Biology subjects

Chen, B.

Publications and source records attributed to Chen, B..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

In situ generated vaccine-like pyroptosome for personalized cancer immunotherapy

The efficacy of in situ cancer vaccination has been hampered by poor spatiotemporal orchestration of multiple key steps of cancer-immunity cycle in most tumours and systemic toxicity related to therapeutic strategies. Here, we report a systemic injectable and pyroptosis-enabled nanoadjuvant (SPEN) that precisely evokes the secretion of vaccine-like pyroptosome in tumour area for eliciting robust anti-tumour immunity. SPEN induces vigorous immunogenic pyroptosis, triggering the efficient release of tumour antigen-rich pyroptosomes, DAMPs, and proinflammatory cytokines. Upon the activatable release of a TLR7/8 agonist into pyroptosome, the generated pyroptosome functions as in situ cancer vaccine for cooperatively activating the cancer-immunity cycle while avoiding systemic toxicity. This in situ vaccine boosts both innate and adaptive immune response, facilitating the eradication of primary tumour and long-lasting cancer prevention. Our findings provide new insights into the rational design of pyroptosis-inducing nanomedicines for boosting the cancer-immunity cycle, thus advancing personalized cancer immunotherapy.

cancer biology↗

bioRxiv2025

KYLO-0603, a novel liver-targeting, thyroid hormone recep-tor-β agonist for the treatment of MASH

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease associated with liver-related complications and death. Kylo-0603 is a novel agonist for the thyroid hormone receptor {beta} (THR-{beta}) that has been developed by merging the structures of three acetylgalactosamine (GalNAc)-modified ASPGR ligands with a triiodothyronine (T3) analog. This unique design allows for both THR-{beta} activation and targeted delivery to hepatocytes, significantly reducing the risk of adversed effects related to increased systemic thyroid hormone-like effects. Additionally, it effectively lowers serum cholesterol by as much as 69.2% and low-density lipoprotein cholesterol (LDL-C) levels by up to 88.2% in the MASH mouse model. Meanwhile, Kylo-0603 was shown to reduce steatosis by up to 1.3 points (P < 0.001), inflammation by 1.8 points (P < 0.0001), and ballooning by 0.8 points (P < 0.01). The non-alcoholic steatohepatitis (NASH) activity score (NAS) decreased by up to 3.7 points (P < 0.0001), and the fibrosis score dropped by 0.6 points (P < 0.05). These findings suggest that Kylo-0603 is effective in enhancing liver tissue NASH status and inhibiting fibrosis progression. In summary, Kylo-0603, as a highly both tissue and target selective and low-toxicity THR-{beta} agonist, shows significant promise for treating MASH and is likely to emerge as a new therapeutic option for patients with this condition.

biochemistry↗

bioRxiv2025

Critical Evaluation of Sphingolipids Detection by MALDI-MSI

The increasing interest in the role of sphingolipids in (patho)physiology has led to the demand for visualization of these lipids within tissue samples (both from animal models and patient specimens) using techniques such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While increasingly adopted, detection of sphingolipids with MALDI-MSI is challenging due to: i) the significant structural variations of sphingolipid molecules, ii) the potential breakdown of the more complex molecules into structurally simpler species which may confound the analysis, and iii) the great difference in levels among sphingolipid classes and subspecies, with the low-abundant ones often being close to the detection limit. In this study, we adopted a multi-pronged approach to establish a robust pipeline for the detection of sphingolipids by MALDI-MSI and to establish best practices and limitations of this technology. First, we evaluated the more commonly adopted methods [2,5-Dihydroxyacetophenon (DHA) or 2,5-Dihydroxybenzoic acid (DHB) matrix in positive ion mode and 1,5-Diaminonaphthalene (DAN) matrix in negative ion mode] using MALDI-MS on reference standards. These standards were used at ratios similar to their relative levels in biological samples to evaluate signal artifacts originating from fragmentation of more complex sphingolipids and impacting low level species. Next, by applying the most appropriate protocol for each sphingolipid class, MALDI-MSI signals were validated in cell culture by modulating specific sphingolipid species using sphingolipid enzymes and inhibitors. Finally, the optimized parameters were utilized on breast cancer tissue from the PyMT mouse model. We report the optimal signal for sphingomyelin (SM) and, for the first time, Sph in DHB positive ion mode (in cells and PyMT tissue), and the validated detection of ceramides and glycosphingolipids in DAN negative ion mode. We document the extensive fragmentation of SM into sphingosine-1-phosphate (S1P) and even more so into ceramide-1-phosphate (C1P) using DAN in negative ion mode and its effect in generating an artifactual C1P tissue signal; we also report the lack of detectable signal for S1P and C1P in biological samples (cells and tissue) using the more suitable DHB positive ion mode protocol.

