Biology subjects

Zhang, M.

Publications and source records attributed to Zhang, M..

At least 217 records · Page 12Linked to original sources

bioRxiv2025

Genome-wide CRISPR screens identify PTGES3 as a novel AR modulator

The androgen receptor (AR) is a critical driver of prostate cancer (PCa). To study regulators of AR protein levels and oncogenic activity, we created the first live cell quantitative endogenous AR fluorescent reporters. Leveraging this novel AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators including HOXB13 and GATA2 and also unexpected top hits including PTGES3, a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell cycle arrest, and cell death in AR-driven PCa models. PTGES3 is not a commonly essential gene, and our data nominate it as a prime PCa therapeutic target. Clinically, analysis of PCa data demonstrate that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, forms a protein complex with AR in the nucleus, regulates AR protein stability in vitro and in vivo and modulates AR function in the nucleus at AR target genes. PTGES3 represents a novel therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.

cancer biology↗

bioRxiv2025

Context-Dependent Variability Of HIF Heterodimers Influences Interactions With Macromolecular And Small Molecule Partners

Hypoxia inducible factors (HIFs) are transcription factors that coordinate cellular responses to low oxygen levels, functioning as an /{beta} heterodimer which binds a short hypoxia response element (HRE) DNA sequence. Prior studies suggest HIF/HRE complexes are augmented by the binding of additional factors nearby, but those interactions are not well understood. Here, we integrated structural and biochemical approaches to investigate several functionally relevant HIF assemblies with other protein, small molecule, and DNA partners. First, we used cryo-electron microscopy (cryo-EM) to establish HIF-1 and HIF-2 self-assemble to form novel "dimer-of-heterodimers" (DoHD) complexes on extended human EPO enhancer sequences, with one heterodimer bound at a canonical HRE site and the second binding in an inverted fashion to an HRE-adjacent sequence (HAS) 8 bp away. Consistent with ARNT PAS-B domains predominating interactions within a DoHD, we found HIF-1 and HIF-2 co-assemble mixed DoHD complexes on the same DNA. Second, we saw that despite the increased complexities of the larger complexes, ligands for the isolated ARNT or HIF-2 PAS-B domains are still capable of binding and disrupting both the heterodimer and DoHD complexes, albeit with variable potencies depending on the ligand. Finally, we combined cryo-EM and hydrogen- deuterium exchange by mass spectrometry (HDX-MS) to show how HIF-1 and HIF-2 heterodimers engage the transforming acidic coiled-coil containing protein 3 (TACC3) coactivator via both ARNT and HIF- subunits, though this was unseen in the larger DoHD. Our findings highlight the importance of both molecular context and dynamics in biomolecular complex formation, adding to the complexities of potential regulation. Significance StatementHypoxia inducible factors (HIFs) are transcription factors that regulate oxygen-dependent cellular processes with implications in certain types of cancers. Current molecular structures of HIFs bound to short DNA fragments provide insights into their function, but leave open questions about how they bind longer natural DNA fragments and interact with small molecules and protein coactivators. Integrating structural and biochemical techniques, we discovered a novel assembly in which two HIFs bind together on a single extended DNA fragment, forming a "dimer-of-heterodimers", which exhibits some differences in ligand and coactivator binding than heterodimers or isolated PAS domains. Our studies highlight how functional contexts can shift structural paradigms and provide greater insight into the mechanisms by which HIFs and similar bHLH-PAS transcription factors operate.

