Biology subjects

Wu, C.

Publications and source records attributed to Wu, C..

At least 127 records · Page 7Linked to original sources

bioRxiv2024

Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

Mechanistic understanding of the immune checkpoint receptor PD1 is largely based on mouse models, but human and mouse PD1 orthologs exhibit only 59.6% identity in amino acid sequences. Here we show that human PD1 is more inhibitory than mouse PD1 due to stronger interactions with the ligands PDL1 and PDL2 and with the effector phosphatase Shp2. A novel motif highly conserved among PD1 orthologs in vertebrates except in rodents is primarily responsible for the differential Shp2 recruitment. Evolutionary analysis suggested that rodent PD1 orthologs uniquely underwent functional relaxation, particularly during the K-Pg boundary. Humanization of the PD1 intracellular domain disrupted the anti-tumor activity of mouse T cells while increasing the magnitude of anti-PD1 response. Together, our study uncovers species-specific features of the PD1 pathway, with implications to PD1 evolution and differential anti-PD(L)1 responses in mouse models and human patients.

immunology↗

bioRxiv2024

Broadly Reactive Anti-VHH Antibodies for Characterizing, Blocking, or Activating Nanobody-Based CAR-T Cells

Production of chimeric antigen receptor T cell (CAR-T) therapies is dependent on the use of antibody reagents to label, isolate, and/or expand T cell products. We sought to create antibody-based tools that directly target the variable domain of heavy-chain only antibodies (VHH or nanobody) used in some CAR molecules. Two murine antibodies were identified which bind to distinct epitopes in the conserved framework regions of llama-derived VHHs, and not to human VH domains. We produced a high-quality dual-clonal anti-VHH antibody product which reacts with over 98% of VHH proteins, regardless of their antigenic specificity. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus could be used for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies potently inhibited VHH-CAR function, blocking CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH antibodies could also be applied for activation and expansion of VHH CAR-T cells, inducing 730-fold mean expansion, >94% CAR purity, with retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen specific activity without functional exhaustion. Overall, these data identify a useful new tool for understanding and manipulating VHH-based CAR-T cells. Funding SourceThis work was funded by the National Research Council Canada Disruptive Technology Solutions Cell and Gene Therapy challenge program, and BioCanRx Declaration of interestsThe anti-VHH antibodies reported here are the subject of a provisional patent application by the National Research Council of Canada

immunology↗

bioRxiv2024

Sidechain chemistry-encoded solid/liquid phase transitions of condensates

Nature effectively leverages multivalent interactions among fundamental building blocks in solvents to create remarkable materials for various purposes. One prominent example is the formation of biomolecular condensates through the phase separation of proteins and nucleic acids. In particular, these condensates play crucial roles in regulating cellular functions and constructing natural materials. During the phase separation, solvents not only provide liquid environments for solvating molecules but play crucial roles in affecting the material properties of condensates. However, it remains controversial in the literature that alcohol molecules, as one type of solvents, can solidify some condensates while also melting others, leading to liquid-to-solid phase transition (LSPT) or solid-to-liquid phase transitions (SLPT), respectively. The mechanism underlying the alcohol-induced solid/liquid phase transitions of condensates remains poorly understood. Here, we combine systematic experimental characterizations with molecular dynamics simulations to demonstrate that the phase transitions of condensates depend on their sidechain chemistry and dominant molecular interactions. Specifically, "hydrophilic" condensates, which consist of many charged sidechains, undergo LSPT by adding alcohols due to strengthened electrostatic interactions. In contrast, "hydrophobic" condensates comprised of abundant aromatic sidechains undergo SLPT with the addition of alcohols because of weakened cation-{pi} and {pi}-{pi} interactions. Importantly, these findings are generally applicable for predicting phase transitions of a wide range of condensates formed by synthetic polyelectrolytes and intrinsically disordered proteins based on their sidechain hydrophobicity or amino acid compositions. Our work not only reconciles a conundrum in the literature but provides a fundamental framework for understanding the responsiveness of condensates to environmental stimuli. These insights are instrumental for developing therapeutic drugs to treat pathological aggregates and engineering stimuli-responsive biomaterials from the perspective of sidechain chemistry and molecular interactions.

