Biology subjects

Wan, Y.

Publications and source records attributed to Wan, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

PCNA Inhibition Enhances the Antitumor Activity of KRAS-Targeted Therapies in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a dismal prognosis. More than 90% of PDAC tumors harbor KRAS mutations, and several KRAS inhibitors, such as off-state, on-state, mutation-specific, and pan-RAS inhibitors, are being tested in preclinical and clinical settings. However, the response to these inhibitors as single agents is less than optimal, indicating the need to identify novel combination therapies to improve treatment outcomes. Proliferating cell nuclear antigen (PCNA) is a ring-shaped clamp protein that regulates DNA replication, repair, and resolution of transcription-replication conflict, which are critical processes for pancreatic cancer survival. AOH1996 is a first-in-class, selective PCNA inhibitor in Phase I trials. Here, we found that AOH1996 treatment is efficacious in various PDAC models in vitro. PCNA and KRAS are predicted to be synthetic lethal partners, and RNA sequencing of AOH1996-treated PDAC cells reveals enrichment of MAPK and PI3K signaling pathways. Combination of AOH1996 with KRAS inhibitors demonstrates strong synergy across KRAS G12C and G12D mutant models. Treatment with a combination of AOH1996 and KRAS inhibitors induces cell cycle arrest and apoptosis in PDAC cells. Robust antitumor activity of AOH1996 in combination with RMC-6236 was observed in PDAC tumoroids. In vivo, the combination of AOH1996 with sotorasib or MRTX1133 reduced tumor growth rates compared to single-agent therapy, with no impact on mouse body weight. Residual tumor analysis showed sustained pERK and Myc inhibition in the combination arm. In conclusion, combination of AOH1996 with KRAS inhibitors is a promising therapeutic strategy for KRAS-driven PDAC, warranting further clinical investigation.

cancer biology↗

bioRxiv2025

Genomic and Transcriptomic Landscapes of MEN1-Wild-Type Low-Grade Metastatic Pancreatic NETs Uncover Key Oncogenic Drivers and Targetable Pathways

Sporadic pancreatic neuroendocrine tumors (pNETs) with wild type MEN1 represent a major yet largely ignored subset whose biology and metastatic potential remain poorly understood. Because metastasis can occur despite low histologic grade and modest mutational burden, we hypothesized that metastatic competence in MEN1-wild-type pNETs reflects quantitative reinforcement of shared oncogenic pathways rather than distinct mutational processes. We profiled 75 primary low-grade pNETs by whole-exome and RNA sequencing, including 25 percent with lymph node and/or liver metastasis, and integrated genomic and transcriptomic data to connect pathway lesions with expression state. Metastatic tumors showed a slight increase in mutation frequency but conserved base-substitution spectra relative to non-metastatic cases, and adverse clinicopathologic features were enriched in Grade 2 disease. Aggregating alterations to pathways revealed broad convergence on canonical networks, with transcriptomic analyses demonstrating cohort-wide enrichment of Calcium, WNT, and KRAS/PI3K-AKT programs in metastasis. Intersection of significantly mutated genes with differentially expressed genes identified a focused 29-gene overlap, including RYR1 and ZNF273, that marks these convergent axes and distinguishes metastatic from non-metastatic tumors. Gene set enrichment confirmed preferential activation of Calcium, WNT, and PI3K-AKT signaling in metastatic tumors, consistent with a network-intensity model of progression. Finally, upstream-regulator analysis (iPathwayGuide) and gene-centric perturbation mapping (Gene2Drug) nominated candidate targeted and repurposable agents predicted to reverse the metastatic expression phenotype and flagged drugs unlikely to provide benefit, yielding a prioritized, testable therapeutic shortlist which includes fasudil and spaglumic acid. Convergent, domain-specific mutational patterns in highly mutated genes such as ZNF273 and CLCA1 define a molecular signature that could stratify metastatic risk in low-grade pNETs. Collectively, our data reframe metastasis in MEN1-wild-type low-grade pNETs as a property of pathway state rather than mutation quantity and provide a translational blueprint for biomarker-guided therapy development focused on Calcium, WNT, and KRAS/PI3K hubs.

