Biology subjects

Ding, X.

Publications and source records attributed to Ding, X..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Transient Poly(ADP-Ribose) Triggers FUS Condensation Hysteresis via a Prion-Like Mechanism

Hysteresis--where a system retains memory of a transient stimulus--is common in signaling but can also arise in intracellular organization. DNA repair foci, a type of biomolecular condensate, are initiated by the short-lived noncanonical nucleic acid poly(ADP-ribose) (PAR). PAR recruits proteins with prion-like domains (PrLDs), such as Fused in Sarcoma (FUS), and initiates their condensation, which persists even after PAR degradation. How FUS transitions from PAR-dependent to PAR-independent condensation remains unclear. Here, we show that PAR binding triggers a conformational switch in FUS, enabling sustained condensation. PAR binds to the C-terminal arginine-rich region of FUS, displacing intramolecular contacts, and exposing the N-terminal PrLD. This conformational opening allows PrLD interactions in trans, stabilizing condensates independently of PAR. FUS thus undergoes a regulated, nucleated conformational conversion--reminiscent of classical prions. This mechanism implies a paradigm of nucleic acid-induced conformational memory that may underlie hysteresis in intracellular organization in health and disease. HIGHLIGHTS[bullet] PAR-initiated FUS condensation follows a bi-modular mechanism involving both FUS termini. [bullet]Simulations predict and experiments confirm PAR disrupts FUS intramolecular contacts. [bullet]Upon condensation, FUS adopts a conformation with its N-terminus open for interactions. [bullet]N-terminal interactions maintain FUS condensation when PAR degrades during DNA repair.

molecular biology↗

bioRxiv2025

Modular Inter-brain Synchrony Network Associated with Social Difficulty in Autism Spectrum Disorder: a Graph Neural Network-Driven Hyperscanning Study

Understanding social difficulties in Autism Spectrum Disorder (ASD) remains challenging due to its neurobiological heterogeneity and the limited ecological validity of conventional neuroimaging methods in capturing dynamic social interactions. Hyperscanning analysis based on functional near-infrared spectroscopy (fNIRS), which measures inter-brain synchrony (IBS) during dyadic interaction, offers a novel avenue to address these challenges. However, prior studies on ASD have reported inconsistent findings, primarily focusing on intra-regional synchronization while overlooking cross-regional network dynamics. To bridge this gap, we proposed an interpretable graph neural network (GNN) model to systematically identify ASD-specific IBS modular network between child-caregiver dyads during naturalistic cooperative puzzle-solving and free-talking tasks. We identified distinctive key IBS sub-networks for the cooperative puzzle-solving task and free-talking task, with the frontal eye field (FEF) of caregivers, the dorsal lateral prefrontal cortex (DLPFC) and the motor region of children highlighted. Furthermore, the key IBS sub-networks were found to be able to predict multiple domains of the core ASD symptoms. By integrating hyperscanning with GNN-driven analysis, this work uncovers task-dependent inter-brain neural mechanisms underlying social difficulties in ASD. These findings advance the field by proposing a data-driven framework to identify IBS biomarkers tied to clinical profiles, paving the way for personalized interventions that integrate computational neuroscience with clinical practice.

neuroscience↗

bioRxiv2025

Developing a versatile technology for agroinfiltration in multiple plants

Agrobacterium-mediated transient transformation methods are widely used in plant science and molecular pharming. However, plant immune response and post-transcriptional gene silencing (PTGS) can compromise gene expression, limiting the adaptability of these methods across plant species. The lack of a robust reporter system further hinders advancements in agroinfiltration technology. To address these challenges, we developed a fungal bioluminescence pathway (FBP)-based reporter system that utilizes caffeic acid to generate self-sustained luminescence. By incorporating NahG and P19, our FBP assay enhances transient transformation efficiency in over 20 plant families, including major crops, vegetables, ornamental plants, and trees. Furthermore, we demonstrate its utility for assessing protein localization, interactions, and transcriptional regulation in multiple plant species, thereby broadening our understanding of gene function throughout the plant kingdom.

