Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 307 records · Page 17Linked to original sources

bioRxiv2025

A Multi-Layered Atlas of Spatial Regulatory Programs and Therapeutic Vulnerabilities in Glioblastoma

Glioblastoma (GBM) is marked by profound spatial heterogeneity. This complexity fuels tumor progression and therapy resistance. Although recent spatial transcriptomics (ST) studies have defined transcriptional niches, or metaprograms, the systems level regulatory logic linking upstream signaling, transcriptional programs, and therapeutic vulnerabilities remains unresolved. Here, we introduce an integrated, multi layered framework that unifies transcription factor and pathway activity inference (STAN and SPAN) with ligand/receptor and drug target mapping, thereby constructing a systems-level regulatory atlas of GBM across 26 tumors. This atlas uncovers spatial niches organized around transcriptional hubs and signaling programs, including hypoxic mesenchymal regions coordinated by HIF1A and SOX2 with mTORC1 signaling, macrophage rich areas driven by STAT3 mediated immune modulation, and astrocyte-like states shaped by lipid metabolic regulators such as SREBF2. By coupling this regulatory atlas to drug target predictions, we identify niche specific therapeutic vulnerabilities, such as VEGF blockade in HIF1A high regions and MAO-B inhibition in mesenchymal states. Finally, we provide an open, interactive web resource to make these data broadly accessible. This work provides a generalizable blueprint for linking spatial regulation to therapeutic hypotheses across diverse human diseases.

bioinformatics↗

bioRxiv2025

GLIS3 Marks a Neural-like Progenitor Cell State that Drives Metastasis in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has a high rate of recurrence and metastasis despite intensive therapy. The neural-like progenitor (NRP) transcriptional program is enriched in residual disease after neoadjuvant chemotherapy and radiotherapy; however, the mechanisms for increased NRP expression in the post-treatment setting remain unclear. Here, we find that NRP signatures are strongly enriched in tissue injury and regeneration, and NRP cancer cells co-express transcription factors involved in pancreatic development. We identify both cell autonomous and ligand mediated mechanisms for inducing NRP expression in vitro. We establish and characterize isogenic mouse organoid overexpression models for transcription factors associated with the NRP, classical, and basal-like states. We discovered that Glis3 is a key NRP-associated transcription factor that drives malignant properties including greater clonogenicity, tumor growth, and metastasis compared to isogenic models for the classical and basal-like states. Our work highlights the emergence of clinically relevant developmental regeneration programs in the post-treatment context.

cancer biology↗

bioRxiv2025

Local translation of circular RNAs is required for synaptic activity and memory

Circular RNAs (circRNAs) are enriched in synapses and implicated in cognitive processes, and recent studies have shown that circRNAs can encode micropeptides, which suggests that there may be novel synaptic proteoforms in the brain that await discovery. Here we report widespread learning-induced local circRNA translation in the prefrontal cortex of male C57BL/6 mice. More than 1500 synapse-enriched circRNAs contain active IRES elements, with 842 interacting with the ribosome and 241 exhibiting direct evidence of activity-induced translation. We discovered a synapse-enriched micropeptide (P1) that is derived from a single exon circRNA, the mRNA host of which encodes an enzyme associated with protein repair. Although P1 is only a third of the size of the full-length protein, it is locally expressed, enzymatically active, and interacts with plasticity-related proteins, including CaMKII. In addition to direct effects on synaptic activity, targeted P1 knockdown impairs whereas its overexpression enhances fear extinction memory. These findings shed new light on the dark proteome in the brain and reveal local circRNA translation as a novel mechanism of plasticity and memory.

neuroscience↗

bioRxiv2025

SomaMutDB 2.0: A comprehensive database for exploring somatic mutations and their functional impact in normal human tissues