cell biology↗

bioRxiv2025

Dietary protein source mediates colitis pathogenesis through bacterial modulation of bile acids

Introductory paragraphEvidence-based dietary recommendations for individuals with inflammatory bowel diseases (IBD) are limited. Red meat consumption is associated with increased IBD incidence and relapse in patients, suggesting that switching to a plant-based diet may limit gut inflammation. However, the mechanisms underlying the differential effects of these diets remain poorly understood. Feeding diets containing plant- or animal-derived proteins to murine colitis models revealed that mice given a beef protein (BP) diet exhibited the most severe colitis, while mice fed pea protein (PP) developed mild inflammation. The colitis-promoting effects of BP were microbially-mediated as determined by bacterial elimination or depletion and microbiota transplant studies. In the absence of colitis, BP-feeding reduced abundance of Lactobacillus johnsonii and Turicibacter sanguinis and expanded Akkermansia muciniphila, which localized to the mucus in association with decreased mucus thickness and quality. BP-fed mice had elevated primary and conjugated fecal bile acids (BAs), and taurocholic acid administration to PP-fed mice worsened colitis. Dietary psyllium protected against BP-mediated inflammation, restored BA-modulating commensals and normalized BA ratios. Collectively, these data suggest that the protein component of red meat may be responsible, in part, for the colitis-promoting effects of this food source and provide insight into dietary factors that may influence IBD severity.

microbiology↗

bioRxiv2025

The metabolic enzyme GYS1 condenses with NONO/p54nrb in the nucleus to spatiotemporally regulate glycogenesis and myogenic differentiation

Accumulating evidence indicates that metabolic enzymes can directly couple metabolic signals to transcriptional adaptation and cell differentiation. Glycogen synthase 1 (GYS1), the key metabolic enzyme for glycogenesis, is a nucleocytoplasmic shuttling protein compartmentalized in the cytosol and nucleus. However, the spatiotemporal regulation and biological function of nuclear GYS1 (nGYS1) microcompartments remain unclear. Here, we show that GYS1 dynamically reorganizes into nuclear condensates under conditions of glycogen depletion or transcription inhibition. nGYS1 complexes with the transcription factor NONO/p54nrb and undergoes liquid-liquid phase separation to form biomolecular condensates, leading to its nuclear retention and inhibition of glycogen biosynthesis. Compared to their wild-type littermates, Nono-deficient mice exhibit exercise intolerance, higher muscle glycogen content, and smaller myofibers. Additionally, Gys1 or Nono deficiency prevents C2C12 differentiation and cardiotoxin-induced muscle regeneration in mice. Mechanistically, nGYS1 and NONO co-condense with the myogenic transcription factor MyoD and preinitiation complex (PIC) proteins to form transcriptional condensates, driving myogenic gene expression during myoblast differentiation. These results reveal the spatiotemporal regulation and subcellular function of nuclear GYS1 condensates in glycogenesis and myogenesis, providing mechanistic insights into glycogenoses and muscular dystrophy.

cell biology↗

bioRxiv2025

Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma

Chemotherapy often kills a large fraction of cancer cells but leaves behind a small population of drug- tolerant persister cells. These persister cells survive drug treatments through reversible, non-genetic mechanisms and cause tumour recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies combined with CRISPR knockout and phenotypic drug screen, we identified that chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically, PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol biosynthesis, which persister cells rely on to maintain homeostasis under chemotherapy- induced oxidative stress and lipid peroxidation. PRDM9 inhibition, combined with chemotherapy, resulted in strong anti-cancer efficacy in preclinical glioblastoma models, significantly enhancing the magnitude and duration of the antitumor response by eliminating persisters. These findings demonstrate a previously unknown role of PRDM9 in promoting metabolic reprogramming that enables the survival of drug-tolerant persister cells.