biochemistry↗

bioRxiv2025

Role of DEPDC1 in Glioblastoma Malignant Phenotype and Radiosensitivity

Radiotherapy has significantly improved survival outcomes in glioblastoma (GBM) patients, particularly when combined with tumor resection and temozolomide chemotherapy. However, radioresistance remains a major obstacle, limiting further advances in prognosis and potential cure. DEPDC1, a gene implicated in various malignant phenotypes, has not yet been investigated for its role in GBM radioresistance, despite suggestive evidence from previous studies. In this study, analysis of the cancer database and GBM tissue microarrays revealed that DEPDC1 expression is significantly elevated in GBM tissues compared to normal brain tissues. Using GBM cell lines with DEPDC1 knockdown, we observed markedly reduced proliferation and motility. Notably, DEPDC1 downregulation significantly enhanced radiosensitivity, as demonstrated by increased radiation-induced apoptosis, G2/M phase arrest, DNA damage, and impaired DNA repair post-radiation, resulting in lower survival rates in clonogenic assays. Mechanistically, these effects may be mediated through DEPDC1-driven upregulation of NF-{kappa}B, supported by tumor sample analysis from our xenograft mouse model. Collectively, our findings suggest that DEPDC1 is not only involved in GBM malignancy but also represents a promising therapeutic target for overcoming radioresistance and developing effective radiosensitizers.

cancer biology↗

bioRxiv2025

Intravenous BCG immunization drives naive CD4+ T cell to Th1 differentiation via CD4+ T cell epigenetic reprogramming

Intravenous (IV) administration of live Bacille Calmette-Guerin (BCG) boosts potent immunity through coordinated interactions of innate immune response and adaptive immune response for BCG antiviral activity. The underlying mechanisms by which BCG initiates CD4+ T response remain incompletely understood. Here, we show that IV administration of BCG drives naive CD4+ T cell differentiation to Th1 cell through epigenetic reprogramming. Single-cell transcriptomic and epigenomic analyses revealed that IV BCG immunization induced metabolic reprogramming in monocytes and increased the proportion of T cells. The immunization induced chromatin openness for transcription factors regulating naive CD4+ T cells differentiation into Th1 cells. Importantly, we observed increases in Stat1, Irf1 and interferon-stimulated genes such as Igtp in Th1 cells, leading to upregulation of interferon related gene expression for antiviral response by BCG immunization. Activated transcription factors like Irf1 in CD4+ T cells promotes Il12rb1 expression to facilitate IL-12 signaling and drive naive CD4+ T cells differentiation into Th1 cells. We showed that BCG immunization reduced respiratory syncytial virus (RSV) infection. Thus, transcription regulation through epigenetic reprogramming plays a critical role in BCG-induced CD4+ T cell differentiation and to the broad antiviral activity. SignificanceBacille Calmette-Guerin (BCG) vaccination confers nonspecific protection against heterologous pathogens. Recent work demonstrated that coordinated interactions of innate immune and persistent CD4+ T cell response plays a critical role in BCG induced antiviral activity. In our study, we investigated BCG immunization on epigenetic reprogramming of CD4+ T cells and found that BCG immunization elicited antiviral activity by provoking T cell proliferation. We observed chromatin openness favoring naive T cell differentiation to Th1 response. Importantly, we observed STAT1 and interferon regulatory factor-1 (Irf1) activation. These findings provide mechanistic insights into BCG-induced Th1 response through epigenetic reprogramming and highlight the potential of BCG in offering a long-term protection against emerging and reemerging pathogens. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/655674v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1c3cb94org.highwire.dtl.DTLVardef@16a8e8forg.highwire.dtl.DTLVardef@b58b88org.highwire.dtl.DTLVardef@dbc994_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIntravenous BCG immunization drives naive CD4+ T cell differentiation to Th1 response; C_LIO_LIBCG alters chromatin accessibility for transcriptional factor expression favoring Th1 response through a Irf1-Il12rb1-IL12 feedback loop; C_LIO_LIThe immunization elicits antiviral activity against RSV infection; C_LI

immunology↗

bioRxiv2025

Integrating Single-Cell Biophysical and Transcriptomic Features to Resolve Functional Heterogeneity in Mantle Cell Lymphoma

