2+, calcineurin, NFAT signaling, loss of contractile gene programs, leading to age-related contractile loss and contractile to synthetic SMC remodeling and gut wall stiffening. Human intestinal SMCs supported conservation of this pathway, and PIEZO1 gain of function carriers showed a trend toward delayed colonic transit. Thus, maladaptive SMC Piezo1 mechanotransduction is a targetable mechanism of aging-associated gut dysmotility.

physiology↗

bioRxiv2026

Inference of self-limiting neutrophil swarming dynamics using Bayesian physics-informed neural networks

Neutrophil swarming is a critical immune response in mammals and fish, in which neutrophils are recruited to inflammatory sites where they coordinate into a swarm that neutralizes pathogens. While excessive swarming can drive prolonged inflammation, a quantitative understanding of swarming dynamics remains limited. We developed a one-dimensional radial reaction-diffusion model of neutrophil swarming with two kinetic parameters, in order to capture the self-limiting swarming dynamics in both murine and human neutrophils in response to different inflammatory stimulus sizes. To ensure that the inverse problem is well-posed, we first performed sensitivity and identifiability analyses. We then developed a physics-informed neural network (PINN) to infer the key parameters governing swarm expansion and self-limitation. To account for uncertainty in noisy experimental measurements, we further extended this framework to a Bayesian PINN (B-PINN), which provides credible intervals for the inferred parameters. Both models were validated against synthetic data generated by numerical simulation and subsequently applied to in vitro experimental data from human and murine neutrophils in response to three bioparticle cluster sizes. The PINN-inferred dynamics show that larger bioparticle clusters are associated with greater cumulative recruitment and larger swarms in both species. The models further reveal species-specific differences in both the amplitude of initial recruitment and the timescale on which it self-limits. Additionally, the B-PINN posterior distributions quantify uncertainty in these species- and cluster size-dependent trends and identify where additional measurements would be most informative. To our knowledge, this is the first application of physics-informed machine learning to model neutrophil swarming dynamics. This framework provides a starting point for systematically comparing recruitment dynamics between human and murine neutrophils and offers guidance for future experimental design.

systems biology↗

bioRxiv2026

Human Cerebral Cortex Organization Characterized by Functional PET-FDG "Metabolic Connectivity"

PurposeIn this study, we characterize the spatiotemporal organization of resting-state metabolic connectivity (RSMC) in the human brain, as measured by [18F]- fluorodeoxyglucose (FDG) functional PET (fPET-FDG). We examine the relationship between RSMC organization and resting-state functional connectivity (RSFC) derived from functional magnetic resonance imaging and other known cortical organizational principles. MethodsResting-state fPET-FDG data from 24 individuals were obtained from a publicly available repository. We characterized local metabolic organization using connectivity-based boundary mapping, with adaptations to account for the low signal-to-noise ratio of fPET-FDG data. We then estimated global metabolic organization through community detection-based network and principal gradient analyses. Furthermore, we examined how metabolic connectivity is shaped by temporal-frequency-specific components of fPET-FDG signal. Finally, we contextualized metabolic organization by relating metabolic gradients to anatomical, functional, and energetic reference measures. ResultsAt the local scale, boundary mapping results indicated structured transitions shaped by a combination of both fast and slow fPET-FDG signals, partly overlapping with RSFC boundary maps. Globally, RSMC analyses revealed a robust metabolic structure organized along a superior-inferior cortical gradient. This pattern remained consistent across network community detection and principal gradient analyses and was primarily driven by low-frequency, minute-scale fPET-FDG dynamics. The identified large-scale metabolic profile aligns closely with several known anatomical and energetic constraints. ConclusionThis study characterizes the spatiotemporal organizational principles of RSMC, deepening insight into the brains energetic framework and providing a basis for future cognitive and clinical investigations of metabolic connectivity organization.

neuroscience↗

bioRxiv2026

Single-cell and spatial transcriptomics unveils key regulators governing cell differentiation for Schistosoma sexual development