cancer biology↗

bioRxiv2025

Hydrogen-bonded organic framework nanotransducers enabled sono-optogenetics for Parkinsonian rats

Cell-type-specific activation of parvalbumin (PV)-expressing neurons in the external globus pallidus (GPe) through optogenetics has shown promise in facilitating long-lasting movement dysfunction recovery in mice with Parkinsons disease. However, its translational potential is hindered by adverse effects stemming from the invasive implantation of optical fibers into the brain. In this study, we have developed a non-invasive optogenetics approach, utilizing focused ultrasound-triggered mechanoluminescent nanotransducers to enable remote photon delivery deep in the brain for genetically targeted neuromodulation. These mechanoluminescent nanotransducers consist of sonosensitized hydrogen-bonded frameworks and chemiluminescent L012, serving as a nanoscale light source through ultrasound-induced cascade reactions. This system offers high ultrasound-triggered brightness and long-lasting light emission, facilitating repeatable deep brain stimulation. Our sono-optogenetics technology demonstrated effective modulation in the mouse motor cortex for limb motion control and activation of PV-GPe neurons for rescuing movement dysfunction over time in dopamine-depleted Parkinsons disease rats. This approach demonstrates the pathway for achieving genetically targeted and non-invasive neuromodulation for long-lasting treatment of Parkinsons disease, towards non-human primate models and clinical applications.

bioengineering↗

bioRxiv2025

Comparative immuno-biology at clinical recognition of early multiple organ dysfunction syndrome in pediatric and adult patients using single-cell transcriptomics

Globally, sepsis remains a major health issue, with Multiple Organ Dysfunction Syndrome (MODS) being a leading cause of mortality. MODS, a severe condition often seen in intensive care units, typically results from infections or trauma and involves complex pathophysiological processes requiring various clinical interventions. Although infections are the main triggers, the mechanisms driving MODS remain unclear. To investigate the transition of sepsis to MODS, we generated a single cell RNA sequencing dataset comprising 86,839 immune cells from pediatric sepsis patients at the clinical onset of MODS patients and age-matched controls, identifying 22 distinct cell types. A cluster of S100 genes, located in the same genomic region, was highly expressed in neutrophils in MODS patients, demonstrating strong diagnostic potential across cohorts (AUC=0.94- 0.99) and potential as therapeutic targets. We found that many B and T cells showed heightened inflammation and increased apoptotic activity during early MODS. Additionally, specific transcription regulators and surface proteins associated with inflammation and S100 regulations were uniquely expressed in MODS. Pseudotime analysis revealed distinct S100 gene expression patterns between controls and MODS. Cell-cell interaction analysis highlighted dendritic cells as key mediators, enhancing communication between plasma cells and V{delta} T cells while activating inflammatory and immunosuppressive pathways. We also analyzed 116,803 immune cells from adult MODS patients, revealing stronger immune dysregulation compared to pediatric MODS, including altered S100 gene expression, and enhanced cell-cell interactions. These findings suggest that S100 genes may serve as a marker for MODS. Furthermore, insights gained from adult MODS could improve our understanding of rare pediatric MODS and contribute to the development of better therapeutics for all MODS patients.

bioinformatics↗

bioRxiv2024

Increased versatility and convenience: advances and strategy optimization of ReMOT-mediated genetic modification in insects

Genetic modification via gene editing has become a widely adopted and demonstrably effective method in functional gene research within entomology. However, the optimal efficiency and simplicity of delivering exogenous guide RNA-clustered regularly interspaced short palindromic repeats-associated protein 9 (gRNA-Cas9) complexes into target tissues are crucial for successful gene editing. The Receptor-Mediated Ovary Transduction of Cargo (ReMOT) strategy, which simplifies the delivery process, target site selection, technical requirements, and delivery cost compared with embryonic microinjection, enabling efficient editing at the germline level, is gaining increasing attention. Although the feasibility and advantages of this technique have been demonstrated in various insect species, further optimization of operational details and the overcoming of further bottlenecks are still required. This review focuses on advances in developing ReMOT as a valuable technology, exploring its applicability, rationale for selecting the ovary as a delivery target site, factors influencing its efficiency, and recommendations for improvement. The versatility and effectiveness of ReMOT make it a promising method for researchers looking to make precise genetic modifications with greater ease and efficiency.