Recent advances in ultra-accurate sequencing technologies have revealed that somatic mutations accumulate across the human lifespan and may contribute to both normal aging and disease. These mutations are highly diverse, often non-recurrent, and functionally heterogeneous, which makes their biological impact difficult to evaluate systematically. Although many studies have profiled somatic mutations in individual tissues or limited cohorts, a centralized and scalable platform that integrates discoveries and supports functional interpretation has been lacking. To address this gap, we present SomaMutDB 2.0 (https://somamutdb.org/SomaMutDB/), a substantially expanded database that catalogs 8.9 million mutations (8.57 million SNVs and 0.29 million INDELs) from 10,852 samples of 607 human subjects across 47 studies. Beyond expanded data coverage, SomaMutDB 2.0 introduces a comprehensive functional annotation framework that applies 22 predictive models, encompassing coding, regulatory, expression-based, and ensemble predictors, to systematically assess mutational impact. Users can browse pre-annotated variants through an interactive interface or upload their own variants for real-time analysis, with results contextualized against all mutations from normal, non-diseased tissues in the database. Together, these advances establish SomaMutDB 2.0 as the most comprehensive resource currently available for characterizing somatic mosaicism and functional interpretation in human health and aging. Graphical AbstractSomaMutDB 2.0 provides an expanded catalog of 8.9 million somatic mutations across 30 tissues, along with pipelines for mutational signature analysis and 22-tool functional annotation that enable user-submitted variant interpretation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/676124v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1c76484org.highwire.dtl.DTLVardef@1984f71org.highwire.dtl.DTLVardef@8789a9org.highwire.dtl.DTLVardef@5ed54a_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

A Human Neuronal Co-Culture System Reveals Early Tumor-Neuron Communication and Targetable Pathways in Glioblastoma

BackgroundGlioblastoma (GB, IDH wild type) is the most aggressive primary brain tumor in adults, with recurrence driven by residual tumor cells that re-establish interaction with surrounding neurons. While neuronal activity is recognized as a driver of GB progression, the earliest neuronal responses to tumor contact remain poorly understood. Existing models rarely capture these acute events or the heterogeneity of responses generated by tumors of different origins, limiting insight into the earliest neuron-tumor interactions. MethodsWe developed a dual-interface human iPSC-derived neuronal culture system to investigate acute neuronal responses to glioblastoma exposure. Neurons were challenged with either established GB cell lines or patient-derived glioblastoma cells (PDGCs). Using quantitative proteomics, high-resolution imaging, and immunological assays, we characterized compartment-specific neuronal changes and mapped activated signaling pathways. We also screened selective inhibitors for their effects on both tumor proliferation and neuronal integrity. FindingsWithin 24 hours of exposure, neurons displayed synaptic remodeling and activation of GB-related signaling cascades. Proteomic analysis of GB exposed neurons revealed enrichment of pathways associated with GB and abnormalities in neuronal circuits. Notably, the U-87MG cell line, but not PDGCs, induced pronounced synaptic disruption, neurite retraction, and MAPK pathway activation, with distinct molecular signatures across neuronal compartments. ERK1/2 and p38 MAPK signaling were differentially activated depending on the GB cells source, correlating with specific structural and functional synaptic alterations. Targeted inhibition of MAPK components significantly suppressed U-87MG proliferation and preserved neuronal architecture. InterpretationWe present a human neuronal culture model that detects and discriminates the earliest neuron-derived responses to glioblastoma from diverse tumor sources. By linking neuronal remodeling to tumor-specific signaling pathways, the platform uncovers both the heterogeneity of neuron-tumor interactions and early, targetable vulnerabilities. This system offers a translational tool to advance understanding of GB recurrence and to guide development of therapies with dual neuroprotective and anti-tumor efficacy. In BriefEarly interactions between glioblastoma and human neurons drive rapid, source-specific synaptic remodeling mediated by compartmental MAPK pathway activation. This neuronal co-culture model identifies distinct profiles of tumor-neuron communication, highlights synaptic vulnerability as a therapeutic axis, and demonstrates that MAPK pathway inhibition yields both neuroprotective and anti-tumor effects. SummaryGlioblastoma (GB) co-opts neuronal circuits to drive tumor progression, yet the earliest neuronal responses that may shape recurrence remain poorly defined. We developed a human iPSC-derived neuronal co-culture model that captures acute communication between neurons and glioblastoma from diverse sources, including serum-adapted cell lines and patient-derived cells. Within 24 hours, glioma-exposed neurons exhibited synaptic remodeling and activation of tumor-associated signaling pathways. High-resolution imaging and proteomics revealed compartment-specific synaptic alterations, with ERK1/2 and p38 MAPK signaling differentially engaged depending on the tumor source, corresponding to distinct structural and functional outcomes. Pharmacologic inhibition of MAPK components both suppressed tumor cells growth and preserved neuronal integrity. By modeling source-dependent and early neuron-tumor interactions, this platform not only identifies MAPK signaling as a critical mediator of synaptic vulnerability but also provides a clinically relevant tool for investigating the mechanisms of glioblastoma recurrence. It offers a framework for developing therapies that target the dual vulnerabilities of tumor progression and circuit remodeling. Highlights O_LIA dual-interface human iPSC-derived neuronal co-culture system models early neuron-glioblastoma (GB) interaction. C_LIO_LISerum-adapted U-87MG, but not serum-free patient-derived GB cells, induces pronounced synaptic disruption and neurite retraction. C_LIO_LIGlobal proteome of neurons reveals GB-associated signatures and neuronal circuit alterations in response to both U-87MG and patient-derived GB cells. C_LIO_LIERK1/2 and p38 MAPK signaling are differentially activated in neuronal compartments, depending on GB source. C_LIO_LIMAPK pathway inhibition suppresses U-87MG proliferation and preserves neuronal integrity, revealing actionable neuroprotective and anti-tumor targets. C_LI