biophysics↗

bioRxiv2024

Molecular mechanisms of uric acid transport by the native human URAT1 and its inhibition by anti-gout drugs

Gout, a common and painful disease, stems from hyperuricemia, where elevated blood uric acid levels lead to urate crystal formation in joints and kidneys. The human urate transporter 1 (hURAT1) plays a critical role in urate homeostasis by facilitating urate reabsorption in the renal proximal tubule, making it a key target for gout therapy. Pharmacological inhibition of hURAT1 with drugs such as dotinurad, benzbromarone, lesinurad, and verinurad promotes uric acid excretion and alleviates gout symptoms. Here we present cryo-electron microscopy structures of native hURAT1 bound with these anti-gout drugs in the inward-open state, and with uric acid in inward-open, outward-open, and occluded states. Complemented by mutagenesis and cell-based assays, these structures reveal the mechanisms of uric acid reabsorption and hURAT1 inhibition. Our findings elucidate the molecular basis of uric acid transport and anti-gout medication action, and provide a structural framework for the rational design of next-generation therapies for hyperuricemia and gout.

biochemistry↗

bioRxiv2024

Development of a Conditional Plasmid for Gene Deletion in Non-Model Fusobacterium nucleatum strains

Fusobacterium nucleatum is an opportunistic pathogen with four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within F. nucleatum. While ATCC 23726 is highly transformable, most other Fusobacterium strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an fdx promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the galK gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species Fusobacterium periodonticum. Incorporating a sacB counterselection marker in this conditional plasmid enabled the deletion of the radD gene in non-model strains. Interestingly, while radD deletion in 23726, 10953, and 21_1A abolished coaggregation with Actinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant F. nucleatum strains, enhancing our understanding of this pathogen. IMPORTANCEFusobacterium nucleatum is implicated in various human diseases, including periodontal disease, preterm birth, and colorectal cancer, often linked to specific strains and reflecting the species genetic and phenotypic diversity. Despite this diversity, most genetic research has centered on the model strain ATCC 23726, potentially missing key aspects of other strains pathogenic potential. This study addresses a critical gap by developing a novel conditional plasmid system that enables gene deletion in genetically recalcitrant strains of F. nucleatum. We successfully deleted genes in the clinical strain CTI-2, the FNA strain 21_1A, and F. periodonticum for the first time. Our findings, particularly the varying behavior of the radD gene production in coaggregation across strains, underscore the complexity of F. nucleatum and the need for broader genetic studies. This work advances our understanding of F. nucleatum virulence at the strain level and provides a valuable tool for future bacterial genetics research.

microbiology↗

bioRxiv2024

Inferring in vivo murine cerebrospinal fluid flow using artificial intelligence velocimetry with moving boundaries and uncertainty quantification

Cerebrospinal fluid (CSF) flow is crucial for clearing metabolic waste from the brain, a process whose dysregulation is linked to neurodegenerative diseases like Alzheimers. Traditional approaches like particle tracking velocimetry (PTV) are limited by their reliance on single-plane two-dimensional measurements, which fail to capture the complex dynamics of CSF flow fully. To overcome these limitations, we employ Artificial Intelligence Velocimetry (AIV) to reconstruct three-dimensional velocities, infer pressure and wall shear stress, and quantify flow rates. Given the experimental nature of the data and inherent variability in biological systems, robust uncertainty quantification (UQ) is essential. Towards this end, we have modified the baseline AIV architecture to address aleatoric uncertainty caused by noisy experimental data, enhancing our measurement refinement capabilities. We also implement UQ for the model and epistemic uncertainties arising from the governing equations and network representation. Toward this end, we test multiple governing laws, representation models, and initializations. Our approach not only advances the accuracy of CSF flow quantification but also can be adapted to other applications that use physics-informed machine learning to reconstruct fields from experimental data, providing a versatile tool for inverse problems.