cancer biology↗

bioRxiv2025

Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

ObjectivesHistone deacetylase 7 (HDAC7), a classical HDAC family member, promotes LPS-inducible glycolysis and inflammatory mediator production in macrophages, innate immune cells that contribute to pathology in metabolic diseases. Here, we investigated myeloid HDAC7 functions in obesity-driven metabolic disease. MethodsWe used gain- and loss-of-function genetic approaches in mice to investigate myeloid HDAC7 functions in hepatic inflammation and metabolic disease, as well as associations with hepatic gene signatures characteristic of advanced chronic liver disease (CLD). ResultsTransgenic expression of Hdac7 in myeloid cells increased liver inflammation and liver mRNA levels of Ccl2 and Il1b, key inflammatory mediators linked to CLD. Liver glycogen levels were also decreased, another feature of CLD. Transgenic expression of Hdac7 in myeloid cells mimicked the hepatic inflammatory phenotype that was observed in mice fed a high fat, high cholesterol, and high sucrose (HFHCHS) diet, an obesity model that mimics some features of metabolic dysfunction-associated steatotic liver disease. In myeloid Hdac7 transgenic mice fed a HFHCHS diet, relative weight gain was increased, fasted glucose levels were elevated and glucose tolerance was dysregulated by comparison to control mice. Conversely, fasted blood glucose levels were reduced and glucose tolerance was improved in myeloid Hdac7-deleted mice on a HFHCHS diet. HDAC7 mRNA levels were also elevated in the livers of people with advanced CLD and spatial transcriptomics revealed that myeloid HDAC7 directs hepatic gene signatures characteristic of advanced CLD. ConclusionsMyeloid HDAC7 contributes to hepatic inflammation and systemic glucose dysregulation in a mouse model of obesity and liver inflammation.

immunology↗

bioRxiv2025

Targeting Glioblastoma Cell State Plasticity for Enhanced Therapeutic Efficacy

Glioblastoma (GBM) is the most common and deadly primary brain cancer, with limited therapeutic options. Treatment failure has been associated with intratumoral heterogeneity and the acquisition of a pronounced mesenchymal-like (MES-L) phenotype after recurrence. Here, we have screened a panel of drugs with diverse mechanisms of action across two patient-derived glioblastoma stem cells (GSCs) to characterize the dynamics of drug-mediated transcriptomic cellular state changes. Our results demonstrate that anti-tumor drugs induce significant but reversible alterations in cellular state distribution at the single-cell level in a drug-specific manner, influencing transitions between mesenchymal and the neurodevelopmental astrocytic-like (AC-L) states. Utilizing barcoded analysis in our recently developed ex vivo glioblastoma cerebral organoid (GLICO) model, we discerned distinct cell state sensitivities to the MES-L enhancing histone deacetylase inhibitor, panobinostat, which are contingent on the inducible modulation of the mesenchymal transcription factor FOSL1. The strategic combination of MES-L enhancing and MES-l suppressing genetic perturbations or drugs significantly increases anti-glioma activity in a strategy we call state-selective lethality. Overall, our findings highlight the critical role of cell state plasticity in the response of GSCs to anti-tumor therapeutic stress and underscore the potential for novel GBM combination drug strategies.

cancer biology↗

bioRxiv2025

Imaging cellular activity simultaneously across all organs of a vertebrate reveals body-wide circuits

