Biology subjects

Zhan, H.

Publications and source records attributed to Zhan, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Robust and accessible in situ sequencing with an open-source automated sequencer

Targeted in situ sequencing underlies many spatial transcriptomic, barcoded connectomic, and multiplexed perturbation techniques. Each experiment requires many cycles of chemical reactions and imaging that are time-consuming and error-prone to perform manually, and few commercial platforms can sequence highly diverse RNA barcodes in situ at cellular resolution across large tissue sections. Here we present Seq-o-Matic, an open-source automated in situ sequencer built entirely from commercially available components, which integrates fluidics, heated incubation, and imaging of up to three continuous 18 mm x 50 mm areas on standard microscope slides. Relative to manual sequencing, Seq-o-Matic reduced hands-on time 6- to 17.5-fold and total run time about 2.6-fold. Across three runs spanning two mouse strains and a neonatal marmoset, sequencing quality, measured by signal retention, channel specificity, and phasing, was similar to that of four manually sequenced mouse brains. In four mouse hemispheres sequenced in separate runs (two C57BL/6J, one NOD, and one WSB), consensus cell types were present in every brain and corresponded to those of a manually sequenced C57BL/6J brain. Within cortical excitatory neurons, the two C57BL/6J brains were more similar to each other than were brains of different strains, suggesting that technical variation between runs was small relative to strain differences. Seq-o-Matic thus makes consistent, high-throughput in situ sequencing accessible to laboratories without specialized in situ sequencing expertise.

genomics↗

bioRxiv2026

Characterization of Congestive Heart Failure Phenotype in a Murine Model of JAK2V617F-positive Myeloproliferative Neoplasm

Background: Murine models are widely used to investigate cardiovascular disease (CVD), yet many established models rely on invasive surgery or genetic manipulation to induce hyperlipidemia and therefore may not fully capture the gradual cardiovascular consequences of mutant hematopoiesis. Tie2-Cre/-FF1/- (Tie2FF1) mice express JAK2V617F mutation in both hematopoietic and endothelial cells and develop spontaneous CVD on a standard chow diet. Methods: We applied a comprehensive, non-invasive cardiovascular phenotyping protocol to 1-year-old Tie2FF1 mice and age-matched controls. Assessments included transthoracic echocardiography, coronary flow reserve by Doppler ultrasound, quantitative analysis of coronary arteriolar structure and perivascular fibrosis, endocardial histology and subendocardial collagen deposition, and histologic evaluation of pulmonary and hepatic congestion. Results: Tie2FF1 mice demonstrated left ventricular dilation, increased cardiac mass, and mildly reduced systolic function without obstructive epicardial coronary disease. Coronary microvascular remodeling was evident by arteriolar narrowing and increased perivascular fibrosis, while coronary flow reserve was markedly reduced in an exploratory cohort. Endocardial endothelial disruption and increased subendocardial fibrosis were also observed. In addition, Tie2FF1 mice showed increased lung and liver weights, pulmonary fluid accumulation, alveolar septal thickening, pulmonary vascular fibrosis, hepatic sinusoidal dilatation, and centrilobular hepatocyte swelling, providing objective evidence of clinically relevant heart failure. Significance: These findings establish Tie2FF1 mice as a spontaneous, non-invasive model with features of coronary microvascular and endocardial endothelial dysfunction and heart failure with mildly reduced left ventricular ejection fraction. The integrated approach described here provides a practical framework for comprehensive murine cardiovascular phenotyping beyond traditional atherosclerosis- and surgery-based models.

pathology↗

bioRxiv2026

Conserved and specialized features of thalamocortical wiring revealed by single-cell projection mapping in mouse and marmoset