Schistosomiasis remains a major global health challenge, primarily due to the large number of egg production by sexually mature parasites. However, the cellular basis and regulatory programs governing sexual development and egg production are poorly characterized. Here, we constructed a dynamic single-cell atlas of Schistosoma japonicum (S. japonicum) covering key development stages of sexual maturation and egg production, and integrated it for the first time with spatial transcriptomics to map tissue-resolved cellular niches. Through bioinformatic analysis and experimental validation, we identified several critical cell populations and regulatory networks that drive parasite development and egg laying. By mapping de novo identified transcription factors (TFs) onto these atlases, we generated a comprehensive spatiotemporal expression profile of TFs and define the regulatory programs of several tissue-specific TFs including Zfp, Fbp3, and Lim. Integrated re-clustering further revealed and validated key regulators of germline stem-cell differentiation in S. japonicum. Finally, comparative analyses with S. mansoni single-cell datasets revealed both conserved and species-divergent cellular plasticity, providing unprecedented insights into their distinct biological traits. Collectively, our comprehensive study establishes a foundational resource for understanding schistosome biology, deciphers regulatory programs underlying cell differentiation and pathogenicity, and accelerates the identification of key molecules involved in sexual development against schistosomiasis.

microbiology↗

bioRxiv2025

Integrated analysis implicates novel insights of NMB into lactate metabolism and immune response prediction in primary glioblastoma

BackgroundGlioblastoma (GBM), the most aggressive primary brain tumor in adults, exhibits profound treatment resistance and poor prognosis. Despite advances in immunotherapy for other cancers, GBM remains refractory, potentially due to its lactate-rich, immunosuppressive tumor microenvironment (TME). While aerobic glycolysis-driven lactate accumulation is known to acidify the TME and impair immune function, the precise mechanisms linking lactate metabolism to immune evasion in GBM remain elusive. MethodsWe integrated multi-omics analyses with machine learning to identify - gene signatures associated lactate metabolism (LM) and Tumor Immune Dysfunction and Exclusion (TIDE) through weighted gene co-expression network analysis (WGCNA). Multiple bioinformatic tools were employed to determine the association of key genes with TME remodeling, immunotherapy response and drug sensitivities. ResultsNeuromedin B (NMB) emerged as a pivotal regulator of lactate-mediated immunosuppression, correlating strongly with LM and TIDE. We found that NMB expression was remarkably correlated with clinical characteristics, immune response, TME remodeling and drug sensitivities. ConclusionThis study demonstrates that NMB is a key cross-factor in metabolic and immune regulation in GBM. It breaks through the traditional perception that metabolism and immunity are mutually exclusive in cancer by promoting oxidative phosphorylation (OXPHOS) metabolism and enhancing anti-tumor immunity through its dual functions. It provides a new direction for the combined intervention of metabolic targeting and immunotherapy, and the strong predictive value of NMB expression for drug response further highlights its clinical application potential in the formulation of personalized treatment strategies for GBM patients.

neuroscience↗

bioRxiv2025

Capturing trophectoderm-like stem cells enables step-wisely remodeling of placental development

The trophectoderm produced from totipotent blastomeres initiates trophoblast development, while placental deficiencies can cause pregnancy disorders. Yet, a culture system that fully recapitulates the entire placenta development is still lacking, greatly limiting related studies. Here, we captured mouse trophectoderm-like stem cells (TELSCs), which can give rise to all trophoblast lineages and be applied to generate trophoblast organoids. We achieved the induction and maintenance of TELSCs from totipotent blastomere-like stem cells or early embryos through a Hippo-YAP/Notch-to-TGF{beta}1 signaling switch. At the molecular level, TELSCs resemble E4.5 trophectoderm and are distinct from all previously known trophoblast-like stem cells. Functionally, TELSCs can generate all trophoblast lineages in both teratoma and chimera assays. We further applied TELSCs to generate trophoblast organoids containing various mature trophoblasts and a self-renewing extraembryonic ectoderm (ExE)-like progenitor population. Interestingly, we observed transiently formed rosette-like structures that rely on Itgb1, which are essential to induce ExE-like progenitors and to generate organoids eventually. Thus, the capture of TELSCs enables comprehensive insights into placental development. HighlightsO_LITELSCs were robustly induced and long-term maintained from TBLCs and early embryos using a newly developed Hippo-YAP/Notch-to-TGF{beta}1 signaling switch strategy C_LIO_LITELSCs resemble E4.5 trophectoderm at the molecular level and generate all trophoblast lineages both in vivo and in vitro functionally C_LIO_LITELSCs can be applied to generate trophoblast organoids containing multiple mature subtypes and a self-renewing ExE-like population C_LIO_LIRosette structure formation that relies on Itgb1 is critical to induce ExE-like progenitors and to eventually form trophoblast organoids C_LI

cell biology↗

bioRxiv2025

Rational design of respiratory syncytial virus dimeric F-subunit vaccines in protein and mRNA forms

