Biology subjects

Liu, P.

Publications and source records attributed to Liu, P..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Directional growth of developing myelin mediated by Wnt gradient and required for proper axon functions

Axon-wrapping myelin sheaths formed by oligodendrocytes are essential for proper functions of the central nervous system. Although much is known about oligodendrocyte development, how the myelin dynamically forms remains unclear. Here we show the preferentially unidirectional extension of developing myelin mediated by Wnt gradient and required for proper axon functions. Using larval zebrafish as an in vivo model, we found that developing myelin in the spinal cord preferentially extends to the anterior end, and this process is dependent on an anterior-to-posterior Wnt4b gradient. Taking advantage of the large size of Mauthner-cell axons, we further showed that disruption of this directional extension impairs the even length distribution of myelin sheaths and faithful transduction of action potentials along the axon, and reduces the reliability of escape behavior. Thus, our study reveals a novel process for precise regulation of myelination, providing a new insight into myelin structuring and functioning.

neuroscience↗

bioRxiv2025

Modeling 2D Spatio-Tactile Population Receptive Fields of the Fingertip in Human Primary Somatosensory Cortex

Tactile fingertip sensations are critical for everyday life. Accordingly, tactile fingertip maps have been extensively studied in human primary somatosensory cortex. However, the fine-grained functional architecture of these maps remains largely unknown. To uncover this architecture, we sought to estimate 2D spatio-tactile population receptive fields (pRFs) of the tip of the index finger in human Brodmann area 3b (BA3b). Using functional magnetic resonance imaging at 7T and submillimeter resolution along with prospective motion correction, we recorded brain responses whilst participants sensed a row of vibrotactile pins sweeping along cardinal axes over a portion of the fingertip. To estimate pRF position and size, we initially fit a 2D Gaussian pRF model to the data, which, however, produced largely implausible pRF estimates. Simulations indicated that this likely occurred because the size of pRFs in BA3b surpasses the portion of the fingertip we stimulated, resulting in an incomplete mapping of pRFs. To address this issue, we constrained the fitting procedure and refined the 2D Gaussian pRF model by keeping pRF size constant. Our results for pRF position then revealed that the ulnar-to-radial axis spanning the fingertip maps onto a superior-to-inferior axis in BA3b. Both the putatively large pRF size (relative to the mapping area) and the pRF position gradient we uncover here appear compatible with receptive field properties quantified in monkeys. Our study provides the first comprehensive investigation into the fine-grained functional architecture of human fingertip maps and brings us one step closer to a thorough understanding thereof.

neuroscience↗

bioRxiv2025

Locus-specific Convergent Evolution and Interchromosomal Rearrangements Contribute Type I Interferon Diversification in Amniotes

Type I interferons (IFN-Is) play essential roles in antiviral immune responses. The extensive diversification of IFN-Is into multiple subtypes and redundant gene copies has posed significant challenges for evolutionary reconstruction. To address this, we developed an integrated analytical pipeline combining the IFN-SCOPE classification model and GENE-GRADE algorithm. Through comprehensive synteny-guided phylogenetic analysis, we identified three evolutionarily conserved IFN-I loci (HACD4, MOB3B, and UBAP2) that maintain chromosomal colocalization across all major amniote lineages - mammals, squamates, and Archelosauria (turtles, crocodilians, and birds). While MOB3B-locus maintains a single IFN-{kappa} ortholog, the HACD4 (IFN-H) and UBAP2 (IFN-U) loci show lineage-specific expansion patterns: IFN-H proliferated in mammals/reptiles but remained single-copy in birds, whereas IFN-U expanded in birds but not in other lineages. Phylogenetic analysis reveals these independently evolved multicopy genes nevertheless cluster into two conserved subgroups (IFN-H2/H1 and IFN-U2/U1), suggesting convergent functional specialization. Within IFN-H and IFN-U clusters, the single-copy IFN-H2 and IFN-U2 genes - positioned at the ancestral ends of their respective genomic arrays - likely represent the progenitor sequences of each locus. Notably, among mammalian IFN-H2 genes, we identified the poorly characterized IFN-{upsilon} (rather than IFN-{beta}) as the ancestral form of mammalian IFN-H subtypes. Furthermore, we identified IFN-I genes at non-canonical loci resulting from interchromosomal duplication events in tortoises and diving ducks and provide clear evidence that interchromosomal duplications contributed to IFN-I gene diversity. These discoveries advance our understanding of the evolutionary mechanisms shaping intronless IFN-I genes in amniotes, and potentially beneficial for developing novel IFN-I based antiviral treatments through comparative immunological approaches.