neuroscience↗

bioRxiv2024

Therapeutically targeting the classical complement pathway with antisense oligonucleotides in Alzheimer's disease

The complement classical pathway (CP) is a key mediator of synapse loss and neurodegeneration in mouse models of Alzheimers (AD) and other neurodegenerative diseases. We analyzed human brain proteomics and found consistent elevations of all CP proteins, but not other complement pathways, in AD patient brains. We performed human genetics analysis that identified a rare variant in the C1S gene within the Finnish population that is associated with AD and we found that a common AD-associated C1S variant correlates with increased C1S protein levels. A targeted assay detected elevated C1S activation in AD patient CSF. Given this specific implication of the CP in AD, we next evaluated the therapeutic approach of targeting the CP in the brain using antisense oligonucleotides (ASOs). To identify promising CP targets for knockdown using ASOs we first tested for rescue of synapse loss in an AD mouse model using heterozygous and homozygous complement knockout mice and examined the relative brain expression levels of different CP genes. Based on these experiments we prioritized C1r, C1s and C4 as promising targets for therapeutic knockdown using ASOs. We then screened for ASOs for each target, evaluating in vitro and in vivo knockdown and toxicity, and identified optimal ASOs targeting C1r, C1s and C4. Experiments with AD model mice demonstrated significant rescue of synapse loss following treatment with C1r, C1s or C4 ASOs. Overall, our findings provide proof of concept for using nucleic acid-based medicine to target the CP in AD and demonstrate the translational potential of this approach.

neuroscience↗

bioRxiv2024

Development of SacB-based Counterselection for Efficient Allelic Exchange in Fusobacterium nucleatum

Fusobacterium nucleatum, prevalent in the oral cavity, is significantly linked to overall human health. Our molecular comprehension of its role in oral biofilm formation and its interactions with the host under various pathological circumstances has seen considerable advancements in recent years, primarily due to the development of various genetic tools for DNA manipulation in this bacterium. Of these, counterselection-based unmarked in-frame mutation methods have proved notably effective. Under suitable growth conditions, cells carrying a counterselectable gene die, enabling efficient selection of rare defined allelic exchange mutants. The sacB gene from Bacillus subtilis, encoding levansucrase, is a widely used counterselective marker partly due to the easy availability of sucrose. Yet, its potential application in F. nucleatum genetic study remains untested. We demonstrated that F. nucleatum cells expressing sacB in either a shuttle or suicide plasmid exhibit a lethal sensitivity to supplemental sucrose. Utilizing sucrose counterselection, we created an in-frame deletion of the F. nucleatum tonB gene, a critical gene for energy-dependent transport processes in Gram-negative bacteria, and a precise knockin of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possible related to the natural competence of F. nucleatum. Post counterselection with 5% sucrose, chromosomal plasmid loss occurred in all colonies, leading to gene alternations in half of the screened isolates. This sacB-based counterselection technique provides a reliable method for isolating unmarked gene mutations in wild-type F. nucleatum, enriching the toolkit for fusobacterial research. IMPORTANCEInvestigations into Fusobacterium nucleatums role in related diseases significantly benefit from the strategies of creating unmarked gene mutations, which hinge on using a counterselective marker. Previously, the galk-based allelic exchange method, while effective, faced an inherent limitation - the need for a modified host. This study aims to surmount this limitation by substituting galK with sacB for gene modification in F. nucleatum. Our application of the sacB-based methodology successfully yielded a tonB in-frame deletion mutant and a luciferase gene knockin at the precise chromosomal location in the wild-type background. The new method augments the existing toolkit for F. nucleatum research and has far-reaching implications due to the easy accessibility to the counterselection compound sucrose. We anticipate its broader adoption in further exploration, thereby reinforcing its critical role in propelling our understanding of F. nucleatum.