All cells in an animal collectively ensure, moment-to-moment, the survival of the whole organism in the face of environmental stressors1,2. Physiology seeks to elucidate the intricate network of interactions that sustain life, which often span multiple organs, cell types, and timescales, but a major challenge lies in the inability to simultaneously record time-varying cellular activity throughout the entire body. We developed WHOLISTIC (WHole Organism Live Imaging System for recording Tissue and IntraCellular activity), a method to image second-timescale, time-varying intracellular dynamics across cell-types of the vertebrate body. By advancing and integrating volumetric fluorescence microscopy, machine learning, and pancellular transgenic expression of calcium sensors in transparent young Danio rerio (zebrafish) and with proof of concept in adult Danionella, the method enables real-time recording of cellular dynamics across the organism. Calcium is a universal intracellular messenger, with a large array of cellular processes depending on changes in calcium concentration across varying time-scales, making it an ideal proxy of cellular activity3. Using this platform to screen the dynamics of most cells in the body, we discovered unexpected responses of specific cell types to stimuli, such as chondrocyte reactions to cold, meningeal responses to ketamine, and state-dependent activity, such as oscillatory ependymal-cell activity during periods of extended motor quiescence. At the organ scale, the method uncovered pulsating traveling waves along the kidney nephron. At the multi-organ scale, we uncovered muscle synergies and independencies, as well as muscle-organ interactions. Integration with optogenetics allowed us to all-optically determine the causal direction of brain-body interactions. At the whole-organism scale, the method captured the rapid brainstem-controlled redistribution of blood flow across the body. Finally, we advanced Whole-Body Expansion Microscopy4 to provide ground-truth molecular and ultrastructural anatomical context, explaining the spatiotemporal structure of activity captured by WHOLISTIC. Together, these innovations establish a new paradigm for systems biology, bridging cellular and organismal physiology, with broad implications for both fundamental research and drug discovery.

neuroscience↗

bioRxiv2025

Genomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam

This article reports an unusual Klebsiella pneumoniae clinical isolate, KpMVR1, resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, and investigates the underlying genetic alterations using comparative genomics and molecular experiments. Resistance to carbapenems and third-generation cephalosporins is increasing in K. pneumoniae globally, restricting therapeutic options. The {beta}-lactam/{beta}-lactamase inhibitor combinations are widely used to circumvent {beta}-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a hospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat profile--KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from another patient in the same hospital--were susceptible to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid genome assembly were conducted for these three isolates. Annotated genome assemblies were compared to identify genetic variation, and mutagenesis experiments were performed to verify predicted functional alterations. All isolates belonged to a novel clone ST8134 and carried blaKPC-2-like alleles (KpMVR1: blaKPC-157; KpMVS1 and KpMVS2: blaKPC-2) in presumptively conjugative plasmids. ISEc68 caused a frameshift mutation in KpMVR1s ompK36 gene, reducing the meropenem-vaborbactam and imipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. In conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence for in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy as well as the need to characterise underlying resistance mechanisms to help assess the potential for onward transmission. Data summaryIllumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) under BioProject accession PRJNA1084250, with BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and SAMN46778011 (KpMVS2). The genome assemblies of these isolates have also been deposited in Pasteur Institutes database for K. pneumoniae species complex (bigsdb.pasteur.fr/klebsiella/) under ids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). Impact statementThis is the first blaKPC-positive K. pneumoniae isolate referred to the UKs national reference laboratory with resistance to three last-resort {beta}-lactam/{beta}-lactamase inhibitor combinations meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, implicating in vivo emergence of this unusual resistance profile during prolonged antimicrobial chemotherapy. This isolate belonged to a novel clone ST8134 and harboured a plasmid-borne blaKPC-2-like allele blaKPC-157. We identified complex genetic alterations in this isolate: chromosomal large deletions, point mutations, and an ISEc68-induced loss-of-function truncation of the ompK36 porin gene. We determined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. pneumoniae to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, aztreonam, aztreonam-avibactam, ceftazidime, ceftazidime-avibactam, and cefiderocol. Our work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor combinations in healthcare and to understand underlying resistance mechanisms for assessing the potential of resistance transmission.

microbiology↗

bioRxiv2025

RNA structural heterogeneity in a eukaryotic cell influences its heat shock response