BackgroundRespiratory syncytial virus (RSV) poses a significant public health threat, particularly to children and the elderly. Two protein-based vaccines and one mRNA vaccine have been approved, all targeting the prefusion conformation of the fusion (F) glycoprotein. However, it has been reported that the F protein transitions to the post-fusion state during storage, resulting in a reduction of the vaccines immunogenicity. MethodsIn this study, we engineered novel pre-F-based antigens to preserve pre-F-specific immunodominant epitopes while eliminating sub-potent ones. Based on this, we constructed a series of single-chain dimers and selected the one with the highest expression yield and melting temperature (Tm). Next, we created a heterodimer, scDimer AB. Structural and protein characterization analyses were conducted to verify our design. All monomeric and scDimer antigens were used to immunize rodent models. Additionally, we prepared the antigens in mRNA form and immunized BALB/c mice. Finally, we combined both antigen forms, administering intramuscular mRNA priming followed by intranasal protein delivery in mice. In all immunization strategies, viral challenges were performed in animals to evaluate the immunologic protective effects. FindingsThrough rational design, we developed a monomeric and two single-chain dimeric (scDimer) proteins with the expected characteristics, including complete II, V, and O epitopes and a partial III epitope. The scDimers elicited stronger binding and neutralizing antibody responses in rodent models compared to the monomer, and they also boosted T cell responses when combined with appropriate adjuvants. After three doses of scDimer immunization, live RSV was barely detectable in the tissues of infected animals. The copies of RNA encoding N-gene were significantly reduced in the immunized groups compared to the PBS-injected control groups. We also engineered mRNA versions of the antigens and verified their protective efficacy in mice. Notably, there were no significant differences between intranasal boost and intramuscular two doses after RSV challenged, suggesting that intranasal boost provided equivalent protection to intramuscular vaccination and could reduce the risk of vaccine-enhanced disease (VED) potentially. InterpretationThe scDimer-based RSV vaccines effectively protected rodents from RSV infections, highlighting their clinical potential. Our antigen design removed certain suboptimal epitope regions, enhancing the efficiency of antigen presentation and increasing the proportion of the most potent pre-F-specific neutralizing antibodies. This approach provides a novel perspective for future vaccine design. FundingNational Key R&D Program of China, National Science Foundation of China, Young Scientists in Basic Research, Chinese Academy of Sciences, and Special Program of China National Tobacco Corporation. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe licensed respiratory syncytial virus (RSV) vaccines were based on RSV pre-F structure. We conducted a PubMed search for articles published in English from databases inception to March 31, 2025, using the search terms "RSV" (interchanged with "respiratory syncytial virus"), "pre-F" (interchanged with "prefusion F") and "vaccine". Our search revealed that most of the articles focus on combining pre-F with RSV G protein or other proteins to form viral-like particles (VLPs) or nanoparticles. The second-largest category of articles examined the antibodies level elicited by the pre-F antigens in animal model or some special cohorts. Other studies addressed topics such as optimization of injection dosage and immunization strategies, methodologies for detecting pre-F-specific antibodies, and the development of mRNA-LNP vaccines. Notably, only 4 articles described novel designs of RSV pre-F antigens, all of which were trimeric and based exclusively on the RSV A subtype F protein sequence. Added value of this studyTo our knowledge, our design is the first single-chain dimer version of RSV pre-F antigen. The engineered antigens demonstrated high yield, thermostability, and immunogenicity comparable to that of DS-Cav1. Additionally, we developed the first chimeric RSV-antigen covering both subtype A and B, thereby broadening the potential protective spectrum. We further validated the immunogenicity of our designs in mRNA form and as a protein-mRNA combination. This research emphasizes the potential of constructing and combining minimized, stable antigen modules, offering a novel approach for future vaccine development. Implications of all the available evidenceIn this study, we present a novel approach for constructing and combining minimized, stable antigen modules for future vaccine design. The strong immunogenicity of our antigens underscores their potential for clinical application. Furthermore, given the structural similarities between the RSV antigen and those of other Pneumoviruses, such as human metapneumovirus (hMPV), our design strategy could be extended to the development of multivalent vaccines.