Intra-tumor heterogeneity impacts disease progression and therapeutic resistance but remains poorly characterized by conventional histologic, immunophenotypic, and molecular approaches. Single-cell biophysical properties distinguish functional phenotypes complementary to these approaches, providing additional insight into cellular diversity. Here we link both buoyant mass and stiffness to gene expression to identify clinically relevant phenotypes within primary mantle cell lymphoma (MCL) cells, employing MCL as a model of biological and clinical diversity in human cancer. Linked measurements reveal that buoyant mass and stiffness characterize B-cell development states from naive to plasma cell and correlate with expression of oncogenic B-cell receptor signaling genes such as BLK and CD79A. Additionally, changes in cell buoyant mass within primary patient specimens ex vivo correlate with sensitivity to Brutons Tyrosine Kinase inhibitors in vivo in MCL and chronic lymphocytic leukemia, another B-cell malignancy. These findings highlight the value of biophysical properties as biomarkers of response in pursuit of future precision therapeutic strategies.

cancer biology↗

bioRxiv2025

Identification and Characterization of PLUTO-201, a Novel Long Non-Coding RNA Associated with Poor Outcomes in Prostate Cancer

Background: Despite extensive investigation, the factors promoting aggressive prostate cancer are poorly understood. In particular, despite a few prominent examples, the role of long non-coding RNAs (lncRNAs) is largely unknown. Methods: We performed a comprehensive analysis of whole-genome transcriptome data to identify differential expression across 1,567 patients with prostate cancer, then correlated differential expression with risk of metastatic recurrence. We characterized the lncRNA most associated with metastasis in vitro and in vivo to investigate the mechanism by which it promotes aggressive prostate cancer. Results: We have identified a novel lncRNA, Prostate Cancer Associated hnRNPK Interacting Transcript (PCAHIT), which is strongly associated with metastasis and poor overall survival in men with prostate cancer. We find that overexpression/knockdown of PCAHIT in pre-clinical models of prostate cancer modulates proliferation rates and markers of an aggressive phenotype through regulation of steroid biosynthesis and expression of the MHC class I complex, driving increased growth in androgen-depleted conditions and decreased susceptibility to T cell-mediated cytotoxicity. We further find that the heterogeneous nuclear ribonucleoprotein hnRNPK directly binds PCAHIT and is indispensable for its activity. Conclusions: Overall, our findings indicate that PCAHIT is a driver of aggressive prostate cancer phenotypes and poor clinical outcomes through suppression of the immune response and increased androgen-independent cancer growth. PCAHIT is a potential biomarker of aggressive disease.

cancer biology↗

bioRxiv2025

Human chronic inflammation is orchestrated by spatially restricted inflammation-activated Dendritic cells

Dendritic cells (DCs) form coordinated networks that orchestrate inflammatory responses across tissues. Upon activation, conventional dendritic cells (DCs) undergo changes in their transcriptome, phenotype, and function, depending on the tissue microenvironment. The role of DC activation during human chronic inflammation has yet to be explored. To address this, we aimed to investigate activated DCs across a range of chronically inflamed human tissues, with a focus on cervical lymph nodes (LNs). We performed scRNAseq and flow cytometry on healthy, acutely, chronically inflamed, and cancer-associated human cervical (LNs) and identified a novel inflammation-associated DC (iaDC) cell state, found only within chronically inflamed LNs. iaDCs were defined by a unique cytokine expression profile (CXCL9, CXCL10, IL1B) and could be phenotypically distinguished from other activated DCs by their elevated expression of CD1C, CD206, CD319, and CD274. Functionally, we found that IFN{gamma} signaling induces DC2s to enter the iaDC cell state and that this process can be abrogated by inhibition of JAK-STAT signaling. Using spatial transcriptomics, we observed LN iaDC residing within a specific chronic inflammatory niche, which was enriched for inflammatory monocytes, NK cells, and effector memory CD4+ and CD8+ T cells. Extrapolating our findings to other chronic inflammation-associated diseases, we similarly observed the emergence of iaDCs within the intestines of treatment-resistant Crohns Disease patients, synovial membranes and lymph nodes of rheumatoid arthritis patients and lungs of sarcoidosis patients. Similarly to the lymph node, iaDCs were found to reside in conserved chronic inflammation-associated spatial niches within the small intestine of affected individuals and displayed equivalent transcriptional characteristics and surrounding cellular neighborhoods. Collectively, these data highlight a previously unexplored role of iaDCs in human chronic inflammatory diseases and propose a conserved spatially restricted iaDC-populated chronic inflammatory niche associated with resistance to therapy. These findings highlight novel avenues to shape inflammatory trajectories by targeting iaDCs and their spatial niches, for example, by inhibiting JAK-STAT signaling.