RNA structure-based gene regulation remains under-explored in eukaryotes. While RNA can form different conformations in solution, the extent to which it folds into structure ensembles and how the different conformations regulate gene expression still needs to be fully understood. We coupled the SHAPE compound NAI-N3 with direct RNA sequencing to identify structure modifications along a single RNA molecule (sm-PORE-cupine). Using a combination of base mapping and direct signal alignment, we boosted the percentage of mappable RNA molecules from direct RNA sequencing. Using Bernoulli Mixture Model (BMM) clustering, we show that we can separate RNA structure ensembles from ligand-bound and unbound riboswitches accurately, identify isoform-specific structure ensembles along the SARS-CoV-2 genome, and determine RNA structure ensembles in the transcriptome of a eukaryote, C. albicans, at yeast (30{degrees}C) and hyphae (37{degrees}C) states. We observed that RNAs are more structurally homogenous at 37{degrees}C compared to 30{degrees}C, are more variable in vivo than in vitro, and show higher homogeneity in 3UTRs than in the coding region. We also identified structure ensembles that are associated with changes in translation efficiency and decay in C. albicans at 30{degrees}C and 37{degrees}C and validated translational changes using reporter assays. Our work shows that single-molecule RNA structure probing using direct RNA sequencing can be applied to diverse transcriptomes to study the complexity and function of RNA structures.

molecular biology↗

bioRxiv2025

A conserved long-range RNA interaction in SARS-CoV-2 recruits ADAR1 to enhance virus proliferation

Long-range RNA-RNA pairing impacts the genome structure and function of SARS-CoV-2 variants. To understand the structure and function relationships of different SARS-CoV-2 variants that have emerged during the COVID-19 pandemic, we performed high-throughput structure probing and modelling of the genomic structures of the wildtype (WT), Alpha, Beta, Delta and Omicron variants of SARS-CoV-2. We observed that genomes of SARS-CoV-2 variants are generally structurally conserved, and that single-nucleotide variations and interactions with RNA binding proteins can impact RNA structures across the viruses. Importantly, using proximity ligation sequencing, we identified many conserved ultra-long-range RNA-RNA interactions, including one that spans more than 17 kb in both the WT virus and the Omicron variant. We showed that mutations that disrupt this 17 kb long-range interaction reduce viral fitness at later stages of its infection cycle, while compensatory mutations partially restore virus fitness. Additionally, we showed that this ultra-long-range RNA-RNA interaction structure binds directly to ADAR1 to alter the RNA editing levels on the viral genome. These studies deepen our understanding of RNA structures in the SARS-CoV-2 genome and their ability to interact with host factors to facilitate virus infectivity.

genomics↗

bioRxiv2025

T cell AIM2 Exacerbates Lung Inflammation in OVA-LPS and HDM-induced Asthma Models

Asthma is a complex airway inflammatory disease characterized by immune dysregulation, with diverse cellular contributors to inflammation that result in airway hyperresponsiveness (AHR) and excessive immune responses. The role of the inflammasome in asthma is conflicted. AIM2 is an innate immune receptor that activates the inflammasome through DNA binding. The clinical relevance of AIM2 in asthma is supported by analysis of blood or endobronchial biopsies from patients with severe asthma, showing increased AIM2 but not NLRP3 expression. We investigated the role of AIM2 in allergic asthma using ovalbumin (OVA)-LPS and house dust mite (HDM)- induced models. AIM2 expression was elevated in lung homogenates and bronchoalveolar lavage fluid (BALF) cells from allergen-induced groups compared to controls. In the OVA-LPS model, deletion of Aim2 led to reduced airway smooth muscle actin expression and DNA damage, contributing to decreased AHR and lung inflammation in whole body Aim2-/- mice. Surprisingly, cell-specific deletion in CD4+ T cells but not regulatory T cells or myeloid cells significantly affected AHR, indicating AIM2 in CD4+ T cells is a main driver of pathogenesis. Additionally, in HDM-induced asthma, Aim2-/- mice also exhibited reduced AHR. Cell-specific deletion reveals that AIM2 in CD4+ T cells promotes AHR and cytokine production, while AIM2 in myeloid cells modulates IgG1 levels and IL-13-producing CD4+ T cells, and the percentage of alveolar macrophages in BALF. These findings reveal that AIM2 differentially regulates HDM-induced allergic asthma through distinct exacerbating roles in T cells and myeloid cells, highlighting its potential as a therapeutic target. SignificanceAllergic asthma is a complex, heterogeneous disease characterized by airway inflammation and hyper-responsiveness. AIM2, an inflammasome activated by DNA, has emerged as a critical regulator of inflammatory diseases. In humans, AIM2 expression is elevated in severe asthma. In vivo studies highlight AIM2 as a key modulator of allergic asthma in both OVA-LPS and HDM- induced models. Surprisingly, its functional relevance in both models is mediated by its intrinsic role in T cells, while a modest role in myeloid cells is restricted to the HDM model. AIM2 promotes airway hyper-responsiveness and lung inflammation through enhancing type II cytokines, immune subpopulations and DNA damage response. These findings suggest AIM2 as a biomarker and therapeutic target for managing asthma, particularly in severe asthma subtypes.