plant biology↗

bioRxiv2025

Diverse activity in prefrontal projections promotes temporal control of action

Prefrontal neurons can have diverse activity during cognitive functions like working memory, attention, and timing; however, the importance of this heterogeneity is unclear. Our goal was to better understand the diversity of prefrontal activity through connectivity. We harnessed circuit-specific tools to capture activity within prefrontal projections during interval timing, an elementary cognitive process that requires working memory for temporal rules and attention to the passage of time to estimate a temporal interval of several seconds. We used human electroencephalography and single neuronal recordings in mice to capture prefrontal activity during interval timing, with major patterns characterized by time-dependent ramping (monotonic changes) over a temporal interval. We then leveraged retrograde viruses to interrogate prefrontal cortex (PFC) projections to the mediodorsal thalamus (PFC-MD) and to the dorsomedial striatum (PFC-DMS). We report three novel results. First, circuit-specific calcium fiber photometry revealed that PFC-MD and PFC-DMS activity encoded distinct temporal signals, with PFC-MD projections ramping down and PFC-DMS ramping up to interval timing response times. Second, circuit-specific inactivation revealed that PFC-DMS inactivation disrupted animals internal estimates of time. Third, circuit-specific single-nucleus RNA sequencing of prefrontal projections revealed distinct transcriptomic profiles between PFC-MD and PFC-DMS projections, with enriched genes for cortical layers and neuromodulators, and specific genes such as Cux2, Camk2n1, Htr4, and Foxp2. These data suggest differences in gene expression and connectivity give rise to the diversity of prefrontal activity during interval timing. These findings advance our fundamental understanding of prefrontal function and dysfunction in human disease.

neuroscience↗

bioRxiv2025

The Drosophila G protein-coupled receptor, GulpR, is essential for lipid mobilization in response to nutrient-limitation

Enteroendocrine cells (EECs) of the intestinal epithelium are major regulators of metabolism and energy homeostasis. This is mainly due to their expression and secretion of enteroendocrine peptides (EEPs). These peptides serve as hormones that control many aspects of metabolic homeostasis including feeding behavior, intestinal contractions, and utilization of energy stores. Regulation of EEP production and release depends largely on EEC-exclusive G protein-coupled receptors (GPCRs) that sense nutrient levels. Here we report the identification of a GPCR expressed principally in EECs, which we have named GulpR due to its role in the response to nutrient stress. We show that GulpR regulates transcription of the EEP Tachykinin (Tk) and that both GulpR and Tk are essential for the transcriptional response that promotes survival of nutrient limitation. Infection with V. cholerae also activates transcription of Tk and lipid mobilization genes. While GulpR is required for activation of Tk transcription during infection, Tk does not play a role in regulation of lipid mobilization genes or survival of infection. Our findings identify a role for GulpR and Tk in survival of starvation and suggest that, although starvation and infection both require significant mobilization of energy stores, the signal transduction systems that regulate the metabolic response to each are distinct. Author Summary.Humans and other animals, including Drosophila, metabolize dietary nutrients such as sugars, lipids, and proteins into polysaccharides, fatty acids, and amino acids, respectively, to generate energy that fuels essential cellular processes like cell division, ion transport, muscle contraction, and more. The ability to adapt to changes in nutrient availability and energy demand is therefore crucial for homeostasis and survival. Nutrient scarcity during starvation and an increased demand for energy during an immune response against pathogenic infection require utilization of the bodys own lipid and glycogen stores. This adaptive response largely relies on the ability of the intestine to sense and respond to a variety of stimuli, including microbes and dietary nutrients. Here, we have identified and characterized a Drosophila melanogaster receptor that is expressed in a rare intestinal cell type. We report that this receptor regulates production of peptide hormones that are known to impact metabolic homeostasis and discover that one of these peptide hormones is crucial for utilization of systemic lipid stores when flies experience starvation but not infection stress. Our findings therefore indicate that activation of lipid mobilization in response to nutrient limitation and infection are regulated via different mechanisms.