genetics↗

bioRxiv2024

Enantioselective photoenzymatic C(sp3)-H functionalization via single-electron oxidation of carbanions

Established strategies for enantioselective hydroalkylation for C(sp3)-C(sp3) bond formation usually require pre-functionalized substrates as radical precursors in both transition-metal and photoenzymatic catalysis. Based on a sequential proton transfer/electron transfer (PT/ET) strategy, here we show a cooperative photoenzymatic system consisting of a flavin-dependent ene-reductase (ER) and an organophotoredox catalyst fluorescein (FI) to achieve atom-economic enantiodivergent hydroalkylation of electron-deficient C(sp3)-H with olefins. Mechanistic studies revealed a pathway for radical intermediate formation via excited-state FI*-induced single-electron oxidation of carbanions under alkaline conditions. The overall catalytic efficiency is enhanced by the electron transfer (ET) between FMNox and FI-*, while the stereoselectivity is controlled by ERs through enantioselective hydrogen atom transfer (HAT). We anticipate that this mode of photoenzymatic catalysis will inspire new pathways for generating free radical intermediates and foster innovative strategies for achieving photoenzymatic new-to-nature reactions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/627421v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@11b6c1eorg.highwire.dtl.DTLVardef@1575346org.highwire.dtl.DTLVardef@1c24a2org.highwire.dtl.DTLVardef@1a93c0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Drip irrigation suppresses methane emissions by reshaping the rhizosphere microbiomes in rice

The rhizosphere microbiomes of rice plants under conventional flood irrigation consist of highly complex consortia of microorganisms and in particular methanogens purportedly associated with methane emissions therein. Controlled irrigation has been proposed as a cultivation method of choice over continuous flooding to reduce water and fertilizer usage in an aerobic environment. However, a systemic understanding of the assembly and function of microbiota in the rhizosphere under drip and flood irrigation remains unclear. Using empirical analyses, we report a significant reduction in methane emissions in controlled irrigation compared to the flooded environment. Genotypic or varietal differences did not influence such methane emissions under conventional flooded cultivation of rice. Using metagenomic sequencing and computational analyses, we provide a deeper understanding of how drip irrigation or continuous flooding affect the root-associated microbiomes in rice. Rhizosphere soil from two different rice varieties, Huanghuazhan and Temasek rice, grown under drip or flood conditions in a greenhouse was collected over 2 months post-transplantation for metagenomic analysis. Our results reveal that drip irrigation favours microbes involved in the nitrifying-denitrifying processes while continuous flooding enriches for methanotrophs and methanogenic archaea. Syntrophic microbiomes associated with methanogenesis were significantly reduced in drip irrigation. Several keystone taxa were evident in the co-occurrence network model related to methanogenic, methanotrophic, nitrifying, sulphur-oxidising and sulphur-reducing activities. Lastly, oxygen availability and redox potential were identified as key drivers that reshape rhizosphere microbiota and the associated metabolic functional differences observed between the two irrigation regimes leading up to the microbial mitigation of climate impact. IMPORTANCEUnlike previous studies in alternate wet-dry irrigation systems, this study characterised the rice microbiomes in a controlled drip irrigation settings where water levels were maintained at low levels and soil remained unflooded throughout the entire season in a greenhouse. A reduction of more than 90% in methane emissions was observed with drip irrigation compared to flood irrigation. Significant correlation was found between levels of methane emitted and mcrA gene copies detected with a Pearson correlation coefficient R of 0.77 and p-value of 2.3e-10. Methanogens are highly abundant in continuous flooded rice soil and are significantly reduced in drip-irrigated soil. Metagenomic profiling indicates that the shifts in microbial diversity under drip irrigation favour nitrifying microorganisms and is likely influenced by increased oxygen availability due to higher soil redox potential.