cancer biology↗

bioRxiv2025

wspA mutation mediated Pseudomonas aeruginosa rugose small colony variant and its hyper-biofilm

Pseudomonas aeruginosa is a major multidrug-resistant pathogen whose biofilm formation complicates treatment. P. aeruginosa rugose small colony variants (RSCVs), characterized by enhanced biofilm production and resistance, pose significant clinical challenges. However, the formation mechanisms remain unclear in clinical strains. Paired clinical P. aeruginosa isolates AR8023-1 (wild-type) and AR8023-2 (RSCV) were collected from urine samples of a neurosurgery inpatient chronologically. The genome comparison was performed using Breseq, and plasmid complementation was performed to construct complemented strain (AR8023-2RifR::wspAAR8023-1). Then, all the aforementioned strains were examined RSCV phenotype, biofilm formation, and motility, and gene expression differences induced by the wspA mutation were analyzed via transcriptome sequencing, followed by quantification of c-di-GMP. In addition, we assessed antimicrobial susceptibility of the strains under both planktonic and biofilm conditions. The results showed that both AR8023-1 and AR8023-2 were identified as ST3420. AR8023-1 exhibited typical smooth morphology, while AR8023-2 displayed a RSCV phenotype. A single SNP difference was identified between the two strains, characterized by a deletion of glutamine at position 289 (CAG triplet deletion) in the wspA gene of strain AR8023-2. The complement isolates restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed a significant upregulation of c-di-GMP metabolic genes in RSCVs (P < 0.001), indicating that the WspA{Delta}289Q mutation activates diguanylate cyclase (DGC) activity, thereby elevating c-di-GMP synthesis. Intracellular c-di-GMP levels were significantly higher in RSCVs than those in WT and complement isolates, respectively (P < 0.001). Consequently, biofilm susceptibility testing demonstrated the {beta}-lactams MBIC of RSCVs was [≥]8-512-fold higher than those of WT and complement isolates. This study identifies a clinical WspA{Delta}289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance. IMPORTANCEP. aeruginosa is a leading multidrug-resistant pathogen whose biofilm formation capability greatly complicates treatment outcomes. This study identifies a novel clinical mutation in the wspA gene (WspA{Delta}289Q) that drives the emergence of rugose small colony variants (RSCVs), a phenotype linked to heightened biofilm production and antimicrobial resistance. We demonstrate that this mutation upregulates cyclic di-GMP synthesis, leading to increased biofilm formation and markedly reduced susceptibility to {beta}-lactam antibiotics under biofilm conditions. These results provide important insights into the genetic basis of RSCV development in clinical settings and highlight the role of c-di-GMP signaling in biofilm-associated resistance. Our findings underscore the need to target c-di-GMP pathways as a potential strategy for combating persistent P. aeruginosa infections.

microbiology↗

bioRxiv2025

Class A PBPs reinforce the septal cell wall following initial synthesis by SEDS-bPBP pairs during bacterial cytokinesis