genetics↗

bioRxiv2025

Vibrio cholerae colonization of the arthropod intestine activates innate immune signaling in enteroendocrine cells via phosphorylation of the nuclear receptor ultraspiracle

The Gram-negative rod Vibrio cholerae causes profuse diarrhea in humans and is found in close association with both terrestrial and aquatic arthropods in the environment. We have previously shown that V. cholerae colonizes the arthropod intestine. Here we show that tryptophan produced by V. cholerae in the arthropod intestine is used by enterocytes to synthesize serotonin and signal to enteroendocrine cells (EECs) to activate the TNF-like immune deficiency pathway IMD. We define the EEC serotonin signaling pathway, which involves a subset of serotonin G protein-coupled receptors, Gq, phospholipase C, and protein kinase C. This pathway culminates in phosphorylation and potentiation of the nuclear receptor ultraspiracle, which binds the sex hormone ecdysone to activate IMD signaling. While IMD signaling increases antimicrobial peptide synthesis in all intestinal cell types, IMD signaling in EECs uniquely activates expression of the enteroendocrine peptide Tachykinin and the V. cholerae colonization factor Peritrophin-15a. We propose that, because V. cholerae secretes a metabolite that activates IMD signaling in EECs, it has evolved to exploit the arthropod intestinal innate immune response to maximize adhesion to the arthropod intestine.

microbiology↗

bioRxiv2025

The role of inhibitory neurons in novelty sound detection in regular and random statistical contexts.

Detecting statistical regularities in sound and responding to violations of these patterns, termed deviance detection, is a core function of the auditory system. In the human brain, studies have shown that deviance responses are enhanced in regular compared to random auditory contexts, but the underlying neuronal circuit mechanisms remain unclear. Here, we examined how inhibitory neurons contribute to context-dependent deviance responses in mouse auditory cortex (AC). Using two-photon calcium imaging in AC of awake head-fixed male and female mice, we recorded neuronal activity during presentation of spectro-temporally rich moving ripple sounds, with deviant ripples embedded in either regular or random ripple sequences. AC neurons exhibited stronger responses to deviant sounds in regular contexts compared to random ones. To identify circuit mechanisms, we selectively inactivated parvalbumin (PV), somatostatin (SST), or vasoactive intestinal polypeptide (VIP) inhibitory neurons during the deviant stimulus presentation. Inactivation of PV and SST neurons broadly increased deviance responses in both contexts. In contrast, VIP inactivation selectively reduced responses to deviant stimuli in the regular, but not random, context, decreasing the context-dependent deviance signal enhancement. At the population level, inactivating all three neuronal subtypes increased detectability of the deviant stimulus, but the effects were context-dependent only for VIP inactivation. These findings reveal a distinct role for VIP neurons in modulating deviance signals based on context regularity, identifying a specific inhibitory neuron type that is critical for context-sensitive auditory processing and predictive coding. SIGNIFICANCE STATEMENTUnderstanding how the brain detects and responds to predictable and unexpected sounds is critical for communication and navigation in complex acoustic environments. Although prior studies have shown that detection of novel sounds is enhanced by regularity in background sound patterns, the underlying circuit mechanisms have remained unknown. Here, we find that neurons in the auditory cortex exhibit stronger deviance responses when the deviant sounds are embedded in regular, predictable sound sequences as compared to random sound sequences. Importantly, we identify a distinct role for specific inhibitory neurons in modulating this context-dependent enhancement. These findings reveal a new insight into how cortical circuits implement predictive coding strategies to optimize sensory processing.

neuroscience↗

bioRxiv2025

A unified eIF1A+ luminal cells-centered hypoxic and "cold" tumor microenvironment promotes PCa progression among different subtypes