microbiology↗

bioRxiv2024

Benchmarking DNA Foundation Models for Genomic Sequence Classification

The rapid evolution of DNA foundation models promises to revolutionize genomics, yet comprehensive evaluations are lacking. Here, we present a comprehensive, unbiased benchmark of five models (DNABERT-2, Nucleotide Transformer V2, HyenaDNA, Caduceus-Ph, and GROVER) across diverse genomic and genetic tasks including sequence classification, gene expression prediction, variant effect quantification, and topologically associating domain (TAD) region recognition, using zero-shot embeddings. Our analysis reveals that mean token embedding consistently and significantly improves sequence classification performance, outperforming other pooling strategies. Model performance varies among tasks and datasets; while general purpose DNA foundation models showed competitive performance in pathogenic variant identification, they were less effective in predicting gene expression and identifying putative causal QTLs compared to specialized models. Our findings offer a framework for model selection, highlighting the impact of architecture, pre-training data, and embedding strategies on performance in genomic and genetic tasks.

genomics↗

bioRxiv2024

Comparative Analysis of Housing Temperature Impact on Heart Failure with Preserved Ejection Fraction in J vs N Strain C57BL/6 Mice

IntroductionHeart failure studies are conducted in preclinical animal models with different genotypic strains, 7-times higher metabolic rate, 5 to 6 times higher heart rate, and are housed in a cold-stressed environment of 23{degrees}C, unlike humans. These differences severely affect how animals respond to interventions, particularly those that lead to the development of metabolic syndrome, such as the two-hit model of diet-induced obesity (DIO) and N-nitro-L-arginine methyl ester (L-NAME) administration. A two-hit model of diet-induced obesity (DIO) and L-NAME administration has been proposed to induce heart failure with preserved ejection fraction (HFpEF) and mimic the hallmarks of metabolic syndrome and inflammation-induced heart failure in humans [1]. However, studies have reported conflicting results using this model. In this study, we examined the influence of mouse strain and environmental temperature on the development of metabolic syndrome and HFpEF using a two-hit model. MethodsEight-week-old, C57BL/6 mice (n=30) from the J and N strains were randomized to receive a high-fat diet (HFD) plus L-NAME versus a regular chow diet; and were randomized to be housed at a regular temperature of 23 {degrees}C versus a thermoneutral temperature of 30 {degrees}C. Glucose tolerance test (GTT, 2g/kg body weight), blood pressure via tail cuff, and echocardiography were conducted at baseline and, then at 5 and 15 weeks. Metabolic phenotyping was conducted at week 15 by using the Promethion Sable System. ResultsOur study revealed the significant effects of housing temperature and strain on the development of metabolic syndrome and HFpEF following the initiation of HFD +L-NAME over 5 and 15 weeks. At 5 weeks, both strains showed thermoneutral housing-induced attenuation of the effects of HFD + L-NAME on blood pressure and glucose tolerance, with the J strain exhibiting reduced diastolic dysfunction. By week 15, thermoneutral housing decreased energy expenditure (EE) and fat oxidation in both strains, while specifically reducing the respiratory exchange ratio (RER)_and glucose oxidation in J strain. Ejection fraction increased in both strains compared with the Chow group, except for J strain at 23 {degrees}C. Notably, physical activity levels remained constant across the groups, suggesting that the observed metabolic changes were not activity related. These findings highlight the complex physiological adaptations of these strains to different housing temperatures. ConclusionsThermoneutral housing conditions elicited strain-specific metabolic and cardiac effects in mice, with the J strain showing more pronounced responses. These findings highlight the critical influence of ambient temperature on experimental outcomes in rodent models, emphasizing the need to consider housing conditions when interpreting the results of metabolic and cardiovascular research.

immunology↗

bioRxiv2024

Structures of H2A.Z-associated human chromatin remodelers SRCAP and TIP60 reveal divergent mechanisms of chromatin engagement