Across mammals, brain regions can duplicate, expand, and diversify, requiring long-range connectivity to accommodate species-specific specializations while preserving globally ordered wiring. This challenge is especially pronounced in primate thalamocortical circuits, where select cortical fields and their thalamic partners have expanded disproportionately. Although gene expression in the thalamus follows broad and conserved gradients, how thalamocortical projections are organized at single-neuron resolution, and how this organization is reshaped by expansion, remain unknown. Here, we investigate thalamocortical projection organization by in situ sequencing and BARseq projection mapping in marmosets and mice. We profiled the gene expression of 1.5 million marmoset neurons and jointly measured gene expression and cortical projections in 708 marmoset and 1,518 mouse neurons that spanned multiple thalamic nuclei. In both species, projections of individual neurons targeted diverse areas that together spanned a large fraction of the cortex. Comparing projections at the single-neuron level and local neighborhood level revealed that marmoset thalamocortical projections were more spatially segregated, producing a point-to-point architecture. Strikingly, this local specialization coexisted with a conserved gradient that predominated over discrete anatomical borders: In the higher-order sensory thalamus of both species, gene expression and projections varied continuously across nucleus borders, and borders had only a small effect on projections. Furthermore, in both marmoset and mouse, gene expression gradients were associated with the anteroposterior locations of cortical targets. These results reconcile discrete nucleus and gradient-based models of thalamic organization and suggest that primate circuit specialization is superimposed on a conserved molecular-projection gradient.

neuroscience↗

bioRxiv2026

MSAgent: An Evidence Grounded Agentic Framework for LLM-driven Scientific Exploration in Mass Spectrometry-based Metabolomics

Mass spectrometry (MS) is a cornerstone high-throughput technology for molecular discovery, yet the reliable elucidation of chemical structures remains a formidable, expert-dependent bottleneck. Currently, achieving a reliable molecular identification from raw mass spectra necessitates a manual assembly--a labor-intensive ordeal of heuristic reasoning and the tedious integration of siloed computational tools, perpetuating a profound throughput gap between rapid data acquisition and the glacial pace of structural annotation. Here we present MSAgent, an autonomous agentic framework that bridges the gap between computational automation and expert intuition by emulating the cognitive logic of human specialists. By orchestrating a MSToolbox of over 50 domain-specific tools via Large Language Models (LLMs), MSAgent dynamically unifies the analytical pipeline into a scalable, evidence-grounded workflow, allowing for intent-aware planning, cross-resources outputs synthesis, and visual mechanistic interpretation within traceable reasoning chains and evidence-backed analytical reports. We evaluated MSAgent across multiple open benchmarks, including the established community challenges - Critical Assessment of Small Molecule Identification (CASMI) 2016/2022, CANOPUS, and LLM-oriented test cases. On CASMI, MSAgent consistently boosts retrieval performance by over 10% MRR across diverse benchmarks while ensuring high reliability--improving or preserving ranks in 95% of cases. For more challenging molecular de novo tasks on CANOPUS, MSAgent builds upon the outputs of baseline models with consistent refinement, yielding over a 40% average gain in Tanimoto similarity for ground-truth recovery. In addition, MSAgent demonstrates remarkable advantages in eliminating the hallucination phenomenon over LLMs without domain tool support, producing better-calibrated confidence (Pearson r = 0.438 vs -0.219 for gpt-4o). It improves exact-match rate by 38.8% over gpt-4o in candidate discrimination tasks, and achieved a 64% success rate in recommending high-quality candidate structures with Tanimoto similarity more than 0.7, where gpt-4o predominantly selected candidates with similarity below 0.3. Our work enables high-throughput mass spectrometry data to be analyzed in an intent-driven and automated manner, lowering the analysis barrier for no-expert to deliver molecular identification result with transparent analytical process, and accelerating discovery in metabolism and related fields by bridging the gap between experimental data acquisition and computational interpretation.

bioinformatics↗

bioRxiv2026

Targeting TPO/MPL Signaling to Mitigate JAK2V617F-driven Cardiac Microvascular Disease