immunology↗

bioRxiv2025

Targeting C42 in PKGIα alleviates diastolic dysfunction in HFpEF

BackgroundIn our exploration of novel therapeutic strategies for treating heart failure with preserved ejection fraction (HFpEF), we focused on cysteine 42 (C42) in cGMP-dependent protein kinase I (PKGI), which plays a crucial role in regulating blood pressure and diastolic relaxation. Our findings demonstrate that targeting C42 on PKGI with urolithin A effectively limits diastolic dysfunction and cardiac remodeling in a multi-hit mouse model of HFpEF, while also improving the kinetics of relaxation and contraction in human-engineered heart tissue. MethodsWe evaluated various polyphenols, including urolithin A, for their capacity to oxidize and activate PKGI. This assessment involved recombinant protein, isolated rat aortic smooth muscle cells, and myography experiments using isolated mesenteric vessels. Additionally, we investigated the ability of urolithin A to stimulate PKGI-dependent phosphorylation of phospholamban (PLN) in neonatal rat ventricular myocytes (NRVMs). A multi-hit mouse model was employed to induce HFpEF in wild-type and C42S PKGI knock-in (KI) mice. Following this, the mice were given vehicle or urolithin A by gavage, and heart function was evaluated using echocardiography. The relevance to human physiology was assessed using human-engineered heart tissue. ResultsThe polyphenols quercetin, fisetin, and urolithin A were shown to induce C42-dependent activation of PKGI. This mechanism of kinase activation led to vasorelaxation, which was attenuated in mesenteric vessels isolated from KI mice. Additionally, urolithin A treatment promoted PKGI-dependent phosphorylation of phospholamban in isolated cardiomyocytes. In a multi-hit model of HFpEF, urolithin A reversed diastolic dysfunction and cardiac remodeling in wild-type mice, but not in KI mice. These findings were shown to be likely relevant to humans, as urolithin A also enhanced the kinetics of relaxation and contraction in human-engineered heart tissue. ConclusionsTargeting C42 in PKGI with urolithin A presents a promising new strategy for the treatment of HFpEF.

biochemistry↗

bioRxiv2025

CRIS: A Centralized Resource for High-Quality RNA Structure and Interaction Data in the AI Era

As interest in RNA-based therapeutics expands, there is a growing demand for RNA structure elucidation and RNA-RNA interactions in both academic and clinical settings. Despite rapid advances in methods for RNA structure determination, the field faces persistent challenges in data reproducibility, quality control, and accessibility, largely due to inconsistencies in data processing and analysis workflows. Concurrently, methodological improvements have generated increasingly complex datasets, which necessitate a standardized framework. Here, we present the Crosslinking-based RNA Interactomes and Structuromes (CRIS) database, a comprehensive resource designed to address these limitations. Among existing experimental and computational approaches for RNA structure characterization, crosslinking-based technologies offer superior reliability, high throughput, and high resolution. CRIS provides rigorously curated datasets, standardized workflows, and user-friendly tools, together with built-in quality metrics and detailed visualization guidance to ensure reproducibility and transparency while pairing seamlessly with existing experimental pipelines. By delivering high-complexity RNA datasets alongside accessible computational tools, CRIS serves as a standardized reference for both new and existing data, facilitating investigation through comparative analyses and providing a training resource for deep learning-based computational exploration. This will enable integration into machine learning workflows for large scale, novel RNA structure discovery.

bioinformatics↗

bioRxiv2025

A nasal vaccine candidate, based on S2 and N proteins from SARS-CoV-2, generates a broad antibody response systemic and at lower respiratory tract.