bioinformatics↗

bioRxiv2025

Efficient Cas9 nuclease-based editing in skeletal muscle via lipid nanoparticle delivery

Gene editing holds great promise for muscular dystrophy treatment, but the rapid evaluation of different editing modalities in skeletal muscle in vivo remains challenging due to lack of simple, effective delivery tools. Here we demonstrate that selective organ targeting (SORT) lipid nanoparticles (LNP) encapsulating optimized Cas9 cargo can facilitate efficient, local delivery to skeletal muscles achieving editing rates [≥]35% and restore protein expression for a proof-of-concept muscular dystrophy target. Interestingly, efficient editing in skeletal muscle was observed despite a strong adaptive immune response to repeat dosing of the Cas9 LNPs. High efficiency editing mediated by LNP-based delivery of Cas9 to skeletal muscle permitted detailed analysis of insertion and deletion (InDel) outcomes in vivo for a set of potential therapeutic target sites, which differed substantially from InDel outcomes observed in proliferating cells in one specific instance. Overall, our findings on enhanced LNP delivery of Cas9, platform-specific immune responses, and differential editing patterns observed between in vitro and in vivo models provide valuable insights that should inform the development of gene editing therapeutics for neuromuscular diseases. One Sentence SummarySORT LNPs permitted efficient Cas9-mediated repair of a pathogenic allele in skeletal muscle in a mouse model of LGMDR7.

molecular biology↗

bioRxiv2025

A synthetic biology toolkit for interrogating plasmid-dependent methylotrophy and enhancing methanol-based biosynthesis of Bacillus methanolicus

Bacillus methanolicus represents a thermophilic methylotroph whose methanol utilization depends on plasmid-encoded genes. It serves as a unique model for deciphering plasmid-dependent methylotrophy and an ideal chassis for low-carbon biomanufacturing using CO2-derived C1 substrates. Despite its evolutionary uniqueness and industrial potential, the lack of synthetic biology tools has hindered both mechanistic understanding and strain engineering. Here, we present a comprehensive synthetic biology platform comprising a high-efficiency electroporation protocol, a CRISPR method enabling robust and multiplex genome editing, diverse neutral loci for gene integration and overexpression, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, host restriction-modification systems, and functional significance of the chromosomal methylotrophic genes through targeted deletion. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR editing, we engineered L-arginine feedback regulation to achieve the first L-arginine biosynthesis from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and low-carbon biomanufacturing.

synthetic biology↗

bioRxiv2025

The β-amyloid oligomer Aβ*56 is associated with Alzheimer's dementia independently of amyloid pathology

The amyloid cascade hypothesis posits that {beta}-amyloid (A{beta}) oligomers (A{beta}o) play a key role in the pathogenesis of Alzheimers disease (AD). A{beta}o are believed to contribute to dementia in AD, and more than a dozen have been identified. While some oligomers are associated with amyloid plaques, others--such as A{beta}*56--are not. It remains unclear whether these plaque-independent oligomers contribute to dementia. A{beta}*56 is a non-fibrillar, sodium dodecyl sulfate-stable, brain-derived type of A{beta}o that impairs memory in mice. We recently confirmed and extended the characterization of A{beta}*56 in AD mouse models and identified two variants, namely A{beta}(40)*56 and A{beta}(42)*56 containing canonical A{beta}(1-40) and A{beta}(1-42), respectively. To examine the role of A{beta}*56 in dementia, we measured A{beta}*56 in elderly individuals--selected for equivalent levels of amyloid plaques--with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD dementia. Using immunoprecipitation (IP), western blotting (WB), size exclusion chromatography, and conformation-sensitive antibodies, we confirmed the presence of A{beta}(40)*56 and A{beta}(42)*56 in human brains. Using IP/WB coupled with densitometry-based semi-quantitative analysis, we found that levels of both A{beta}*56 variants were elevated in AD dementia compared to NCI and MCI and correlated inversely with various measures of memory and cognition. Furthermore, mediation analyses showed that these associations were mediated by phosphorylated tau at serine 202 and threonine 205 (ptau 202/205)-containing neurofibrillary tangles. These findings support an association between A{beta}*56 and AD dementia independently of amyloid pathology. The results are significant because they suggest that targeting A{beta}*56 in patients could provide additional cognitive benefits beyond those achieved by current anti-A{beta} therapies that focus on removing amyloid plaques.