BackgroundIndividuals with JAK2V617F-mutant myeloproliferative neoplasms or clonal hematopoiesis of indeterminate potential have a markedly increased risk of cardiovascular disease, yet the mechanisms by which mutant blood cells drive vascular and cardiac dysfunction remain incompletely understood. Although the thrombopoietin (TPO) receptor MPL is central to hematopoiesis and is expressed in vascular endothelial cells (ECs), its role in JAK2V617F-associated cardiovascular complications is unknown. Methods and ResultsWe generated chimeric mice with JAK2V617F-mutant blood cells and wild-type endothelium by bone marrow transplantation and challenged them with a high-fat/high-cholesterol diet to model cardiometabolic stress. These mice developed a distinct cardiovascular phenotype characterized by microvascular disease, increased left ventricular mass, and relatively preserved left ventricular ejection fraction. Histological analysis revealed coronary arteriole stenosis, perivascular fibrosis, reduced microvascular density, and endocardial injury, without evidence of epicardial coronary stenosis or myocardium infarction. Single-cell RNA sequencing revealed activation of inflammatory, stress-response, and endothelial-to-mesenchymal transition gene signatures in ECs, most prominently within the endocardial ECs. Immunohistochemistry identified MPL expression predominantly in endocardial ECs. TPO/MPL signaling was upregulated in endocardial ECs in mice with JAK2V617F-mutant hematopoiesis, and treatment with an anti-MPL neutralizing antibody markedly improved cardiac pathology, restored endocardial integrity, and increased coronary microvascular density despite persistent systemic inflammation. ConclusionsJAK2V617F-mutant hematopoiesis induces coronary microvascular dysfunction. Endocardial ECs represent a key cellular target under cardiometabolic stress, and endocardial MPL signaling constitutes a potential targetable pathway in JAK2V617F-associated cardiovascular disease. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/715884v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@15dcebdorg.highwire.dtl.DTLVardef@1b00a2eorg.highwire.dtl.DTLVardef@6c6f98org.highwire.dtl.DTLVardef@1128f75_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIJAK2V617F-mutant hematopoiesis induces cardiac microvascular disease C_LIO_LIMPL is expressed in endocardial ECs and MPL inhibition restores endocardial integrity and improves cardiac microvascular function C_LI

cell biology↗

bioRxiv2026

Data-Driven Retrieval of Effective Point Spread Functions for Super-Resolution Optoacoustic Imaging

Localization-based super-resolution techniques have revolutionized biomedical imaging by surpassing classical diffraction limits. However, their performance is fundamentally constrained by distortions of the point spread function (PSF) induced by the system and the sample, which are particularly prominent in the case of spatially under-sampling. Here we introduce a data-driven effective point spread function retrieval (DEPR) method that directly learns continuous, field-dependent system responses from experimental point source datasets. Through statistical aggregation of thousands of targets and iterative self-supervised refinement, DEPR captures spatially variant imaging characteristics in situ without prior assumptions or external calibrations. When integrated into the localization optoacoustic tomography (LOT) pipeline, DEPR achieves accurate sub-pixel localization despite under-sampled conditions, thus enhancing resolution while reducing computational burden. We demonstrate its efficacy by in vivo imaging of the murine brain microvasculature using microparticle contrast agents in the first (NIR-I) and second (NIR-II) near-infrared windows, achieving significant improvement in data utilization efficiency and substantial reduction of gridded artifacts compared to conventional approaches. The method resolves vascular structures with [~]41 m separation across a [~]3 mm imaging depth range. This framework addresses fundamental challenges shared across diverse localization-based imaging modalities, offering a robust and generalizable strategy for high-precision imaging in complex biological systems.

neuroscience↗

bioRxiv2025

Genetic complementation reveals structure-function links in nodavirus RNA replication complex crowns