microbiology↗

bioRxiv2024

Cardiolipin-mimic lipid nanoparticles without antibody modification delivered senolytic in-vivo CAR-T therapy for inflamm-aging

mRNA-based in vivo CAR T cell engineering offers advantages over ex vivo therapies, including streamlined manufacturing and transient expression. However, current delivery requires antibody-modified vehicles with manufacturing challenges. In this study, inspired by cardiolipin, we identified a cardiolipin-like di-phosphoramide lipid that improved T cell transfection without targeting ligands, both in vivo and in vitro. The T cell-favored tropism is likely due to the lipids packing, shape, and rigidity. Encapsulating circular RNA further prolonged mRNA expression in the spleen and T cells. Using PL40 lipid nanoparticles, we delivered mRNA encoding a CAR targeting the senolytic and inflammatory antigen urokinase-type plasminogen activator receptor (uPAR), alleviating uPAR-related liver fibrosis and rheumatoid arthritis (RA). Single cell sequencing in humans confirmed uPARs relevance to senescence and inflammation in RA. To further enhance clinical translation, we screened and humanized scFvs against uPAR, establishing PL40 mRNA encoding a circular human uPAR CAR, with potential for treating aging-inflamed disorders. One Sentence SummaryWeve developed a unique class of Cardiolipin-mimic lipids that facilitate mRNA delivery to T cells in vivo without the need for antibody modification, enhancing the treatment of liver fibrosis and rheumatoid arthritis through circular CAR uPAR RNA and propelling the clinical application of humanized CAR against human uPAR.

bioengineering↗

bioRxiv2024

Multi-purpose controllable protein generation via prompted language models

Deep learning is increasingly powerful for designing proteins that meet structural and functional requirements. However, most existing methods follow a conventional pipeline: first defining a backbone structure and then generating sequences consistent with it. This approach, which encodes all design goals indirectly through structures, restricts flexibility and struggles to address multiple, complex design objectives simultaneously. We present PROPEND, a multi-purpose protein sequence design method based on the "pre-train and prompt" framework. We show PROPENDs broad utility and accuracy both in silico and in vitro by directly controlling multiple properties through the prompt of backbones, blueprints, functional tags, and their combinations. For the five sequences tested with in vitro experiments, PROPEND achieved a maximum functional recovery of 105.2%, significantly outperforming the classical design pipelines 50.8%.

synthetic biology↗

bioRxiv2024

Activation of hypothalamic-pontine-spinal pathway promotes locomotor initiation and functional recovery after spinal cord injury in mice

The hypothalamus is critical for regulating behaviors essential for survival and locomotion, but how it integrates internal needs and transmits locomotion commands to the spinal cord (SC) remains unclear. We found that glutamatergic neurons in lateral hypothalamic area (LHA) are essential for regulating motivated locomotor activity. Using single-neuron projectome analysis, trans-synaptic tracing, and optogenetic manipulation, we showed that LHA facilitates motivated locomotion during food seeking via pontine oral part (PnO) projection neurons, rather than direct SC projections or indirect stress signaling via medial septum and diagonal band. Activating PnO-SC projection neurons also initiated locomotion. Importantly, LHA-PnO projection neurons were crucial for regulating locomotor recovery following mouse spinal cord injury (SCI). Closed-loop deep brain stimulation (DBS) of LHA via gating by motor cortex signals markedly promoted long-term restoration of hindlimb motor functions after SCI. Thus, we have identified a hypothalamic-pontine- spinal pathway and the stimulation paradigm for potential therapeutic intervention after SCI.

neuroscience↗

bioRxiv2024

Heat Acclimation in Mice Requires Preoptic BDNF Neurons and Postsynaptic Potentiation

Heat acclimation (HA) is a key adaptive response in mammals to repeated heat exposure, essential for fitness and survival1-3. HA improves cardiovascular function, thermal comfort, and exercise capacity4, 5. However, the lack of a genetically tractable model has hindered understanding of the molecular and neural mechanisms underlying HA. Here, we show that 10 days of daily 38{degrees}C exposure lowers core body temperature (Tcore) and reduces anxiety during subsequent heat exposures in mice. HA increases brain-derived neurotrophic factor (BDNF) expression in the medial preoptic area (MPO). BDNF-expressing MPO neurons (MPOBDNF) show increased intrinsic heat sensitivity after HA. These neurons orchestrate downstream targets in the dorsomedial hypothalamus (DMH) and rostral raphe pallidus (rRPa) to mediate HA effects. BDNF, acting through its receptor tropomyosin-related kinase B (TrkB) in the DMH, facilitates the anxiolytic effect of HA by enhancing excitatory synaptic connections between MPOBDNF and DMH neurons. This study provides new insights into HA mechanisms, setting the stage for future research on heat stress reduction and exercise optimization.