immunology↗

bioRxiv2025

RNA structure directs RNA partitioning and is actively disrupted inside stress granules to enable cellular recovery

RNA structures play important roles in liquid-liquid phase separation. However, how it is regulated during stress response and stress granule formation is still under studied. Here, we performed in vivo RNA structure probing before and after sodium arsenite treatment, and in stress granules. While RNAs generally become more double-stranded upon stress, they maintain their single-strandedness inside stress granules. We showed that RNA single-strandedness enables increased inclusion inside stress granules and that stress granule-enriched RNAs form fewer intra- and intermolecular RNA-RNA interactions. Additionally, several RNA binding proteins including SRSF1 are enriched in differential structure regions. eCLIP analysis revealed that SRSF1 binds to single-stranded regions along RNAs, and increased SRSF1 binding enabled better inclusion of RNAs in stress granules, whereas depletion of SRSF1 decreased stress granule formation under mild oxidative stress. We also observed the active unwinding of RNAs inside stress granules regulated by helicases, including DDX3X, and showed that inhibition of DDX3X results in slower dissolution of stress granules during recovery. Our study reveals the existence of multiple mechanisms to maintain RNA single-strandedness inside stress granules and to allow reversibility of stress granule formation, highlighting the importance of regulating RNA structure to enable cellular plasticity and stress response.

molecular biology↗

bioRxiv2025

The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

Brain metastatic cells undergo metabolic adaptations, such as increased reliance on oxidative phosphorylation. Integrating proteomic and transcriptomic profiling of patient samples revealed a consistent upregulation of the carnosine dipeptidase-1 (CNDP1) in brain versus extracranial metastases. Carnosine is an abundant metabolite in brain and muscle, known to exert anti-proliferative effects on cancer cells. Here, we demonstrate that CNDP1 inhibition suppresses both the establishment and maintenance of melanoma brain metastasis while its ectopic expression is sufficient to confer brain metastatic potential to poorly metastatic cells. CNDP1 suppression results in activation of the Integrated Stress Response via Heme-Regulated Inhibitor Kinase and reprogrammed translation towards preferential expression of mitochondrial and survival transcripts. We further show that CNDP1 upregulation supports mitochondrial activity by limiting the levels of its substrate carnosine, a copper ionophore, thus protecting metastatic cells from carnosine-induced copper toxicity. Our studies reveal a novel metabolic adaptation during brain metastasis, which can be leveraged for therapeutic purposes.

cancer biology↗

bioRxiv2025

Evidence of a novel sublineage of Streptococcus agalactiae in elephants from zoo populations in Germany