Positive-strand RNA viruses replicate their RNA genomes in virus-induced, membrane-bounded organelles. As first found for nodaviruses, the necked cytosolic portals of these organelles bear ringed "crown" complexes of viral RNA replication proteins that drive synthesis, capping and release of new RNA genomes. Nodavirus crowns contain two 12-mer rings of viral protein A with C-proximal polymerase domains stacked. In the basal ring, protein As N-proximal RNA capping domains form a central, toroidal floor, while in the apical ring these domains extend radially outward. A third protein A conformation provides a putative central Pol domain interacting with the viral dsRNA replication intermediate in a vesicle beneath the crown. Protein As multiple conformations likely differentially contribute to crown assembly, RNA template recruitment, (-) and (+) strand synthesis, RNA capping, and progeny RNA release. Protein As high copy numbers may provide robustness to these processes. To test such concepts, we combined mutational, complementation and functional analyses. Strong complementation between null mutants in protein As polymerase and RNA capping active sites showed that they operate in independent protein A copies, likely at distinct sites. Thus, neither function is required in all protein A copies, nor are both required in any single copy. Lack of complementation between mutants in distinct RNA capping steps implied that major RNA capping steps must be performed in the same protein. Although RNA polymerase and capping activity were not required in all protein A subunits, none of a series of deletions across these domains were complementable, showing the importance of structural and other requirements for crown assembly, etc.. Surprisingly, RNA replication was more sensitive to depleting the fraction of subunits retaining protein As C-terminal intrinsically disordered region than polymerase or capping activity. These and other results reveal and illuminate the cooperative, interdependent nature of protein As diverse functions. Author summaryPositive-strand RNA viruses represent the largest genetic class of viruses and include human, animal, and plant pathogens causing major agricultural, economic, and environmental consequences. Using no DNA intermediates to multiply their RNA genomes, these viruses modify cellular membranes into novel, infection-specific RNA replication organelles. Emerging results show that RNA replication proteins encoded by many or most of these viruses assemble into ringed, crown-like viral protein complexes gating portals to these compartments. We previously revealed that nodavirus crowns contain two stacked 12-mer rings of viral replicase protein A, which contains polymerase, RNA capping and other domains. The nodavirus experiments reported here are among the earliest explorations in cells to illuminate the functions and interactions of such multi-domain RNA replication proteins in the context of their highly multimeric crowns. Critical questions include whether all domains and interactions are required in all protein A conformations, whether protein A multiplicity might provide dose-responsive redundancy for any crown functions, or whether defects in individual protein copies might inhibit or even poison operation of the entire crown. The results have significant implications for positive-strand RNA virus biology and thus for efforts toward virus control and beneficial uses.

microbiology↗

bioRxiv2025

Sensory neuronal mTORC1 signaling establishes neuroimmune memory that initiates allergic immunity

Environmental allergens are enriched in protease activity, a feature that directly activates cutaneous sensory neurons, triggering itch and Substance P release, which promotes Th2-skewing CD301b+ dendritic cell migration and initiates allergic immunity. However, allergens are typically encountered as repeated, subthreshold exposures, and how these cumulatively induce sensitization is unknown. We identify a sensory neuron-intrinsic mechanism of neuroimmune memory that enables this process. Initial activation by protease allergens induces sustained mTORC1 signaling and PGC-1-associated mitochondrial remodeling, establishing a metabolically primed state that amplifies neuroimmune responses upon allergen re-exposure. This heightened state, independent of adaptive immunity, drives enhanced itch, increased CD301b+ dendritic cell migration, and augmented Th2 differentiation. Disrupting neuronal mTORC1 signaling or mitochondrial stability abrogates this amplification while leaving primary responses intact. Importantly, this mechanism generalizes across distinct protease allergens, revealing mTORC1-driven metabolic reprogramming in sensory neurons as a form of innate neuroimmune memory underlying allergen cross-sensitization and polysensitization.

immunology↗

bioRxiv2025

Computational modeling of optogenetic control in human atrial tissue: The role of fibrosis type and illumination strategy