Since the beginning of the COVID-19 pandemic various groups around the world have intensively worked in the development of vaccine candidates against SARS-CoV-2. Several vaccines have been approved in the past years, the majority are based on the Spike or RBD proteins and employs parenteral administration routes. Considering the recent history of Coronavirus zoonotic events, causing serious human health problems, the generation of vaccines with a broad scope of protection and the potential to cut / reduce the transmission remains in the spotlight. The current global pandemic preparedness initiatives have promoted also the preclinical evaluation of a new group of Coronavirus vaccines. In the present work a nasal vaccine candidate based on two highly conserved Sarbecovirus proteins, S2 and nucleocapsid (N), is evaluated in two different mice strains. The vaccine preparation, containing a CpG ODN as adjuvant, was able to generate high antibody titers against both antigens, in sera and bronchoalveolar lavages. This antibody response results cross-reactive to S2 from SARS-CoV-1 and MERS-CoV, and to N from SARS-CoV-1. However, a very low neutralizing capacity was found in the sera of the immunized mice when a pseudoviral system assay was used. On the other hand, the vaccine preparation induces, at systemic compartment, IFN {gamma} secretion, and a marked IgG2a response, specific against both proteins; a profile consistent with the development of a Th1 pattern. Although further evaluations should be done, including protection assays, the demonstrated cross-reactivity level and mucosal response constitutes promising features of this vaccine candidate.

immunology↗

bioRxiv2025

TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

Unconventional protein secretion (UcPS) exports diverse signal peptide-lacking cargoes, yet its cargo selectivity remains poorly understood. Here, we identify TMED proteins as key regulators of vesicle-dependent UcPS, mediating selective cargo release via translocation into secretory carriers. TMED proteins act as translocators, facilitating cargo passage across lipid bilayers with assistance from HSP90 chaperones and partial cargo unfolding. Selectivity arises during translocation, where TMED cytoplasmic tails bind specific cargoes. The ER-Golgi intermediate compartment (ERGIC) is essential for TMED-mediated translocation and release. TMED homo-oligomerization enhances translocation, while hetero-tetramerization inhibits it. ERGIC localization promotes homo-oligomerization, which is further stabilized by cargo binding, forming a feed-forward mechanism to enhance translocation. These findings establish TMED proteins as central regulators of cargo diversity in UcPS, with their oligomerization and subcellular localization modulating translocation efficiency.

cell biology↗

bioRxiv2025

Brain-computer interface training fosters perceptual skills to detect errors

Accurate perception of visuo-motor errors is essential for perceptual and sensorimotor learning so that corrective actions are performed timely to maintain stability and goal-directed behavior. However, a key challenge remain: conventional, behavioral perceptual training -- typically based on response accuracy feedback-- is limited in improving sensitivity to small, subtle errors. While prior approaches have focused on modulating sensory regions to enhance perceptual learning, we propose an alternative approach: targeting a cognitive neural marker, namely the error positivity (Pe), a component of the error-related potential (ErrP) that primarily originates in the anterior cingulate cortex (ACC), a key region involved in decision-making. We hypothesize that the Pe, which reflects conscious awareness of errors, serves as a modifiable neural correlate of error perception. To address the performance bottlenecks seen in conventional, behavioral perceptual training, we show that real-time feedback on ErrP presence or absence during perceptual training over five longitudinal days enhances the Pe component and improves participants ability to detect small visuo-motor errors --an outcome not achieved through conventional, behavioral perceptual training. These findings offer new neurophysiological insights into error perception and learning, and establish ErrP-based BCI interventions as a promising tool for accelerating perceptual learning, particularly in contexts where subtle error detection is critical.

bioengineering↗

bioRxiv2025

Disentangling complex language contact and admixture in the broad Gansu-Qinghai region