Bacterial cytokinesis requires the coordinated action of septal peptidoglycan (PG) synthases to ensure efficient and accurate septum formation. Two major types of synthases contribute: SEDS-bPBP and bifunctional class A PBPs (aPBPs). How their activities are organized in space and time has remained unclear. Using Deinococcus radiodurans, which often contains both constricting and closed septa in the same cell, we show that septal PG synthesis proceeds through distinct steps at different regions. Primary synthesis at the leading edge by SEDS-bPBP pairs drives constriction, whereas aPBPs act later at the lagging edge and in closed septa to thicken the wall, enhance cross-linking, and reinforce septal integrity. At the single-molecule level, both synthase types move directionally to perform processive PG synthesis at distinct sites. We also observed spatiotemporal separation of SEDS-bPBP and aPBP synthases in Staphylococcus aureus, suggesting that secondary septal PG synthesis is a conserved mechanism securing successful cytokinesis.

microbiology↗

bioRxiv2025

Endothelin Signaling via EDNRB receptor Reduces Proliferation and Promotes Proneural-to-Mesenchymal Transition in Gliomas

Diffuse gliomas are incurable primary brain tumors encompassing three histo-molecular subtypes: glioblastomas (GB), astrocytomas, and oligodendrogliomas. The latter two harbor IDH1 mutations and exhibit slower progression than glioblastomas. Diffuse gliomas are composed of highly plastic tumor cells capable of transitioning between astrocyte-like, oligodendrocyte-like, progenitor-like, and mesenchymal-like states, driven by genetic alterations and microenvironmental cues. The proneural-to-mesenchymal transition (PMT), associated with increased malignancy, is notably influenced by cytokines in the tumor microenvironment. Endothelin cytokines (ET-1, ET-2, ET-3), primarily secreted by vascular cells, regulate not only vascular tone but also astrocyte and neural stem cell proliferation via the G-protein-coupled receptors EDNRA and EDNRB. Prior studies using serum-cultured glioma lines suggested pro-proliferative effects of endothelins; however, such models poorly recapitulate the in vivo glioma context. In this study, we comprehensively revisited endothelin signaling - covering receptor expression, regulation, downstream pathways, and cellular responses-using eleven serum-free, patient-derived glioma lines (glioblastomas, IDH-wt and IDH-mutant oligodendrogliomas and astrocytomas), along with primary tumor samples. Multi-omics and electrophysiological analyses revealed EDNRB as the predominant receptor, enriched in astrocyte-like cells, upregulated by BMPs or growth factor withdrawal, and downregulated by interferons, IL-6 cytokines, endothelins, and Hippo/YAP activation. In contrast, EDNRA was expressed by a perivascular tumor subpopulation and induced by Notch signaling in glioblastomas but not in IDH1-mutant cells. Functionally, endothelins reduced proliferation across all models while promoting migration and PMT. Mechanistically, EDNRB activation increased intracellular Ca{superscript 2} and activated ERK, STAT3, and apamin-sensitive SK2/SK3 potassium channels. These findings identify endothelin signaling as an important regulator of glioma cell plasticity and behavior. HighlightsO_LIEDNRB is the predominant endothelin receptor expressed in glioma cells, with a small subset of tumor cells expressing EDNRA in close proximity to blood vessels C_LIO_LIEndothelin signaling reduces proliferation while promoting cell migration and Proneural-to-Mesenchymal transition C_LIO_LIEndothelin activates downstream Ca2+, K+, ERK, and STAT3 signaling pathways C_LIO_LIEDNRB expression is both positively and negatively regulated by inflammatory cytokines and the Hippo/YAP1 pathway, whereas EDNRA is upregulated by Notch signaling and hypoxia C_LI

cancer biology↗

bioRxiv2025

Transcontinental Spread of HPAI H5N1 from South America to Antarctica via Avian Vectors

Our study has for the first time identified H5N1 strains (clade 2.3.4.4b, genotype B3.2) in brown skuas from the Fildes Peninsula, South Shetland Islands, Antarctica. These findings indicate that highly pathogenic avian influenza H5N1 viruses is now actively circulating in Antarctic ecosystems, representing a significant expansion of its geographic range.

genomics↗

bioRxiv2025

ABCDscores: An R package for computing summary scores in the ABCD Study(R)