Atrial fibrosis promotes atrial fibrillation by stabilizing spiral waves, with focal fibrosis causing strong anchoring that resists termination. Optogenetics offers a low-energy alternative for rhythm control, but its efficacy in human atrial tissue remains under investigation. Here, we developed a two-dimensional computational model of human atrial tissue expressing the light-sensitive protein GtACR1, coupled with fibroblasts, to simulate spiral wave dynamics under varying degrees (0%-100%) and types (focal vs. diffuse) of fibrosis. We examined the effects of subthreshold illumination area (0%-100%) and spatial intensity profiles (uniform, parabolic, linear, exponential) on termination efficiency, and tested low-intensity periodic stimulation for wave drift induction. Results show that the minimum light area required for termination increases with fibrosis degree. At 25%-50% fibrosis, focal substrates require larger illumination areas than diffuse ones, confirming stronger anchoring. Crucially, the relationship between illumination area and termination time is non-monotonic. An "optimal light range" exists: smaller areas fail to eliminate the wave, while excessively large areas suppress wavefront collisions, prolonging termination. In diffuse fibrosis, low-intensity periodic illumination successfully induces drift and boundary collision, enabling self-termination. In contrast, in focal fibrosis, the spiral wave core remains anchored, and drift cannot be initiated by modulating stimulation frequency or intensity alone. Our findings demonstrate that fibrosis type and extent critically influence optogenetic control efficacy. The existence of an optimal illumination strategy highlights the need for spatially tailored interventions. These results provide a mechanistic basis for developing individualized, low-energy optogenetic defibrillation protocols. Author summaryAtrial fibrillation, a common heart rhythm disorder, is often sustained by rotating electrical waves in the heart muscle. Scarring (fibrosis) in the atria can anchor these waves, making them harder to eliminate. Optogenetics -- a technique that uses light to control genetically modified heart cells--offers a promising low-energy approach to stop these dangerous rhythms. However, how different patterns of scarring affect the success of optical interventions remains unclear. In this study, we developed a computational model of human atrial tissue expressing a light-sensitive protein (GtACR1) to investigate how two major types of fibrosis--localized "focal" scars versus widespread "diffuse" scarring -- influence the effectiveness of light-based rhythm control. We found that focal fibrosis strongly anchors spiral waves, requiring larger illuminated areas for termination, while diffuse fibrosis allows more efficient disruption with smaller light zones. Crucially, we discovered an "optimal light range": too little light fails to stop the wave, but too much can paradoxically prolong termination by suppressing wavefront collisions. Furthermore, we show that low-intensity periodic light can induce drift and self-termination in diffuse fibrotic tissue, but fails in focal substrates due to structural anchoring. Our findings highlight that one-size-fits-all optical strategies are suboptimal, and that personalized illumination protocols--tailored to individual fibrosis patterns--could enable safer, lower-energy defibrillation in the future.

physiology↗

bioRxiv2025

The Immune-Modulatory Function of Megakaryocytes in the Hematopoietic Niche of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by dysregulated megakaryopoiesis and neoplastic hematopoietic stem cell (HSC) expansion. Using a murine model with MK-specific JAK2V617F expression, we establish an MPN aging model where mutant MKs drive HSC expansion and a progressive decline in wild-type HSC function. Compared to wild-type MKs, JAK2V617F MKs exhibit heightened inflammation and innate immune activation with aging, including increased antigen presentation, elevated pro-inflammatory cytokines, skewed T cell populations, and impaired T cell functions in the marrow niche. Enhanced MK immunomodulatory function is linked to mutant cell expansion and MPN progression in a chimeric murine model with co-existing wild-type and JAK2V617F mutant HSCs. LINE-1 (long-interspersed element-1), a retrotransposon linked to innate immune activation and aging, is upregulated in mutant MKs during aging in murine models. We validated that LINE-1-encoded protein ORF1p is expressed in marrow MKs in 12 of 13 MPN patients but absent in control samples from patients undergoing orthopedic surgery (n=5). These findings suggest that MKs reprogram the marrow immune microenvironment, impairing normal HSC function while promoting neoplastic expansion in MPNs. LINE-1 activation in mutant MKs may be a key driver of immune dysregulation in MPNs. Key PointsO_LIJAK2V617F mutant MKs reprogram the marrow immune microenvironment to promote neoplastic HSC expansion in MPNs. C_LIO_LILINE-1 activation in diseased MKs triggers chronic inflammation and immune dysfunction in MPNs. C_LI

cancer biology↗

bioRxiv2025

Bisphosphonates Trigger Anti-Ageing Effects Across Multiple Cell Types and Protect Against Senescence