PCa is a malignancy with high heterogeneity arises from both tumor microenvironment (TME) and histological subtypes. To achieve superior clinical efficacy, it is imperative to identify unified progression drivers within such heterogeneity in PCa, thereby enabling the development of more consistent and effective therapeutic strategies. In this study, we applied imaging mass cytometry to stain 39 proteins on 71 tissues comprising para-cancer, low-grade acinar adenocarcinoma (LgPAC), high-grade PAC (HgPAC), intraductal carcinoma (IDC) and ductal adenocarcinoma (DAC) tissues, obtaining the spatial proteomic landscape of 345,233 single cells. We discovered a hypoxic eIF1A+ luminal epithelial (LE) cluster exhibiting both translational activation and immunosuppressive properties, which was enriched in high-risk PCa including HgPAC, IDC and DAC, and associated which tumor progression and unfavorable prognosis. Additionally, eIF1A+ LE orchestrates the formation of an immunologically "cold" TME, characterized by the low infiltration of anti-cancer immune cells including PD1- T cells CD163- macrophages. Through in vivo validation and investigator-initiated study, we further found that Homoharringtonine, an inhibitor targeting protein translation process, effectively inhibited PCa growth, alleviated hypoxic microenvironment, and enhanced immune cell infiltration. This study is the first to apply spatial proteomics to delineate consistent molecular features across histological subtypes, providing transformative insights for clinical management of PCa.

cancer biology↗

bioRxiv2025

A pennycress transparent testa 8 knockout mutant has drastic changes in seed coat anatomy and chemical compositions

Pennycress is a winter annual intermediate crop with approximately 30% seed oil content suitable for producing biofuels. Here, we evaluated seed development, anatomy, and agronomically relevant traits of a transparent testa 8 knockout mutant (tt8-2bp) generated by CRISPR genome editing to improve seed quality. We performed histochemical analyses on wild-type and tt8-2bp seeds at different developmental stages. No visible anatomical defects were observed in embryos and endosperm of tt8-2bp seeds. However, tt8-2bp seed coats completely lost proanthocyanidins which were accumulated in an inner integument cell layer and in the thickened cell wall of an outer integument cell layer of wild-type seed coats. Based on spatial metabolomic and solid-state NMR analyses, tt8-2bp seed coats had decreased aromatic compounds and cell wall polysaccharides compared to wild-type seed coats. Additionally, tt8-2bp seeds had reduced seed coat dry weights and increased embryo dry weights compared to wild-type seeds, indicating changes in macronutrient partitioning during seed development. Mature tt8-2bp seeds exhibited increased imbibition rates and seed coat permeability to water-soluble molecules, suggesting a higher seed coat hydrophilicity than wild-type seeds. In conclusion, we did not find defects in tt8-2bp mutant seeds that were unfavorable agronomically, supporting that TT8 is an attractive target for pennycress domestication. HighlightHistochemical analyses of pennycress seeds revealed a complete loss of proanthocyanidins in tt8-2bp seed coats accompanied by increased seed imbibition rates and seed coat permeability compared to wild-type seeds.

plant biology↗

bioRxiv2025

Kidney cortex macrophages prevent the formation of mineral stones by reabsorbing phosphate from the urine

Resident macrophages are important in maintaining tissue homeostasis by meeting specialized physiological demands and mitigating the unique stresses each tissue endures. In the kidney, urine is formed in a process of glomerular filtration and tubular reabsorption. Supersaturation of mineral solutes such as calcium phosphate poses a persistent challenge to the kidney; if left unchecked, kidneys stones form, which will cause tubular obstruction and even kidney failure. However, cellular mechanisms of preventing kidney stone formation remain incompletely understood. In this study, we found that resident macrophages in kidney cortex distinguishably expressed phosphate transporter SLC34A1. These cells extend transtubular protrusions and were capable of reabsorbing phosphate from the urine through the SLC34A1, a function previously only attributed to tubular epithelial cells. Depletion of Slc34a1 from kidney cortex macrophages led to increased urinary phosphate excretion, disrupting body phosphorus balance. More importantly, loss of macrophage-mediated phosphate reabsorption from the urine resulted in a drastic over-deposition of calcium phosphate microcrystals in kidney tubules, particularly in the late proximal tubules of inner cortex where intratubular calcium concentration is high. This also accelerated mineral stone formation. Exposure to mineral crystals stimulated cortex macrophages to upregulate SLC34A1 expression, a response mechanistically driven by lysosomal disruption. As such, cortex macrophages employed phosphate reabsorption as a proactive strategy to prevent mineral stone formation.