immunology↗

bioRxiv2025

A case for slow-wave morphology as a functional biomarker of gastric disease

AimUnderlying bioelectrical slow waves are critical for regulating gastric motility, and abnormal spatiotemporal slow-wave dysrhythmias are associated with a range of gastrointestinal disorders. However, the definition and role of the morphology of gastric slow-wave signals have remained limited. This study aimed to investigate the potential of gastric slow-wave morphology as an actionable biomarker. MethodsData were repurposed from a study where, following ethical approval, a control cohort (n=9) and a pathological cohort of patients with chronic unexplained nausea and vomiting (CUNV; n=8) underwent intra-operative high-resolution serosal electrical mapping (96-256 electrodes, 4.0-5.2 mm spacing). Slow waves were identified using validated software, and spatiotemporally averaged waveforms were compared between cohorts. These waveforms were replicated in a computational model of gastric slow-wave propagation to explore potential functional implications. ResultsThe slow-wave morphology of the CUNV cohort exhibited a more gradual recovery stroke compared to controls, which manifested as an increase in the normalized recovery stroke area [0.206 (95% CI: 0.169-0.247) vs. 0.134 (95% CI: 0.106-0.166); p=0.011]. Computational modeling showed that these morphological differences could drive spatial slow-wave dysrhythmias. Considering the evident functional importance of gastric slow-wave morphology, we highlighted the three typical morphological features: 1) rapid, brief upstroke, 2) downstroke, and 3) biphasic recovery stroke. ConclusionAltogether, this study presents a case for gastric slow-wave morphology as a biomarker of gastric health and disease and lays a foundation for the standardization of future slow-wave morphology research. PRACTITIONER POINTSO_LIAbnormal spatial slow-wave propagation is associated with a range of gastrointestinal motility disorders, but morphology has had limited consideration. C_LIO_LISlow-wave morphology differs between cohorts of healthy controls and patients with chronic unexplained nausea and vomiting. C_LIO_LIMultiscale mathematical modeling indicates that a disruption to the slow-wave recovery stroke may contribute to spatial disorganization. C_LI

physiology↗

bioRxiv2025

Aird-MSI: a high compression rate and decompression speed format for mass spectrometry imaging data

Mass spectrometry imaging has emerged as a pivotal tool in spatial metabolomics, yet its reliance on the imzML format poses critical challenges in data storage, transmission, and computational efficiency. While imzML ensures cross-platform compatibility, its lower compressed binary architecture results in large file sizes and high parsing overhead, hindering cloud-based analysis and real-time visualization. This study introduces an enhanced Aird compression format optimized for spatial metabolomics through two innovations: (1) a dynamic combinatorial compression algorithm for integer-based encoding of m/z and intensity data; (2) a coordinate-separation storage strategy for rapid spatial indexing. Experimental validation on 47 public datasets demonstrated significant performance gains. Compared to imzML, Aird achieved a 70% reduction in storage footprint (mean compression ratio: 30.03%) while maintaining near-lossless data precision (F1-score = 99.26% at 0.1 ppm m/z tolerance). For high-precision-controlled datasets, Aird accelerated loading speeds by 15-fold in MZmine. The Aird format overcomes crucial bottlenecks in spatial metabolomics by harmonizing storage efficiency, computational speed, and analytical precision, reducing I/O latency for large cohorts. By achieving near-native feature detection accuracy, Aird establishes a robust infrastructure for translational applications, including disease biomarker discovery and pharmacokinetic imaging.

bioinformatics↗

bioRxiv2025

Apolipoprotein E ε4 exacerbates microglia-mediated complement-dependent synapse loss caused by neuronal Tpk deficiency