Language evolution in the Gansu-Qinghai (GQ) region provides a key perspective for understanding cultural development along the Silk Road. Previous genetic and archaeological studies revealed complex multiethnic interactions shaped by migration and sociocultural exchange. However, the lack of structured linguistic data and computational tools for studying language contact has limited rigorous analysis of sociocultural evolution in this area. Here, we presented a new hybrid dataset of phonological and morpho-syntactic features from languages sampled across the GQ region. We introduced a computational framework to assess language contact and admixture, allowing us to quantify interaction among GQ languages and trace their origins. Our results showed that GQ languages have distinct contact patterns in their phonological and morphosyntactic systems, with some languages exhibiting clear evidence of mixture. Using a new statistical method, Trait Sharing Test (TS-TEST), based on tree topology, we identified significant influences from Sinitic, Tibetan, Mongolic, and Turkic languages in shaping GQ linguistic diversity. These findings highlight the GQ region as a linguistic convergence zone on the Silk Road and provide a foundation for quantitative research on language contact and admixture. Our work strengthens the linguistic perspective on cultural evolution in the GQ region and supports future interdisciplinary studies integrating languages, genes, and material cultures.

evolutionary biology↗

bioRxiv2025

Microfluidic Droplet-Assisted Single-Cell Raman Sorting for High-Throughput Screening of Functional Lignin-Degrading Microbes

Lignin, a major component of plant biomass, poses significant challenges for sustainable valorization due to its structural recalcitrance. Traditional microbial screening methods are hindered by culturability limitations and time-consuming workflows. This study introduces a novel Microfluidic Droplet-assisted Single-Cell Raman sorting system (MD-SCR sorting) to address these bottlenecks. By integrating droplet microfluidics with single-cell Raman spectroscopy (SCRS), the platform enables non-destructive, high-throughput screening of lignin-degrading microbes directly from environmental samples. A deuterium tracing strategy was developed, where microbial strains were cultured in lignin media containing 30% D2O. Raman detection of C-D bond vibrations (2040-2300 cm-1) allowed in situ identification of active degraders within 12 hours. The MD-SCR system achieved precise single-cell encapsulation, cultivation, and sorting using a custom-designed three-layered microfluidic chip. Validation experiments demonstrated strong correlation (R2=0.92) between %C-D values (quantifying deuterium assimilation) and traditional shake-flask degradation efficiency. Two lignin-degrading fungi (Pleurotus ostreatus G5 and P. eryngii PB) exhibited distinct C-D peaks, while non-degrading controls showed no deuterium incorporation. Laser intensity optimization (5% power) ensured spectral fidelity with >95% cell viability. The platforms label-free, culture-independent workflow facilitates rapid functional screening of unculturable microbes, overcoming limitations of fluorescence-based methods. This technology not only advances lignin bioconversion by enabling targeted isolation of high-efficiency degraders but also establishes a scalable framework for exploring complex microbiomes. By bridging metabolic phenotyping with single-cell resolution sorting, the MD-SCR system offers transformative potential for sustainable resource utilization and environmental biotechnology.

microbiology↗

bioRxiv2025

Heme-NO dilates arteries via mobilization of NO moieties from a vascular intracellular NO store

Nitrosyl heme (heme-NO) has recently emerged as a surrogate signaling entity for nitric oxide (NO). However, questions remain about how heme-NO signals across the cell membrane. Herein, we test the hypothesis that heme-NO signals as a nitrodilator that vasodilates by mobilizing NO moiety from a nitrodilator-activated intracellular NO store (NANOS) in the vasculature. We identify a novel mechanism for glutathione-catalyzed formation of a model compound, alb-heme-NO, and determine glutathione (GSH) as a ligand in its structure. Heme-NO complexes with plasma proteins in blood and, as such, it is impermeable to red blood cells or the vascular wall. Alb-heme-NO-mediated vasodilation, both in vitro and in vivo, is attenuated by prior depletion of the NANOS and potentiated by NANOS supplementation. Incubation with alb-heme-NO induces efflux of NO moieties from arteries. Additionally, the role of nitrosyl hemoglobin (HbNO) in mediating NO bioactivity export from erythrocytes is challenged. In conclusion, heme-NO functions as an extracellular nitrodilator via activation of the intracellular NANOS.