immunology↗

bioRxiv2025

Cytosolic transport of citrate protects nutrient-austere pancreatic cancer from ferroptosis

Pancreatic cancer (PDAC) cells experience nutrient starvation in a poorly perfused tumor microenvironment. Metabolic dependencies that protect PDAC cells from detrimental oxidative stress in a nutrient-restricted niche represent as tumor-specific targets. While the role of mitochondria in supporting energy production and biosynthetic requirements of cells has been well investigated, their contribution to maintaining intracellular redox homeostasis when PDAC cells are exposed to nutrient deprivation is unknown. Our results demonstrate that cytosolic transport of citrate via SLC25A1 confers a survival advantage to PDAC cells by protecting them from ferroptosis, a well-established iron-dependent cell death mechanism, under nutrient-limited conditions. Employing selective SLC25A1 inhibitor or targeting mitochondrial OXPHOS dramatically reduced GPX4 expression and PDAC cell viability. Rescuing GPX4 expression with the products of both ACLY and ACO1-dependent pathways uncovered their critical role in conferring survival advantage under metabolic stress. Importantly, exogenous expression of GPX4 reversed redox imbalance and metabolic discordance resulting from the lack of SLC25A1 activity, indicating the requirement of citrate-induced GPX4 expression to support mitochondrial health and function. As observed with cultured cells under nutrient limitation, SLC25A1 function was revealed to be indispensable in pancreatic tumor microenvironment, and the reduced growth, due to the lack of SLC25A1 activity, was rescued with antioxidant NAC in preclinical models of PDAC. Lastly, SLC25A1 suppression was accompanied by elevated glutamine metabolism, and combination therapy with pharmacologic inhibitors of SLC25A1 and glutaminase inhibitor CB-839 dramatically suppressed tumor growth, highlighting this combinatorial approach as a potential therapeutic strategy in PDAC.

cancer biology↗

bioRxiv2025

Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo

Understanding how cells control their biophysical properties during development remains a fundamental challenge. While cytoplasmic macromolecular crowding affects multiple cellular processes in single cells, its regulation in living animals remains poorly understood. Using genetically encoded multimeric nanoparticles for in vivo rheology, we discovered that C. elegans tissues maintain distinct cytoplasmic biophysical properties that differ from those observed across diverse systems, including bacteria, yeast species, and cultured mammalian cells. We identified two conserved mechanisms controlling cytoplasmic macromolecular diffusion: ribosome concentration, a known regulator of cytoplasmic crowding, works in concert with a previously unknown function for the giant KASH protein ANC-1 scaffolding the endoplasmic reticulum. These findings reveal mechanisms by which tissues establish and maintain distinct cytoplasmic biophysical properties, with implications for understanding cellular organization across species. One-Sentence SummaryLiving tissues maintain unique intracellular biophysical properties under the control of cytoplasmic constraints and crowding.

biophysics↗

bioRxiv2025

Flaviviruses Hijack HuR to Suppress CNPY3: A Novel Pathway for Immune Evasion and Therapeutic Intervention

Flaviviruses represent a serious global health threat, infecting millions annually with high rates of cross-infection among different flaviviruses, yet no broad-spectrum antiviral therapy currently targets these pathogens. Through biological big data analysis, we observed a significant downregulation of Canopy FGF Signaling Regulator 3 (CNPY3), a positive regulator of defense responses, in flavivirus-infected patients, with the level of downregulation correlating with infection severity. This observation was validated in cellular and animal models. Mechanistically, we demonstrate that flaviviruses hijack the RNA-binding protein Human antigen R (HuR) to destabilize CNPY3 mRNA via adenine-uridine-rich elements (AREs), thus downregulating CNPY3 expression and promoting viral replication. Our findings identify CNPY3 as a novel antiviral mediator essential for the TLR-mediated type I interferon (IFN-I) pathway in defense against flaviviruses. In vitro, CNPY3 overexpression reduced DENV and ZIKV replication, confirming its antiviral role. To explore therapeutic potential, we developed lipid nanoparticles (LNPs) encapsulating CNPY3 mRNA (LNP-CNPY3), and early administration in flavivirus-infected mice improved survival, reduced viral loads, and attenuated neuroinflammation by enhancing antiviral and interferon responses. Together, these findings establish CNPY3 as a critical antiviral target and support LNP-CNPY3 as a promising therapeutic approach against flavivirus infections.