neuroscience↗

bioRxiv2024

H2S remodels mitochondrial ultrastructure and destabilizes respiratory supercomplexes

Mitochondrial form and function are intimately interconnected, responding to cellular stresses and changes in energy demand. Hydrogen sulfide, a product of amino acid metabolism, has dual roles as an electron transport chain substrate and complex IV (CIV) inhibitor, leading to a reductive shift, which has pleiotropic metabolic consequences. Luminal sulfide concentration in colon is high due to microbial activity, and in this study, we demonstrate that chronic sulfide exposure of colonocyte-derived cells leads to lower Mic60 and Mic19 expression that is correlated with a profound loss of cristae and lower mitochondrial networking. Sulfide-induced depolarization of the inner mitochondrial membrane activates Oma1-dependent cleavage of Opa1 and is associated with a profound loss of CI and CIV activities associated with respirasomes. Our study reveals a potential role for sulfide as an endogenous modulator of mitochondrial dynamics and suggests that this regulation is corrupted in hereditary or acquired diseases associated with elevated sulfide. Significance StatementHydrogen sulfide is a product of host as well as gut microbial metabolism and has the dual capacity for activating respiration as a substrate, and inhibiting it at the level of complex IV. In this study, we report that chronic albeit low-level sulfide exposure elicits profound changes in mitochondrial architecture in cultured human cells. Disruption of mitochondrial networks is reversed upon removal of sulfide from the growth chamber atmosphere. Sulfide-dependent depolarization of the inner mitochondrial membrane is associated with loss of cristae and respiratory supercomplexes. Our study reveals the potential for sulfide to be an endogenous regulator of mitochondrial ultrastructure and function via modulation of electron flux and for this process to be corrupted in sulfide dysregulated diseases.

cell biology↗

bioRxiv2024

Connectomic Organization of the Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN), the mammalian master clock, is a special structure dedicated to the time computation. However, a connectomic understanding of the nucleus as a whole is lacking. Using serial section electron microscopy, we reconstructed multiscale and multimodal SCN communication networks. Intra-SCN synaptic network consists of 9,566 morphologically similar neurons and 4.3 million synapses, organized into multiscale circuitries that interact with SCN-traversing axon fascicles. Strikingly, SCN neurons engage in unique soma-soma ephaptic interaction, forming 2,038 electrotonic integrative units with the largest overlapping the light-responsive area (LRA). SCNs paracrine modality contains 47,396 m3 dense core vesicles, with notable scarcity in the LRA, suggesting cross-modal coordination and functional integration. These distinct network features provide comprehensive insights into the system architecture of the mammalian circadian clock.

neuroscience↗

bioRxiv2024

DEMINERS enables clinical metagenomics and comparative transcriptomic analysis by increasing throughput and accuracy of nanopore direct RNA sequencing

Nanopore direct RNA sequencing (DRS) advances RNA biology but is limited by relatively low basecalling accuracy, low throughput, yet high RNA input and costs. Here we introduce a novel DRS toolkit, DEMINERS, which integrates an RNA multiplexing experimental workflow, a machine-learning barcode classifier based on Random Forest and a novel basecaller built on an optimized convolutional neural network providing an additional species-specific training module. With the increased accuracy in barcode classification and basecalling, DEMINERS can demultiplex up to 24 samples and the required RNA input and running time are both substantially reduced. We demonstrated the applications of DEMINERS in clinical metagenomics, cancer transcriptomics and parallel comparison of transcriptomic features in different biological conditions, revealing altered airway microbial diversity in COVID-19 and a potential role of m6A in increasing transcriptomic diversity in glioma and the mature blood-stage of malaria parasites. Overall, DEMINERS is a simple, robust, high-throughput DRS method for accurately estimating transcript levels, poly(A) lengths, and mutation and RNA modification heterogeneity at single-read level, with minimal sequencing biases.

bioinformatics↗