physiology↗

bioRxiv2025

TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

Lysosomal dysfunction occurs in many neurodegenerative diseases, including Parkinsons disease, and activating TFEB to enhance lysosomal biogenesis is a promising therapeutic strategy. To understand TFEB physiology in cells of the brain, we characterised TFEB expression using iPSC-derived models, and transcriptomic analysis of human and mouse brain tissue. Surprisingly, TFEB expression at the RNA and protein level was restricted to glia, whereas the related transcription factor, TFE3, was expressed ubiquitously. We identified HDAC1/2/3 as transcriptional repressors of neuronal TFEB and found the brain-penetrant HDAC inhibitor ACY-738 derepressed TFEB expression and enhanced TFE3 nuclear translocation in iPSC-dopaminergic neurons (iPSC-DaNs). We delineated the role of each transcription factor by genetic manipulation in iPSC-DaNs to reveal divergent roles in which TFEB activates mitochondrial biogenesis, whereas TFE3 enhances lysosomal biogenesis. Finally, we show TFE3 activation corrects the lysosomal dysfunction associated with GBA-N370S and SNCA-Triplication mutations in Parkinsons patient-derived iPSC-DaNs, demonstrating therapeutic utility in neurodegeneration.

cell biology↗

bioRxiv2025

Spatial proteomics with 100+ markers with Highly Multiplexed MALDI-IHC

Highly multiplexed, antibody-based protein imaging of tissue has yielded many insights but does not yet allow the routine analysis of more than 50 markers simultaneously. By leveraging the ability of Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging to resolve hundreds of m/z peaks without spectral overlap, MALDI-IHC with photocleavable mass-tag (PCMT)-labelled antibodies offers the possibility to dramatically increase the number of markers that can be imaged. Here, we demonstrate 100+ plex MALDI-IHC imaging and describe our development of gutenTAG, an open-source analysis pipeline for the resulting highly multiplex data. We validated a panel of 133 tumor-ecosystem-focused antibodies, show that it captures expected histopathology in diverse tissues, and demonstrate reproducibility of the method. We further applied a 120-plex subset of this panel to samples from an endometrial cancer cohort, again capturing expected histopathology and identifying novel spatial structures between TCGA molecular subtypes. Our work demonstrates the feasibility of simultaneously detecting hundreds of protein markers in a reproducible and scalable manner.

systems biology↗

bioRxiv2025

Dysregulated Proline Metabolism Contributes to Subretinal Fibrosis in Neovascular AMD: Therapeutic Potential of Prolyl-4-Hydroxylase Inhibition

Subretinal fibrosis, a major cause of irreversible vision loss in neovascular age-related macular degeneration (nAMD), is driven by excessive deposition of extracellular matrix such as collagens. While proline metabolism is known to play a critical role in collagen biosynthesis and fibrosis, its involvement in subretinal fibrosis remains unclear. Here, we characterized the progression of fibrovascular lesions in JR5558 mice, observing significant molecular alterations as early as 4 weeks of age and phenotypic changes by 8 weeks. Transcriptomic and metabolomic analyses revealed elevated levels of 4-hydroxyproline, an essential component of collagen, alongside significant alterations of other fibrosis-related pathways. P4HA1, a catalytic subunit of prolyl-4-hydroxylase essential for 4-hydroxyproline biosynthesis, was prominently expressed in fibrotic lesions in retinas of JR5558 and two-stage laser-induced murine models, as well as human eyes with nAMD. Targeting P4HA1 with the small-molecule inhibitor diethyl pythiDC significantly attenuated fibrovascular lesion growth in the JR5558 murine models and reduced collagen turnover in human retinal pigment epithelium cells. Combining diethyl pythiDC with aflibercept had a stronger antifibrotic effect than monotherapies in JR5558 mice. These findings suggest a key contribution of proline metabolism, particularly proline hydroxylation, in subretinal fibrosis. Inhibiting P4HA1 with diethyl pythiDC inhibited fibrosis in the models we studied, offering a novel therapeutic strategy. Further research is warranted to explore the potential benefits of combining existing anti-angiogenic therapies with drugs that inhibit proline metabolism for the management of nAMD-associated fibrosis.

neuroscience↗