The ABCD Study(R) is the largest longitudinal research study of brain development and child health across the United States. The study generates a rich multimodal dataset, including file-based raw and derivative data and a curated tabulated dataset comprising data from a broad spectrum of research domains. Historically, summary scores in the tabulated dataset were computed using the electronic data capture system REDCap or external scripting solutions. However, this approach was rigid, unreliable, and lacked transparency around how scores were computed. Here, we introduce ABCDscores, an R software package that is used by the study to compute most non-proprietary summary scores included in the tabulated ABCD data resource and allows for customization to meet specific research needs. ABCDscores provides a flexible, open-source solution that uses R for transparent and reproducible scoring. The package is built to apply standard scoring methods across different types of assessments, ensuring consistent and accurate results even with large datasets. By simplifying complex scoring procedures into a user-friendly process, ABCDscores streamlines research workflows, reduces errors, and saves researchers valuable time. The package aims to improve analyses of data from the ABCD Study(R) and can serve as a model for the development of similar tools and a standardized framework for other research studies, fostering new discoveries and innovation in the field.

bioinformatics↗

bioRxiv2025

The MEK inhibitor trametinib incurs mitochondrial injury and induces innate immune responses in the mouse heart

Trametinib (Trm) is a highly selective MEK inhibitor that potently and persistently abrogates ERK1/2 activation. Trm initially was used to treat BRAF V600E-mutated melanoma but its FDA-approved indications are expanding rapidly. Trm generally is well tolerated but it can cause dose-limiting cardiomyopathy and heart failure. Here we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo, Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part through blunted activity of Electron Transport System Complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial Damage-Associated Molecular Patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations but this lesion was reversed by expression of a phosphomimetic STAT3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trms anti-cancer efficacy.

physiology↗

bioRxiv2025

A benchmark of the somatic mutation landscape using single-cell and single-molecule whole-genome sequencing

Accurate detection of somatic mutations in noncancerous cells is critical for studying somatic mosaicism, a process implicated in aging and multiple chronic diseases. However, single-cell and single-molecule DNA sequencing platforms differ in their error profiles, coverage biases, and sensitivity to specific mutation types, complicating cross-platform comparisons. Here, we present in vitro and in silico benchmarks to quantify true-positive and false-positive rates in single-cell whole-genome sequencing using Single-Cell Multiple Displacement Amplification, and in single-molecule sequencing using Nanorate Sequencing (NS) and whole-genome NS (WGNS). Using standard cell lines, we show that all three methods detect single-nucleotide variants (sSNVs) and small insertions and deletions (sINDELs) with high accuracy, but differ in genomic coverage and susceptibility to artifacts. Method-specific biases influence mutational signatures and hotspot detection. Applying results of the benchmark to IMR-90 fibroblasts, we estimate higher in vitro mutation rates using NS than expected from in vivo data, consistent with potential replication stress and culture-associated DNA damage. Overall, our study highlights the substantial impact of sequencing platform-specific biases on somatic mutation detection and interpretation, and lays the foundation for standardized, cross-platform-comparable analyses of somatic mosaicism in normal human tissues.

genomics↗

bioRxiv2025

Prior photon measurement density function constructed in a standard channel space for functional near-infrared spectroscopy

Photon measurement density function (PMDF) determines the measurement locations and their associated sensitivity to the hemodynamic fluctuations of an fNIRS channel, which is essential for pre-experiment optode arrangement and post-experiment signal analysis/results interpretation in fNIRS studies. However, obtaining the exact PMDF relies on individual MR images, which are unavailable in common fNIRS studies. Previous studies alternatively adopt the PMDF derived from single head templates, i.e., template-PMDF, which ignores the anatomical difference between the template and the participants, as well as the anatomical variability across participants. Here, we propose a methodological framework using a standard channel space to construct a prior-PMDF from a structural MRI database for arbitrarily given channel locations. We demonstrate the utility of the prior-PMDF in estimating channel-wise fNIRS imaging parameters, such as subject-specific PMDF, sensitivity in intracerebral and extracerebral tissue compartments, and sensitivity to regions defined by brain atlas. The performance of the prior-PMDF tested on an independent dataset was compared with the template-PMDF. The results show that the prior-PMDF significantly surpasses the template-PMDF.

bioengineering↗

bioRxiv2025

Structural Compression and Entorhinal Vulnerability: Linking Tentorial Adjacency to Tau Burden and Dementia Progression