Streptococcus agalactiae research primarily centers on investigating human and bovine infections, although this pathogen also can be carried and cause infections in a wider range of animal species. Moreover, infections with S. agalactiae are posing significant health implications and, also, recent studies are highlighting a potential zoonotic risk. Despite the comparatively frequent isolation of S. agalactiae from elephants, only a few reports document infections in wild and zoo populations. We performed a comparative genomic analysis of 24 elephant isolates from three different zoos in Germany, to achieve a comprehensive characterization. Elephant isolates showed pronounced phylogenetic divergence from isolates of other host species, while also forming clusters based on their zoo of origin and their genotypes (MLST profiles). Capsular serotypes could not be identified for the majority of the isolates (n=20/24). Several genes, associated exclusively with the elephant host may underlie the pathogens capacity to improve its survival and virulence across varied ecological niches. This study not only deepens our understanding of S. agalactiae across diverse species and environments but also represents the first whole-genome sequencing characterization of S. agalactiae isolates from elephants, helping to expand our knowledge about infections in exotic animals.

bioinformatics↗

bioRxiv2025

DNA repair functions are essential for bacterial stress defense but render antibiotic tolerant sub-populations susceptible to ciprofloxacin and gentamicin upon prolonged treatment

The unresponsiveness of bacterial tolerant subpopulations to antibiotics has been attributed to physiological dormancy triggered by environmental stresses. In this work, we showed that protein and DNA synthesis activities in tolerant subpopulations that formed during nutrient starvation remained at a high level and only dropped gradually during the course of six days-starvation. Interestingly, upon decline of these activities, the tolerant subpopulations became susceptible to antibiotics that target protein and DNA synthesis, which was found to be required to support DNA repair functions essential for prolonged survival of the tolerant subpopulation. The increasing susceptibility of the bacterial tolerant subpopulation to antibiotics was also due to diminishing energy production, antioxidant defense and efflux functions, which resulted in weakening defense and further inhibition of synthesis and functions of DNA repair and other defense proteins. These findings confirm that bacterial tolerant subpopulations remain physiological active, and open new opportunities for combating bacterial tolerance with existing antibiotics.

microbiology↗

bioRxiv2025

Multicilia dynamically transduce Shh signaling to regulate choroid plexus functions

Choroid plexus is a major site for cerebrospinal fluid (CSF) production, characterized by a multiciliated epithelial monolayer that regulates CSF production. We demonstrate that defective choroid plexus ciliogenesis or Intraflagellar transport yields neonatal hydrocephalus, at least in part, due to increased water channel Aqp1 and ion transporter Atp1a2 expression. We demonstrate choroid plexus multicilia as sensory cilia, transducing both canonical and non-canonical Shh signaling. Interestingly, it is the non-canonical Shh signaling that represses Aqp1 and Atp1a2 expression by Smo/Gi/cAMP pathway. Choroid plexus multicilia exhibit unique ciliary ultrastructure, carrying features of both primary and motile cilia. Unlike most cilia that elongate during maturation, choroid plexus ciliary length decreases during development, causing a decline of Shh signaling intensity in developing choroid plexus, a derepression of Aqp1 and Atp1a2, and ultimately, an increased CSF production. Hence, developmental dynamics of choroid plexus multicilia dampens the Shh signaling intensity to promote CSF production.

developmental biology↗

bioRxiv2024

Time-resolved fluorescent proteins towards fluorescence microscopy in the temporal and spectral domains

Fluorescence microscopy has been widely applied in life sciences. While steady-state fluorescence intensity is mostly used, the time-resolved detection mode relying on fluorescence lifetime remains underexplored. Herein, we present a family of time-resolved fluorescent proteins (TRFP). Using a strategy that regulates lifetime without affecting spectra of FPs, we have developed different TRFPs that cover the visible spectrum and a wide range of lifetime. TRFPs are employed in the temporal-spectral resolved microscopy, allowing the simultaneous imaging of 9 different proteins in live cells and correlation of multiple activities to cell cycles. Finally, TRFPs are integrated into multiplexing super-resolution microscopy to concurrently visualize 4 proteins in live cells, acquiring a remarkable 50 nm spatial resolution using lifetime as signal. TRFPs represent a transformative toolset towards fluorescence microscopy in both the temporal and spectral domains. Beyond the multiplexing imaging, TRFPs could introduce unprecedented opportunities of system complexity and quantitative accuracy into biological research.