microbiology↗

bioRxiv2024

D-glucuronyl C5-epimerase binds to EGFR to suppress kidney fibrosis

Renal tubular cells may actively participate in fibrosis processes leading to end-stage renal failure. However, which molecule play pivotal role in the fibrogenesis is still vague. Glucuronyl C5-epimerase (Hsepi, gene name, Glce) is a key enzyme that catalyzes biosynthesis of Heparan sulfate (HS) chains attached to HS proteoglycan which are ubiquitously located on cell membrane. Homozygous Glce-/- mice may cause embryonic lethality and multi-organ defects. However, whether Glce plays a key role in kidney fibrosis is unknown. Here, we show that Glce expression is significant attenuated in kidneys of patients with renal fibrosis and the animal models. Further study shows that renal tubular-specific Glce deletion in mice exacerbate kidney fibrosis while AAV-mediated overexpressing of Glce in UUO-treated mice may ameliorate kidney fibrosis associated with epithelial-mesenchymal transition (EMT) progress via the TGF-{beta}/Smad2/3 signaling pathway. Mechanism study demonstrates that Glce protein may bind to EGFR to inactivate EGFR/ERK signaling and further impede TGF-{beta}/Smad-driven EMT and renal fibrosis in Glce-/- and the wild type mice. Interestingly, the anti-fibrosis function is independent of Glce enzymatic activation. These data uncover a novel function for Glce which plays a key role in kidney tissues against fibrosis.

pathology↗

bioRxiv2024

In vivo engineered quadrivalent CAR-macrophages break tumor barrier,remodel TME and overcome tumor heterogeneity through antigen spreading

CAR-macrophages have shown promising prospect in treating solid tumors. Ex vivo engineered CAR-macrophages face the challenge of low transfection efficiency, long preparation cycle, and limited cell number. Here, we designed two novel CAR molecules, GPC3-CAR-Super IL-2 and FAP-CAR-{bigtriangleup}TGF{beta}RII from the perspective of targeting tumor cells and improving tumor microenvironment, and generated quadrivalent CAR-macrophages in vivo for treating solid tumors by the LNP-mRNA system. We found that in vivo engineered quadrivalent CAR-macrophages can strongly activate tumor immunity and achieve complete tumor regression without significant side effects. Mechanically, in vivo engineered quadrivalent CAR-macrophages broke down physical barriers around the tumor constructed by CAFs and significantly promoted infiltration and expansion of CD8+ T cells. Moreover, the transiently formed CAR-macrophages in vivo are sufficient to form long-lasting T cell memory which can effectively prevent tumor recurrence. Most importantly, in vivo engineered CAR-macrophages also stimulated T cell memory against antigen-negative tumor cells through antigen spreading, which might effectively prevent the immune escape of heterogeneous tumor cells. Overall, we developed a platform of in vivo CAR-macrophages with dual roles as a tumor-killing effector cell and a recurrence-preventing vaccine.