Alzheimers disease (AD) is a growing public health crisis. The disease is defined neuropathologically by accumulation of amyloid-{beta} plaques and neurofibrillary tangles (NFTs) composed of abnormal tau protein in the brain. Early neurofibrillary degeneration in the entorhinal cortex (EC) is a hallmark of AD and a critical initiating event in the hierarchical pathoanatomical progression. However, the factors triggering initial tau deposition in the EC remain unclear. We propose a novel biomechanical cascade hypothesis, positing that the unique anatomical inferomedial positioning of the EC, including proximity to the tentorial incisura (TI) and other skull base structures, renders it susceptible to very mild yet persistent age-related mechanical stress, analogous to the effects of repetitive mild traumatic brain injury, triggering tau pathology. To test this hypothesis, we developed a method to quantify Entorhinal-Tentorial (EC-TI) proximity and applied it to multimodal imaging data from the Alzheimers Disease Neuroimaging Initiative (ADNI; n=47). Based on this neuroanatomical contact coefficient (NCC), participants were heuristically stratified into high (n=24) and low (n=23) adjacency groups. When controlling for other risk factors, tau PET signal in the EC predicted conversion from mild cognitive impairment to AD only in the high-adjacency group (LLR p=0.009, tau PET in EC p=0.036). These findings identify EC-TI proximity as a novel and anatomically grounded biomarker of AD progression risk. More broadly, they suggest a previously unrecognized biomechanical contribution to the initiation of tau pathology in aging and sporadic AD, opening new avenues for early detection, risk stratification, and mechanistically targeted prevention strategies.

neuroscience↗

bioRxiv2025

The anti-phage mechanism of a widespread trypsin-MBL module

Protease mediate activation of immune effectors is a conserved mechanism across life. This study identified a widespread and co-evolving trypsin-MBL module as a core effector in diverse bacterial immune systems (e.g., Hachiman, Argonaute, AVAST), protecting host via abortive infection. Focusing on Hachiman-trypsin-MBL, we demonstrated that, the protease activity of trypsin*HamAB is inhibited in the presence of ATP and MBL is an autoinhibited DNase with two inserted loops blocking the catalytic pocket. Upon infection, the HamB helicase senses invading nucleic acids and catalyzes the hydrolysis of ATP, activating the associated trypsin-like protease that cleaves specifically at two non-canonical KSS motif of MBL and releases its DNase activity. Cryo-EM structures of activated trypsin*HamAB bound with DNA reveal DNA binding and ATP hydrolysis lead to the release of the trypsin-like domain, enabling activation. Our work provides a comprehensive understanding on the mechanism of widely distributed trypsin-MBL module, wherein proteolytic activation of a toxic nuclease enables robust bacterial immunity while preventing self-toxicity through multi-layered control.

biochemistry↗

bioRxiv2025

Mycobacterium tuberculosis, Mycobacterium kansasii and Rhodococcus equi induce macrophage necroptosis in the presence of a caspase inhibitor acting on a non-canonical target(s)

Macrophages are the predominant cell type infected by Mycobacterium tuberculosis (Mtb) in tuberculosis (TB). Death of Mtb-infected macrophages promotes tissue pathology and releases Mtb to infect other cells, suggesting that inhibiting the death of Mtb-infected macrophages could be an adjunctive treatment of TB. Prospects for such an intervention depend on identifying the molecular pathways leading to cell death. We previously reported that the death of Mtb-infected mouse macrophages in vitro depends on type I interferon (IFN) and that the ensuing upregulation of cis-aconitate decarboxylase (ACOD1; IRG1) contributes to cell death by exacerbating Mtb-induced lysosomal membrane permeabilization. Here we report that death of Mtb-infected primary mouse macrophages in vitro became necroptotic and die faster in the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD) acting on a target other than caspase-8. Macrophages infected with Mycobacterium kansasii and Rhodococcus equi likewise underwent z-VAD-dependent necroptosis. In C57BL/6 mice, which are relatively TB-resistant, we saw no impact of MLKL deficiency on bacterial burden or pulmonary pathology. In contrast, in Sp140-/- mice on the C57BL/6 background, which express high levels of type I IFN after Mtb infection and develop necrotic pulmonary lesions, MLKL-deficiency reduced bacterial burden and pathology after high-dose infection. This report illustrates that off-target action(s) of a caspase-8 inhibitor can switch the cell death pathway to necroptosis in macrophages infected with various Gram-positive pathogens. In turn, this opens the possibility that pathophysiologic circumstances may lead to inhibition of the same target(s) that z-VAD inhibited in our studies. That may be what allows MLKL to exacerbate tuberculosis in mice that are prone to formation of necrotic lesions. Author SummaryTuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), led to about 1.3 million deaths in 2023. The rapid emergence of drug resistance and slow pace of new drug development prompt attention to adjunctive host-directed therapies, an approach that relies on a thorough and detailed understanding of host-Mtb interactions. Mtb infection of macrophages can kill them, promoting tissue pathology and releasing of Mtb to infect other cells. Here we find that a pan-caspase inhibitor z-VAD hastened the death of Mtb-infected macrophages and converted the mode of cell death to RIPK1-RIPK3-MLKL-dependent necroptosis. However, the functional target(s) of z-VAD-FMK and the activation mechanism of RIPK3 differ from the canonical pathways. The pan-caspase inhibitor also promoted rapid, necroptotic death of macrophages infected with Mycobacterium kansasii and Rhodococcus equi. We further found that MLKL-deficiency in Sp140-/- mice on the C57BL/6 background resulted in less weight loss, lower Mtb burdens and mitigation of lung pathology after high-dose Mtb infection. Our findings suggest that necroptosis can contribute to TB pathogenesis in some circumstances. Further identification of the death pathway-switching mechanism could deepen our understanding of host-Mtb interactions and aid in the design of adjunctive host-directed therapies.