H2A.Z is a conserved histone variant that is localized to specific genomic regions where it plays important roles in transcription, DNA repair, and replication. Central to the biochemistry of human H2A.Z are the SRCAP and TIP60 chromatin remodelers, homologs of yeast SWR1 which catalyzes ATP-dependent H2A.Z exchange. Here, we use cryo-electron microscopy to resolve six structural states of the native SRCAP complex, uncovering conformational intermediates interpreted as a stepwise path to full nucleosome engagement. We also resolve the structure of the native TIP60 complex which consists of a structured core from which flexibly tethered chromatin binding domains emerge. Despite the shared subunit composition, the core of TIP60 displays divergent architectures from SRCAP that structurally disfavor nucleosome engagement, suggesting a distinct biochemical function.

molecular biology↗

bioRxiv2024

Nuclear FAK aggravates CD8+ T cell exhaustion via the SP1-IL-6 axis in colorectal cancer

Nuclear abnormalities such as nuclear deformation are hallmarks of many diseases, including cancer. Accumulating evidence suggests that the dense and mechanically stiff tumor microenvironment promotes nuclear deformation in cancer cells. However, little is known about how nuclear deformation in neoplastic cells regulates immune exhaustion in the tumor microenvironment. Here, we found that lamin A/C-mediated nuclear stiffening in neoplastic cells promotes the nuclear translocation of phosphorylated focal adhesion kinase (pFAK), which is strongly correlated with the heterogeneity and exhaustion of CD8+ T cells within the spatial context of the tumor microenvironment in human colorectal cancer. Mechanistically, we revealed that increased nuclear tension within tumor cells promotes pFAK nuclear translocation, where nuclear pFAK was found to regulate SP1/IL-6-mediated T-cell exhaustion and the transcription of proinflammatory cytokines/chemokines. Pharmacological inhibition or disruption of pFAK nuclear translocation enhanced antitumor immune responses and synergistically potentiated PD-1 and TIM-3 immunotherapy by increasing CD8+ T-cell cytotoxicity and restoring exhaustion in preclinical models of colorectal cancer. These findings highlight the pivotal role of nuclear tension-mediated pFAK translocation into the tumor cell nucleus in regulating CD8+ T-cell exhaustion, suggesting that pFAK is a promising target for advancing cancer immunotherapy.

immunology↗

bioRxiv2024

Phase Separation of Actin-Binding Protein TPM4 Fuels Glycolysis under Hyperosmotic Stress

Actin homeostasis is fundamental for cell structure and consumes a large portion of cellular ATP. It has been documented in the literature that certain glycolytic enzymes can interact with actin, indicating an intricate interplay between the cytoskeleton and cellular metabolism. Here we report that hyperosmotic stress triggers actin severing and subsequent phase separation of the actin-binding protein TPM4. TPM4 condensates glycolytic enzymes such as HK2, PFKM, and PKM2, and adhere to and wrap around actin filaments. Notably, the condensates of TPM4 and glycolytic enzymes are enriched of NADH and ATP, suggestive of their functional importance in cell metabolism. At cellular level, actin filaments assembly is enhanced upon hyperosmotic stress and TPM4 condensation, while depletion of TPM4 impaired osmolarity-induced actin reorganization. At tissue level, co-localized condensates of TPM4 and glycolytic enzymes are observed in renal tissues subjected to hyperosmotic stress. Together, our findings suggest that stress-induced actin perturbation may act on TPM4 to organize glycolytic hubs that tether energy production to cytoskeletal reorganization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/602822v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@110c357org.highwire.dtl.DTLVardef@188a519org.highwire.dtl.DTLVardef@f4062org.highwire.dtl.DTLVardef@14c1a61_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Blebs regulate phosphoinositides distribution and promote cell survival through the Septin-SH3KBP1-PI3K axis