bioengineering↗

bioRxiv2024

Adaptation of retinal discriminability to natural scenes

Sensory systems discriminate between stimuli to direct behavioral choices, a process governed by two distinct properties - neural sensitivity to specific stimuli, and neural variability that importantly includes correlations between neurons. Two questions that have received extensive investigation and debate are whether visual systems are optimized for natural scenes, and whether correlated neural variability contributes to this optimization. However, the lack of sufficient computational models has made these questions inaccessible in the context of the normal function of the visual system, which is to discriminate between natural stimuli. Here we take a direct approach to analyze discriminability under natural scenes for a population of salamander retinal ganglion cells using a model of the retinal neural code that captures both sensitivity and variability. Using methods of information geometry and generative machine learning, we analyzed the manifolds of natural stimuli and neural responses, finding that discriminability in the ganglion cell population adapts to enhance information transmission about natural scenes, in particular about localized motion. Contrary to previous proposals, correlated noise reduces information transmission and arises simply as a natural consequence of the shared circuitry that generates changing spatiotemporal visual sensitivity. These results address a long-standing debate as to the role of retinal correlations in the encoding of natural stimuli and reveal how the highly nonlinear receptive fields of the retina adapt dynamically to increase information transmission under natural scenes by performing the important ethological function of local motion discrimination.

neuroscience↗

bioRxiv2024

Airway applied IVT mRNA vaccine needs specific sequence design and high standard purification that removes devastating dsRNA contaminant

The development of next-generation mucosal mRNA vaccines is promising but extremely challenging. Major efforts have been focused on optimizing delivery systems, whereas it is still unknown whether the intrinsic quality of IVT mRNA significantly impacts the potency of airway inoculated mRNA vaccines. Here, we systematically demonstrate the mucosal mRNA vaccine requires a higher standard of purification and tailor-designed sequence to fulfil its potency compared to the parenteral route inoculated counterpart. We found double strand RNA (dsRNA) contaminants are prone to trigger innate immunoreaction in the airway that activates the mRNA degradation mechanism, thereby diminishing the mRNA expression and subsequent antigen-specific immune responses. To address these challenges, we developed a strategy that combines optimized untranslated regions (UTRs) screened from endogenous genes of pulmonary cells with affinity chromatography-based purification which removes almost all the dsRNA contaminants. The optimized mRNA administered via the airway route not only demonstrates superior protein expression (30-fold increase) and reduces inflammation in the lung, but also promotes robust immunity comprising significantly elevated systemic, cellular, and mucosal immune responses, which is in stark contrast to intramuscular injected counterpart that displays less pronounced benefits. Our findings offer new insight into the development of mucosal mRNA therapeutics from an overlooked but crucial perspective of optimizing mRNA components.

synthetic biology↗

bioRxiv2024

InTraSeq: A Multimodal Assay that Uncovers New Single-Cell Biology and Regulatory Mechanisms

Single-cell RNA sequencing (scRNA-seq) has revolutionized cell biology by enabling the profiling of transcriptomes at a single-cell resolution, leading to important discoveries that have advanced our understanding of cellular and tissue heterogeneity, developmental trajectories, and disease progression. Despite these important advances, scRNA-seq is limited to measuring the transcriptome providing a partial view of cellular function. To address this limitation, multimodal scRNA-seq assays have emerged, allowing for the simultaneous measurement of RNA expression and protein. Intracellular Transcriptomic and Protein Sequencing (InTraSeq), a novel multimodal scRNA-seq technology described here, enables the concurrent measurement of mRNA, surface markers, cytoplasmic proteins, and nuclear proteins within individual cells through oligo-barcoded antibodies. This method offers a comprehensive approach to studying cellular function by combining RNA and protein profiling from the same sample and utilizing a relatively simple protocol. The InTraSeq method enables researchers to expand their view of critical intracellular protein expression including post-translational modifications (PTMs) and transcription factors, allowing for the identification of novel cellular subtypes and states that may be obscured by RNA-based analyses alone. This is particularly valuable in understanding the heterogeneity of cell populations and identifying distinct functional states. In this report, we used InTraSeq to characterize the complex cellular states and regulatory mechanisms during Th17 cell differentiation. We simultaneously profiled RNA and protein expression in over 85,000 cells, capturing transcriptional changes, changes in protein expression and the dynamics of signaling pathways at a high resolution. Our results revealed novel insights into Th17 cell differentiation, including the identification of key regulatory factors and their target genes. By simultaneously measuring mRNA, extra and intra-cellular proteins, signaling proteins, and PTMs, InTraSeq offers a comprehensive understanding of cellular processes and enables the identification of novel regulatory mechanisms.

cell biology↗