microbiology↗

bioRxiv2025

Hierarchical coding of local and global positions in episodic memory for naturalistic sequences

Life unfolds continuously across multiple timescales, yet how the brain encodes hierarchical event sequences remains poorly understood. Using magnetoencephalography, we investigated the neural coding of hierarchical positions in naturalistic sequence memory. Participants viewed video sequences with a nested structure and later recalled their serial order. Behavioral analyses revealed transposition errors for items occupying identical global or local position. Multivariate classification and subspace analyses revealed separable neural representations of global and local positions. Local positions were better decoded in high-dimensional space, whereas global positions exhibited greater pattern separability in low-dimensional space. Brain regions encoding global positions exhibited longer neural timescales and reduced sensitivity to low-level event transitions compared to regions encoding local positions. These findings support a parallel hierarchical organization of content and context, a novel framework that enables integration of item and order across hierarchical levels and provides principles for hierarchical representations in both human memory and artificial intelligence. HighlightsO_LIHierarchical position coding in sequence memory for naturalistic stimuli C_LIO_LIThe neural representations of global and local positions are separable C_LIO_LIGlobal and local position codes are low and high dimensional, respectively C_LIO_LIGlobal position regions have longer timescales than local position regions C_LI

neuroscience↗

bioRxiv2025

Novel Function of Arylalkalamine N-acetyltransfe- rase-1 in Modulating Host Coagulation and Blood Feeding in Aedes aegypti

During blood feeding, mosquitoes introduce substantial quantities of salivary proteins into their hosts to facilitate feeding. Arylalkylamine N-acetyltransferase-1 (aaNAT1) is a salivary protein prominently produced in the salivary glands of Aedes aegypti. Previous research has demonstrated that mosquitoes with AaaaNAT1 mutations fail to efficiently inhibit host blood coagulation; however, the precise regulatory mechanisms remain unknown. This study identified the primary metabolites and proteins interacting with AaaaNAT1 through integrated transcriptomic, proteomic, and metabolomic analyses. Combined with isothermal titration calorimetry (ITC), immunohistochemistry, ELISA, platelet function assays, and coagulation tests, we further established that AaaaNAT1 binds norepinephrine, reducing host platelet aggregation and thereby limiting activation of the host coagulation cascade and fibrinolytic system. Additionally, behavioral investigations showed that AaaaNAT1 knockdown in Aedes aegypti significantly prolonged the time required for female mosquitoes to locate a host and successfully obtain blood, while also markedly reducing blood consumption. This effect may stem from the inability of aaNAT1-depleted mosquitoes to effectively suppress host blood aggregation during feeding, as well as the downregulation of octopamine following AaaaNAT1 knockdown. Therefore, this study reveals a novel role for aaNAT1 distinct from its known functions in pigmentation and immunity, advancing our understanding of aaNAT1s role in insects.

molecular biology↗