Bisphosphonates (BPs) have been the major class of medicines used to treat disorders of excessive bone loss for over five decades. Recently it has been recognized that BPs may also have additional significant beneficial extra-skeletal effects. These include a reduction of all-cause mortality and of conditions commonly linked to ageing, such as cancer and cardiovascular disease. Here we show that bisphosphonates co-localize with lysosomal and endosomal organelles in non-skeletal cells and stimulate cell growth at low doses. In vivo spatial transcriptomic analysis revealed differentially expressed senescence markers in multiple organs of aged BP-treated mice, and a shift in cellular composition toward those of young counterparts. Similarly, a 5000-plex plasma proteome analysis from osteopenic patients before and after BP-treatment showed significant alterations in [~]400 proteins including GTPase regulators and markers of senescence, autophagy, apoptosis, and inflammatory responses. Furthermore, treatment with BPs protected against the onset of senescence in vitro. Proteome-wide target deconvolution using 2D thermal profiling revealed novel BP-binding targets (PHB2, ASAH1), and combined with RNA- and ATAC-seq of BP-treated cells and patient data, suggests downstream regulation of the MEF2A transcription factor within the heart. Collectively, these results indicate how BPs may beneficially modify the human plasma proteome, and directly impact multiple non-skeletal cell types through previously unidentified proteins, thereby influencing a range of pathways related to senescence and ageing.

cell biology↗

bioRxiv2024

Selective expansion of motor cortical projections in the evolution of vocal novelty

Deciphering how cortical architecture evolves to drive behavioral innovations is a long-standing challenge in neuroscience and evolutionary biology. Here, we leverage a striking behavioral novelty in the Alstons singing mouse (Scotinomys teguina), compared to the laboratory mouse (Mus musculus), to quantitatively test models of motor cortical evolution. We used bulk tracing, serial two-photon tomography, and high-throughput DNA sequencing of over 76,000 barcoded neurons to discover a specific and substantial expansion ([~]200%) of orofacial motor cortical (OMC) projections to the auditory cortical region (AudR) and the midbrain periaqueductal gray (PAG), both implicated in vocal behaviors. Moreover, analysis of individual OMC neurons projection motifs revealed preferential expansion of exclusive projections to AudR. Our results imply that selective expansion of ancestral motor cortical projections can underlie behavioral divergence over short evolutionary timescales, suggesting potential mechanisms for the evolution of enhanced cortical control over vocalizations--a crucial preadaptation for human language.

neuroscience↗

bioRxiv2024

Spontaneously regenerative corticospinal neurons in mice

The spinal cord receives inputs from the cortex via corticospinal neurons (CSNs). While predominantly a contralateral projection, a less-investigated minority of its axons terminate in the ipsilateral spinal cord. We analyzed the spatial and molecular properties of these ipsilateral axons and their post-synaptic targets in mice and found they project primarily to the ventral horn, including directly to motor neurons. Barcode-based reconstruction of the ipsilateral axons revealed a class of primarily bilaterally-projecting CSNs with a distinct cortical distribution. The molecular properties of these ipsilaterally-projecting CSNs (IP-CSNs) are strikingly similar to the previously described molecular signature of embryonic-like regenerating CSNs. Finally, we show that IP-CSNs are spontaneously regenerative after spinal cord injury. The discovery of a class of spontaneously regenerative CSNs may prove valuable to the study of spinal cord injury. Additionally, this work suggests that the retention of juvenile-like characteristics may be a widespread phenomenon in adult nervous systems.

neuroscience↗

bioRxiv2024

Self-organizing glycolytic waves fuel cell migration and cancer progression

Glycolysis has traditionally been thought to take place in the cytosol but we observed the enrichment of glycolytic enzymes in propagating waves of the cell cortex in human epithelial cells. These waves reflect excitable Ras/PI3K signal transduction and F-actin/actomyosin networks that drive cellular protrusions, suggesting that localized glycolysis at the cortex provides ATP for cell morphological events such as migration, phagocytosis, and cytokinesis. Perturbations that altered cortical waves caused corresponding changes in enzyme localization and ATP production whereas synthetic recruitment of glycolytic enzymes to the cell cortex enhanced cell spreading and motility. Interestingly, the cortical waves and ATP levels were positively correlated with the metastatic potential of cancer cells. The coordinated signal transduction, cytoskeletal, and glycolytic waves in cancer cells may explain their increased motility and their greater reliance on glycolysis, often referred to as the Warburg effect.