biochemistry↗

bioRxiv2025

SLC44A1 deficiency impedes myelin development in the central nervous system

Axon-wrapping myelin sheath is essential for the proper functioning of the central nervous system (CNS). Defects in myelination occur in various neurodevelopmental disorders. Human deficiency of the solute carrier 44A1 (SLC44A1) causes a new type of childhood-onset neurodegeneration with leukoencephalopathy. Currently, there is no effective treatment for this devastating disease, and little is known about its pathological mechanisms. In this study we identified that SLC44A1 is enriched in oligodendrocytes and required for myelin development in the CNS of zebrafish and rodents, which is crucial for normal motor coordination and cognitive function. Defective oligodendroglial maturation and myelinogenesis can be visualized by in vivo time-lapse imaging in the spinal cord of zebrafish with Slc44a1b deficiency. Mechanistically, SLC44A1 deficiency disrupts the expression of genes involved in phosphatidylcholine production pathway, and subsequently inhibiting phospholipid biosynthesis and disturbing the lipid composition of myelin sheaths. More importantly, supplementation with citicoline, a natural choline metabolite, restores developmental myelination in SLC44A1-deficient animals. These findings demonstrate that SLC44A1 is essential for CNS myelination and citicoline supplementation represents a potential therapeutic strategy to address the developmental hypomyelination.

neuroscience↗

bioRxiv2025

Noninvasive ultrasound targeted modulation of calcium influx in splenic immunocytes potentiates antineoplastic immunity attenuating hepatocellular carcinoma proliferation

The spleen, as the largest immune organ, plays a pivotal role in modulating immune responses, particularly in the context of carcinogenesis and tumor progression. Non-pharmacological manipulation, particularly splenic ultrasound stimulation (SUS), has demonstrated significant immunomodulatory efficacy in alleviating chronic inflammatory diseases, suggesting its potential to revitalize splenic immunocompetence suppressing tumor proliferation, yet remains underexplored. This study applied low-frequency pulsed focused ultrasound (FUS) noninvasively stimulating the spleen (FUS sti. spleen) to investigate the efficacy in enhancing antitumor immunity and suppressing hepatocellular carcinoma (HCC). The results showed that FUS sti. spleen significantly suppressed tumor proliferation, achieving a suppression rate of >70% for H22-HCC and >83% for Hepa1-6-HCC, along with significantly prolonged survival. Comprehensive flow cytometry, single-cell RNA sequencing (scRNA-seq) and cytokine analyses demonstrated that SUS profoundly reshaped the splenic and intratumoral immune landscape, specifically activating cytotoxic CD8+ T cells and NK cells while suppressing immunosuppressive cell populations. Mechanistically, FUS facilitated calcium influx in splenic immunocytes, activating multiple signaling pathways, such as TNF, NF{kappa}B, MAPK, HIF-1, and ErbB, thereby counteracting tumor-driven immunosuppressive polarization while potentiating robust immune activation that impedes malignant progression and neoplastic proliferation. Leveraging above insights, we developed spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STNDs@Ca{superscript 2}), which, upon FUS stimulation, undergo cavitation-mediated controlled release of Ca{superscript 2}, further amplifying immunocyte activation and tumor suppression, achieving a remarkable H22-HCC suppression rate of over 90%. This study highlights the therapeutic potential of ultrasound-mediated splenic immunomodulation, both as a standalone intervention and in synergy with STNDs@Ca{superscript 2}, as a novel and noninvasive strategy for cancer immunotherapy.

biophysics↗

bioRxiv2025

A cross-week analysis of urinary extracellular vesicles after respiratory tract exposure intervention identifies systemic signaling changes -- a pilot study of isocyanate-exposed workers

Extracellular vesicles (EVs) from various biofluids have demonstrated potential as prognostic and diagnostic biomarkers in several diseases. However, their utility in early stages of disease development has not been extensively investigated. Additionally, given their role in intercellular communication, decoding the landscape of underlying cell-to-cell signaling in a clinical or subclinical condition based on EV-specific analyses has so far remained challenging. Here, we demonstrate the utility of urinary EVs for cross-week biomonitoring and for capturing the perturbation of underlying organismal signaling in a small well-characterized group of workers with inhalational exposure to methylene diphenyl diisocyanate (MDI) in fabric coating factory. Workers wore organic vapor cartridge respirators during the week to eliminate respiratory exposures. Spot urine was collected before and after the respirator intervention - pre-RESP and post-RESP, respectively. Following extensive characterization of EVs isolated from the urine samples, the relative enrichment of EV-specific RNAs and proteins and their associated biological processes were determined between pre-RESP and post-RESP samples. Distinct EV-specific RNA and protein signatures between the two groups strongly correlated with established biological and immune-related processes involved in asthma and lung inflammation. Using single cell transcriptomics data from the human cell atlas, EV-mRNAs highly enriched in pre-RESP, but not post-RESP, samples revealed global immune dysregulation and enrichment of associated cell types, such as monocytes, macrophages, and neutrophils. The findings indicate that urinary EVs and their contents can be utilized for biomonitoring of lung health and can reveal the effects of exposure on the underlying organismal signaling. One Sentence Summary: Urinary extracellular vesicles and their cargo hold great potential to monitor lung health and changes in systemic signaling in inhalational exposures.