Thiamine pyrophosphokinse-1 (TPK) is a key enzyme that converts thiamine to functional thiamine diphosphate (TDP). TPK insufficiency and hence TDP reduction in neurons induced by amyloid-{beta} deposition and diabetes, an independent risk factor of Alzheimers disease (AD), recapitulate multi-pathophysiological features in the brain of mice, similar to those in human AD. Apolipoprotein E {varepsilon}4 allele (APOE4) is the most well-known genetic risk factor for AD. Clinical trials by boosting TDP using benfotiamine, a derivative of thiamine that significantly elevates TDP level in human encrythrocytes, have shown the inferior clinical efficacies in APOE4 carriers compared to non-APOE4 carriers. Clarifying the relationship between APOE4 and TPK expression and multi-pathophysiological characteristics of AD induced by Tpk deficiency in neurons is imperative. Here, we find that humanized APOE4 didnt directly affect Tpk expression of mice, but markedly aggravates behavior abnormalities of Tpk-cKO mice. Pathologically, the Tpk-cKO mice with humanized APOE4 knock-in (AE-cKO mice) exhibit more synapse loss than the mice with only humanized APOE4 knock-in and the Tpk-cKO mice. Transcriptomics and pathologic analysis identified that APOE4 promoted the overactivation of microglia and the transition of microglia to a disease-associated and phagocytosis state via a complement-mediated pathway. Further, the C3aR antagonist significantly repressed microglia phagocytosis and synaptic elimination of the AE-cKO mice. Our results demonstrate that APOE4 exacerbates behavior dysfunction of Tpk-cKO mice through microglia-mediated complement-dependent synaptic elimination. These findings provide important insights into the role of APOE4 in the pathogenesis of AD.

neuroscience↗

bioRxiv2024

Prediction and design of transcriptional repressor domains with large-scale mutational scans and deep learning

Regulatory proteins have evolved diverse repressor domains (RDs) to enable precise context-specific repression of transcription. However, our understanding of how sequence variation impacts the functional activity of RDs is limited. To address this gap, we generated a high-throughput mutational scanning dataset measuring the repressor activity of 115,000 variant sequences spanning more than 50 RDs in human cells. We identified thousands of clinical variants with loss or gain of repressor function, including TWIST1 HLH variants associated with Saethre-Chotzen syndrome and MECP2 domain variants associated with Rett syndrome. We also leveraged these data to annotate short linear interacting motifs (SLiMs) that are critical for repression in disordered RDs. Then, we designed a deep learning model called TENet (Transcriptional Effector Network) that integrates sequence, structure and biochemical representations of sequence variants to accurately predict repressor activity. We systematically tested generalization within and across domains with varying homology using the mutational scanning dataset. Finally, we employed TENet within a directed evolution sequence editing framework to tune the activity of both structured and disordered RDs and experimentally test thousands of designs. Our work highlights critical considerations for future dataset design and model training strategies to improve functional variant prioritization and precision design of synthetic regulatory proteins.

genomics↗

bioRxiv2024

New role of cardiomyocyte Bmal1 in the regulation of sex-specific heart transcriptomes

It has been well established that cardiovascular diseases exhibit significant differences between sexes in both preclinical models and humans. In addition, there is growing recognition that disrupted circadian rhythms can contribute to the onset and progression of cardiovascular diseases. However little is known about sex differences between the cardiac circadian clock and circadian transcriptomes in mice. Here, we show that the the core clock genes are expressed in common in both sexes but the circadian transcriptome of the mouse heart is very sex-specific. Hearts from female mice expressed significantly more rhythmically expressed genes (REGs) than male hearts and the temporal pattern of REGs was distinctly different between sexes. We next used a cardiomyocyte-specific knock out of the core clock gene, Bmal1, to investigate its role in sex-specific gene expression in the heart. All sex differences in the circadian transcriptomes were significantly diminished with cardiomyocyte-specific loss of Bmal1. Surprisingly, loss of cardiomyocyte Bmal1 also resulted in a roughly 8-fold reduction in the number of all the differentially expressed genes between male and female hearts. We conclude that cardiomyocyte-specific Bmal1, and potentially the core clock mechanism, is vital in conferring sex-specific gene expression in the adult mouse heart.

physiology↗

bioRxiv2023

Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion

Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not- rejected after transplantation in-vivo using single-cell RNA sequencing and immunophenotyping. Non- rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection- associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate mediators, identified Mif (macrophage migration inhibitory factor) as a regulator of immune rejection. Mif knockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data. Statement of significanceHere, we uncover the association of Mif expression with tumor growth and aggressiveness, specifically in low ITH tumors. These findings could facilitate the development of new strategies to treat patients with homogeneous, high-MIF expressing tumors that are unresponsive to immune checkpoint therapy.