Cells rely on substrate adhesion to activate diverse signaling pathways essential for survival. In the absence of proper adhesion, cells undergo anoikis, a form of programmed cell death. Poorly attached cells often exhibit rounded morphologies and form small round protrusions called blebs. While the role of blebs in amoeboid migration is well-documented, recent studies have highlighted their function in anoikis resistance with the help of curvature-recognized proteins. However, little is known about whether the most abundant membrane components-phospholipids function in blebs-facilitated cell survival. Here, we found enriched PI(3,4,5)P3 and diluted PI(4,5)P2 at the bleb membrane, compared to non-bleb plasma membrane regions. Our experimental results showed that both phosphoinositides (PIs) have restricted diffusion pattern between the bleb and non-bleb membranes. Subsequently, we reveal SH3KBP1 to play a crucial role in recruiting PI3K and organizing the Septin-SH3KBP1-PI3K complex at the bleb necks. This process then contributes to differential PIs distribution and anoikis resistance. These novel insights into PIs dynamics elucidate their role in cell blebbing, cancer cell behavior and metastasis, presenting potential targets for new therapeutic interventions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/602823v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@63455eorg.highwire.dtl.DTLVardef@15f2092org.highwire.dtl.DTLVardef@535a26org.highwire.dtl.DTLVardef@43b48e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Live-cell single-molecule dynamics of eukaryotic RNA polymerase machineries

Eukaryotic gene expression in the nucleus is orchestrated by three RNA polymerases (RNAP-I, -II, and -III) and associated factors1,2. Despite extensive biochemical, genomic, structural, and imaging studies, the real-time dynamics of these transcription complexes remain obscure. Here, we employ single-molecule tracking in living yeast to assess the physiological kinetics of over 50 representative proteins encompassing all three RNAP machineries. Components of RNAPI and RNAPIII pre-initiation complexes (PICs) engage in long-lived interactions on chromatin, reflecting their roles for constitutive rRNA and tRNA synthesis, in contrast to the transient RNAPII PIC. We further report the dynamics of key components across the RNAPII transcription cycle2-5--factors for upstream regulation, elongation, histone modification, RNAPII C-terminal domain (CTD) modification, RNA processing, and termination--revealing unprecedented insights into the temporal landscape of RNAPII transcription. Strikingly, many elongation factors, previously thought to travel processively with RNAPII, display transient residence times, suggesting highly dynamic interactions rather than constant association. Systematic screening of RNAPII-associated factors shows that truncation of RNAPII-CTD substantially reduces U1 snRNP residence time and decreases intron retention in ribosomal protein genes, providing insights into how CTD length influences co-transcriptional splicing. Our findings establish a framework for dynamic chromatin interactions of RNA polymerase machineries in living cells.

biophysics↗

bioRxiv2024

Identification of P-type ATPase as a bacterial transporter for host-derived small RNA

Host-derived small RNAs represent a finely tuned host response to maintain the host-microbial homeostasis. Among these, an emerging class known as transfer RNA-derived small RNA (tsRNA) has been implicated in modulating microbial-host interaction. Our previous study showed that when challenged with an oral pathobiont, Fusobacterium nucleatum (Fn), an immortalized human oral keratinocyte cell line releases certain Fn-targeting tsRNAs that selectively inhibit the growth of Fn via their ribosome-targeting function. We also revealed the sequence- and energy-dependent uptake of tsRNAs by Fn. However, the mechanism underlying the tsRNA uptake at the molecular level remains elusive. In this study, using RNA affinity pull-down assay in combination with Mass Spectrometry, we identified a putative P-type ATPase transporter (PtaT) in Fn, which binds Fn-targeting tsRNAs in a sequence-specific manner. AlphaFold 3 simulation provides further evidence supporting the specific binding between PtaT and tsRNA compared to the scrambled control and the DNA counterpart. Through targeted mutagenesis and phenotypic characterization, we demonstrated the important role of PtaT in the uptake and antimicrobial capacity of tsRNAs against Fn in both ATCC 23726 type strain and a clinical tumor isolate (Fn CTI). Furthermore, global RNA sequencing and label-free Raman spectroscopy revealed the phenotypic differences between Fn wild type and PtaT-deficient mutant, highlighting the functional significance of PtaT in purine and pyrimidine metabolism. Collectively, our work identifies a bacterial transporter for tsRNAs and provides critical information for a fundamental understanding of how the host-generated tsRNAs specifically interact with its targeted bacteria at the molecular level.