biochemistry↗

bioRxiv2024

CD62L-selected umbilical cord blood universal CAR T cells

Umbilical cord blood (UCB) T cells exhibit distinct naive ontogenetic profiles and may be an attractive source of starting cells for the production of chimeric antigen receptor (CAR) T cells. Pre-selection of UCB-T cells on the basis of CD62L expression was investigated as part of a machine-based manufacturing process, incorporating lentiviral transduction, CRISPR-Cas9 editing, T-cell expansion and depletion of residual TCR{beta} T cells. This provided stringent mitigation against the risk of graft versus host disease (GVHD), and was combined with simultaneous knockout of CD52 to enable persistence of edited T cells in combination with preparative lymphodepletion using Alemtuzumab. Under compliant manufacturing conditions, two cell banks were generated with high levels of CAR19 expression and minimal carriage of TCR{beta} T cells. Sufficient cells were cryopreserved in dose-banded aliquots at the end of each campaign to treat dozens of potential recipients. Molecular characterisation captured vector integration sites and CRISPR editing signatures and functional studies, including in vivo potency studies in humanised mice, confirmed anti-leukaemic activity comparable to peripheral blood-derived universal CAR19 T cells. Machine manufactured UCB derived T cells banks offer an alternative to autologous cell therapies and could help widen access to CAR T cells.

molecular biology↗

bioRxiv2023

Electrostatic modulation of signaling at cell membrane: Waveform- and time-dependent electric control of ERK dynamics

Different exogenous electric fields (EF) can guide cell migration, disrupt proliferation, and program cell development. Studies have shown that many of these processes were initiated at the cell membrane, but the mechanism has been unclear, especially for conventionally non-excitable cells. In this study, we focus on the electrostatic aspects of EF coupling with the cell membrane by eliminating Faradaic processes with dielectric-coated microelectrodes, and show that the ERK signaling pathway of epithelial cells (MCF10A) can be both inhibited and activated by AC EF with different amplitude thresholds, peaking times and refractory periods. Interestingly, the ERK responses were sensitive to the waveform and timing of EF stimulation pulses, depicting the characteristics of electrostatic and dissipative interactions. Blocker tests and correlated changes of active Ras on the cell membrane with ERK signals indicated that both EGFR and Ras were involved in the rich ERK dynamics induced by EF. We propose that the frequency-dependent dielectric relaxation process could be an important mechanism to couple EF energy to the cell membrane region and modulate membrane protein-initiated signaling pathways, which can be further explored to precisely control cell behavior and fate with high temporal and spatial resolution.

biophysics↗

bioRxiv2023

Unlocking the Role of Endothelial MPL Receptor and JAK2V617F Mutation: Insights into Cardiovascular Dysfunction in MPNs and CHIP

Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential (CHIP) are at a significantly higher risk of cardiovascular diseases (CVDs). Endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in many MPN patients. Here, we investigated the impact of endothelial JAK2V617F mutation on CVD development using both transgenic murine models and human induced pluripotent stem cell lines. Our findings revealed that JAK2V617F mutant ECs promote CVDs by impairing endothelial function and undergoing endothelial-to-mesenchymal transition (EndMT). Importantly, we found that inhibiting the endothelial thrombopoietin receptor MPL suppressed JAK2V617F-induced EndMT and prevented cardiovascular dysfunction caused by mutant ECs. These findings propose that targeting the endothelial MPL receptor could be a promising therapeutic approach to manage CVD complications in patients with JAK2V617F-positive MPNs and CHIP. Further investigations into the impact of other CHIP-associated mutations on endothelial dysfunction are needed to improve risk stratification for individuals with CHIP.

cell biology↗

bioRxiv2023

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help develop novel therapeutic approaches for hematopoietic disorders.

cell biology↗