cancer biology↗

bioRxiv2023

Rationally designed multimeric nanovaccines using icosahedral DNA origami for molecularly controlled display of SARS-CoV-2 receptor binding domain

Multivalent antigen display on nanoparticles can enhance the immunogenicity of nanovaccines targeting viral moieties, such as the receptor binding domain (RBD) of SARS-CoV-2. However, particle morphology and size of current nanovaccines are significantly different from those of SARS-CoV-2. Additionally, surface antigen patterns are not controllable to enable the optimization of B cell activation. Herein, we employed an icosahedral DNA origami (ICO) as a display particle for SARS-CoV-2 RBD nanovaccines. The morphology and diameter of the particles were close to those of the virus (91 {+/-} 11 nm). The surface addressability of the DNA origami permitted facile modification of the ICO surface with numerous RBD antigen clusters (ICO-RBD) to form various antigen patterns. Using an in vitro screening system, we demonstrate that the antigen spacing, antigen copies within clusters and cluster number parameters of the surface antigen pattern all impact the ability of the nanovaccines to activate B cells. Importantly, the optimized ICO-RBD nanovaccines evoked stronger and more enduring humoral and T cell immune responses in mouse models compared to soluble RBD antigens. Our vaccines activated similar humoral immunity and slightly stronger cellular immunity compared to mRNA vaccines. These results provide reference principles for the rational design of nanovaccines and exemplify the utility of DNA origami as a display platform for vaccines against infectious disease.

bioengineering↗

bioRxiv2023

Aβ-aggregation-generated blue autofluorescence illuminates senile plaques, complex blood and vascular pathologies in the Alzheimer's disease

Senile plaque blue autofluorescence in the Alzheimers disease (AD) was discovered around 40 years ago, however, its impact on AD pathology is not fully examined. We analyzed senile plaques with immunohistochemistry and fluorescence imaging on AD brain pathological sections and also the A{beta} aggregation process in vitro in test tubes. In DAPI or Hoechst staining experiments, the data showed that the nuclear blue fluorescence could only be correctly assigned after subtracting the blue autofluorescence background. The plaque cores have very strong blue autofluorescence which is roughly 2.09 times of average DAPI nuclear staining and roughly 1.78 times of average Hoechst nuclear staining. The composite flower-like structures formed by Cathepsin D lysosomal staining wrapping dense core blue fluorescence should not be considered as central-nucleated neurons filled with defective lysosomes since there was no nuclear staining in the plaque core when the blue autofluorescence was subtracted. Furthermore, the dense cores were shown to be completely lack of nuclear signals by PI staining. The A{beta} aggregation assay indicated that both A{beta} self-oligomers and A{beta}/Hemoglobin (Hb) heterocomplexes had significant blue autofluorescence. However, the blue autofluorescence intensity was not always proportional to the intensity of A{beta} immunostaining. The majority of aggregates in the A{beta}/Hb incubations were sensitive to Proteinase K (PK) digestion while the rest were PK resistant. The blue autofluorescence of A{beta} aggregates not only labels senile plaques but also illustrates red blood cell aggregation, hemolysis, CAA, vascular amyloid plaques, vascular adhesion and microaneurysm. In summary, we conclude that A{beta}-aggregation-generated blue autofluorescence is an excellent amyloid pathology marker in the senile plaques, blood and vascular pathologies in the Alzheimers disease.

cell biology↗

bioRxiv2023

Re-evaluating the nuclear staining on pathological sections in Alzheimer's disease

A paper recently published on Nature Neuroscience, asserting that lysosome leakage induces neuronal cell death and the formation of senile plaques in Alzheimers disease. The study believed that flower-like perikaryal rosettes formed by packed autophagic vacuoles around central nuclei, termed "PANTHOS" neurons, are responsible for the senile plaque formation. However, all of the nuclei staining in this paper based on images in the blue fluorescence channel. We did detailed studies on the senile plaque blue autofluorescence and found that senile plaque blue autofluorescence could be easily mistaken as nuclear staining. The blue "nuclear" staining in this paper is probably inaccurate since it was not verified in red or green fluorescence channels and their "nuclei"-related data might be mis-interpreted.

neuroscience↗