microbiology↗

bioRxiv2024

Macrophages facilitate interclonal cooperation-induced tumor heterogeneity and malignancy by activating the innate immune signaling

Tumor heterogeneity is a common hallmark of cancer and is considered a major cause of treatment failure and relapse, yet it remains poorly understood how various types of cells communicate within the tumor microenvironment (TME) to regulate tumor progression in vivo. Here we establish a tumor heterogeneity model in Drosophila eye epithelium by mutating the tricellular junction protein M6 in cells surrounding RasV12benign tumors and dissect the in vivo mechanisms underlying interclonal cooperation-induced malignancy by utilizing sophisticated genetic techniques in conjunction with single-cell RNA sequencing (scRNA-seq). Our findings reveal that loss of M6 facilitates the malignant transformation of neighboring RasV12 tumors by activating the Toll signaling, the innate immune response pathway. Notably, inhibiting Toll signaling impedes tumor progression, whereas its activation synergistically promotes RasV12 tumor malignancy by inactivating the Hippo pathway. Mechanistically, RasV12 tumors surrounded by M6 mutant clones lead to increased recruitment of hemocytes, which are the equivalent of macrophages in Drosophila, in a JNK pathway-dependent manner. Consequently, these tumor-associated macrophages secrete the Spatzle (Spz) ligand, which subsequently activates the Toll receptor within the RasV12 tumors, thereby triggering tumorigenesis. In summary, our study elucidates the complex in vivo interactions between genetically distinct oncogenic cells and between tumors and macrophages, shedding light on how macrophages exploit the innate immune signaling within tumors to regulate tumor heterogeneity and promote tumor progression. Significance statementIntratumoral heterogeneity profoundly affects cancer development and treatment in human tumors. The intricate nature of tumor cells and the presence of diverse cell types pose challenges to uncovering in vivo mechanisms responsible for heterogeneity. Our Drosophila tumor heterogeneity model reveals that fruit fly macrophages promotes both tumor heterogeneity and malignancy. Following recruitment by tumor cells, these macrophages secrete the ligand Spz to activate the Toll signaling pathway within tumor cells, which subsequently inactivates the Hippo pathway to drive tumorigenesis. Our study highlights the crucial role of hemocytes as intermediaries in coordinating tumor heterogeneity and facilitating intercellular communication between different cells within the TME.

cancer biology↗

bioRxiv2024

A Zero-Inflated Hierarchical Generalized Transformation Model to Address Non-Normality in Spatially-Informed Cell-Type Deconvolution

Oral squamous cell carcinomas (OSCC), the predominant head and neck cancer, pose significant challenges due to late-stage diagnoses and low five-year survival rates. Spatial transcriptomics offers a promising avenue to decipher the genetic intricacies of OSCC tumor microenvironments. In spatial transcriptomics, Cell-type deconvolution is a crucial inferential goal; however, current methods fail to consider the high zero-inflation present in OSCC data. To address this, we develop a novel zero-inflated version of the hierarchical generalized transformation model (ZI-HGT) and apply it to the Conditional AutoRegressive Deconvolution (CARD) for cell-type deconvolution. The ZI-HGT serves as an auxiliary Bayesian technique for CARD, reconciling the highly zero-inflated OSCC spatial transcriptomics data with CARDs normality assumption. The combined ZI-HGT + CARD framework achieves enhanced cell-type deconvolution accuracy and quantifies uncertainty in the estimated cell-type proportions. We demonstrate the superior performance through simulations and analysis of the OSCC data. Furthermore, our approach enables the determination of the locations of the diverse fibroblast population in the tumor microenvironment, critical for understanding tumor growth and immunosuppression in OSCC.

genomics↗