pharmacology and toxicology↗

bioRxiv2025

ProteoAutoNet: high-throughput co-eluted protein analysis with robotics and a multi-database search strategy

Co-fractionation mass spectrometry (CF-MS) enables large-scale profiling of endogenous protein-protein interactions. Protein complexes identified by CF-MS using different databases are typically integrated. However, this integration uses varying cutoffs, leading to inconsistencies in protein complex identification. Here, we present ProteoAutoNet, a robotic experimental platform integrating multi-database search strategy into machine learning models, for high-throughput CF-MS analysis. This workflow increases the throughput of sample processing from protein complex to peptide by about two times. We then applied this workflow to map protein interaction networks in thyroid cancer cell lines, identifying significantly upregulated proteasome and prefoldin complexes in lung metastatic follicular thyroid carcinoma cell line FTC238 compared to normal thyroid cell line Nthy-ori 3-1. Notably, we identified a novel protein interaction network comprising PFAS, TGM2, and HK1 that was significantly upregulated in the papillary thyroid carcinoma cell line TPC-1. ProteoAutoNet provides an improved approach for investigating protein-protein interactions and uncovering novel networks, driving advancements in high-throughput proteomics research.

biochemistry↗

bioRxiv2025

ACE2 utilization of HKU25 clade MERS-related coronaviruses with broad geographic distribution

Dipeptidyl peptidase-4 (DPP4) is a well-established receptor for several MERS-related coronaviruses (MERSr-CoVs) isolated from humans, camels, pangolins, and bats (1-6). However, the receptor usage of many genetically diverse bat MERSr-CoVs with broad geographical distributions remains poorly understood. Recent studies have identified angiotensin-converting enzyme 2 (ACE2) as an entry receptor for multiple merbecovirus clades. Here, using viral antigen and pseudovirus-based functional assays, we demonstrate that several bat merbecoviruses from the HKU25 clade previously thought to utilize DPP4 (7), employ ACE2 as their functional receptor. Cryo-electron microscopy analysis revealed that HsItaly2011 and VsCoV-a7 recognize ACE2 with a binding mode sharing similarity with that of HKU5 but involving remodeled interfaces and distinct ortholog selectivity, suggesting a common evolutionary origin of ACE2 utilization for these two clades of viruses. EjCoV-3, a strain closely related to the DPP4-using MERSr-CoV BtCoV-422, exhibited relatively broad ACE2 ortholog tropism and could utilize human ACE2 albeit suboptimally. Despite differences in entry mechanisms and spike proteolytic activation compared to MERS-CoV, these viruses remain sensitive to several broadly neutralizing antibodies and entry inhibitors. These findings redefine our understanding of the evolution of receptor usage among MERSr-CoVs and highlight the versatility of ACE2 as a functional receptor for diverse coronaviruses. SignificanceRecent studies unexpectedly revealed that several merbecoviruses convergently evolved ACE2 receptor usage with distinct binding modes across three continents, challenging the dogma that DPP4 is their primary receptor. Here, we demonstrate that HKU25 clade MERS-related coronaviruses broadly distributed across Eurasia utilize ACE2 as host receptor through a binding mode shared with HKU5, challenging prior findings. These findings reveal a prevalence of ACE2 usage in diverse MERS-related coronaviruses in bats and show that EjCoV-3 is preadapted to use human ACE2, suggesting a potential for spillover. Our data provide a blueprint of host receptor barrier determinants which will facilitate global surveillance and development of countermeasures against these poorly characterized merbecoviruses.

microbiology↗

bioRxiv2025

Targeting Non-Catalytic Sites of SRC Sensitizes the Efficacy of SRC Kinase Inhibitors in Solid Tumors