biochemistry↗

bioRxiv2024

Boosting of CAR-T cells with rhabdovirus is limited by type I interferon and rapid contraction

Rhabdovirus vaccines that encode tumour-associated antigens are potent boosting agents for adoptively transferred tumour-specific T cells. Employing rhabdovirus vaccines to boost adoptively transferred T cells relies on a priori knowledge of tumour epitopes, isolation of matched epitope-specific T cells, and a personalized vaccine, which limit clinical feasibility. Here, we investigated a universal strategy for boosting transferred tumour-specific T cells where boosting is provided through a chimeric antigen receptor (CAR) that is paired with a vesicular stomatitis virus (VSV) vaccine encoding the CAR-target. Boosting CAR-engineered tumour-specific T cells with paired VSV vaccines was associated with robust T cell expansion and delayed tumour progression in syngeneic models. CAR-T cell expansion and anti-tumour function was enhanced by blocking IFNAR1. However, vaccine-boosted CAR-T cells rapidly contracted and antigen-positive tumours re-emerged. In contrast, when the same T cells were boosted with VSV encoding antigen that stimulates through the TCR, the adoptively transferred T cells displayed improved persistence, tumour-specific endogenous cells expanded in parallel, and tumour cells carrying the antigen target were completely eradicated. Our findings underscore the need for further research into CAR-mediated boosting, how this differs mechanistically from TCR-mediated boosting, and the importance of engaging endogenous tumour-reactive T cells to achieve long-term tumour control.

immunology↗

bioRxiv2024

Identification and characterization of UBA5 inhibitors

UBA5 is a critical E1-activating enzyme in the UFMylation pathway, a post-translational modification process implicated in neurodegenerative diseases and cancers. In this study, we developed a high-throughput screening (HTS) assay to identify inhibitors of UBA5 from a library of blood-brain barrier (BBB)-permeable compounds. The assay, based on the AMP-Glo kit, enabled the identification of five novel UBA5 inhibitors belonging to three distinct chemical scaffolds with low micromolar IC50 values. These inhibitors demonstrated selectivity for UBA5 over other E1 enzymes, such as UBA1, and showed efficacy in inhibiting endogenous UFMylation in HEK293T cells. The identified inhibitors not only provided valuable tools for studying UFMylation but also represent potential therapeutic candidates for diseases associated with dysregulated UFMylation, such as Alzheimers disease and cancer. Further optimization of these compounds is necessary to enhance their potency and pharmacokinetic properties. Our findings highlight UBA5 as a promising therapeutic target and pave the way for the development of selective inhibitors that can modulate UFMylation in human diseases.

biochemistry↗

bioRxiv2024

Deficiency of Hv1 Proton Channel Enhances Macrophage Antigen Presentation and Anti-Tumor Responses in Glioma

In the tumor microenvironment of glioblastoma, myeloid cells act as a double-edged sword--they are a major cellular component modulating the immune response while presenting a potential therapeutic target. Our study highlights the voltage-gated proton channel Hv1, predominantly expressed in myeloid cells, as a crucial regulator of their physiological functions. We discovered a strong correlation between increased Hv1 expression and poor prognosis in glioblastoma patients. Depleting Hv1 significantly extended survival in a mouse model of glioma. Employing multiple novel transgenic mouse lines, we demonstrated that Hv1 is upregulated in response to tumor presence, with glioma-associated macrophages as the principal contributors. Specifically, we identified that Hv1 in infiltrating macrophages as the major driver of survival phenotype differences. Through a combination of in vivo two-photon imaging, spectral flow cytometry, and spatial transcriptomic sequencing, we found that Hv1 depletion leads to reduced macrophage infiltration and enhanced antigen presentation, ultimately fostering a stronger adaptive immune response. These findings establish the Hv1 channel as a crucial new immune regulator within the brain tumor milieu, offering a promising target for reprogramming macrophage function to combat glioblastoma.

neuroscience↗