The non-receptor tyrosine kinase SRC promotes the progression of hematologic malignancies and numerous solid tumors. Several SRC kinase inhibitors have been approved for the treatment of hematologic malignancies. However, drug resistance to sole ATP pocket-targeted kinase inhibition, coupled with low potency for solid tumors and insufficient selectivity, has limited the clinical application of SRC kinase inhibitors. The non-catalytic functions of kinases have provided crucial mechanisms underlying kinase inhibitor resistance, thereby presenting new opportunities for discovery SRC-targeted strategies. In this study, we discovered that upon abrogation of phosphorylation by SRC kinase inhibitors, non-catalytic SRC promotes the transcription of the oncogenes TRIB3 and SPC24 transcription by binding to their promoter sequence. Simultaneously, it interacts with TRIB3 and prevents their proteasome-mediated degradation by the E3 ligase CHIP, ultimately inducing resistance to SRC kinase inhibitors. The peptide TS1-2, blocking SRC/TRIB3 interaction in non-catalytic regions, inhibits the tumor progression of pancreatic ductal adenocarcinoma (PDAC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), and breast invasive carcinoma (BRCA), and enhances the efficacy of SRC kinase inhibitors in solid tumors. This study provides the insights of non-catalytic functions of SRC, validates the tumor-promoting function and mechanism of nuclear accumulation of SRC/TRIB3 following SRC kinase inhibitor treatment, identifies SRC/TRIB3 interaction as a potential non-catalytic target, and offers the candidate peptide TS1-2 as a potential combination strategy for overcoming SRC kinase inhibitor resistance. SignificanceThe scaffolding functions of SRC reduced the efficacy of SRC kinase inhibitors, and targeting SRC/TRIB3 non-catalytic functions synergizes the therapeutic effects of SRC kinase inhibitors in SRC-dependent solid tumors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/638389v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@17aa618org.highwire.dtl.DTLVardef@12e86eeorg.highwire.dtl.DTLVardef@19c8d82org.highwire.dtl.DTLVardef@10e6c46_HPS_FORMAT_FIGEXP M_FIG C_FIG SRC kinase inhibitors block myristoylation-dependent membrane location and catalytic phosphorylation of SRC. Phosphorylated SRC activates STAT1 and thereby represses TRIB3 transcription. Accumulated non-catalytic SRC promotes TRIB3, SPC24 transcription. Upregulated TIRB3 interacts with SRC to reduce CHIP-mediated ubiquitin-proteasomal degradation. The peptide TS1-2 disrupts TRIB3/SRC complex by targeting Non-kinase regions of SRC, consequently enhancing the therapeutic efficacy of SRC kinase inhibitors.

cancer biology↗

bioRxiv2025

Myocardial cGMP-PKG1alpha dysregulation contributes to VT pathogenesis in type II diabetes and metabolic syndrome.

BackgroundType-II diabetes (DMII) and metabolic syndrome increase ventricular arrhythmia and sudden cardiac death risk. ObjectivesTo identify signaling mechanisms through which DMII and metabolic syndrome promote ventricular tachycardia (VT). MethodsWe performed ventricular programmed stimulation on leptin receptor mutant (Db/Db) mice with DMII, high fat high sucrose (HFHS)-fed mice with metabolic syndrome, and cGMP-dependent Protein Kinase 1 (PKG1) leucine zipper mutant (LZM) mice, which do not have DMII or metabolic syndrome but have disrupted PKG1 signaling. ResultsDuring ventricular programmed stimulation, Db/Db and HFHS-fed mice displayed increased VT and T-wave alternans. Cardiomyocytes from these mice displayed early afterdepolarizations. Both models demonstrated decreased heart rate response to parasympathetic inhibition, indicating autonomic dysfunction. cGMP, which mediates cardiac parasympathetic stimulation, was reduced in LVs of Db/Db and HFHS-fed mice. Conversely, cGMP augmentation with soluble guanylate cyclase stimulation (riociguat) or phosphodiesterase 5 inhibition (sildenafil) reduced VT inducibility. PKG1 LZM mice had normal autonomic responsiveness, but excess VT inducibility. Db/Db, HFHS, and LZM mice each demonstrated hyperactivated myocardial glycogen synthase kinase3{beta} (GSK3{beta}). Further, GSK3{beta} inhibition with TWS119 abolished inducible VT in these mice. Diastolic cytosolic Ca2+ reuptake slope decreased in cardiomyocytes from all models, while GSK3{beta} inhibition with TWS119 reversed this effect. Phospholamban (PLB), which inhibits sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a-mediated Ca2+ reuptake, was hyperactivated/hypophosphorylated in HFHS-fed and LZM mice, and this was reversed by TWS119. ConclusionsThese findings identify cGMP reduction as driving GSK3{beta} hyperstimulation, calcium dyshomeostasis, and VT in DMII and metabolic syndrome. Pharmacological modulation of these pathways opposes VT pathogenesis.

molecular biology↗

bioRxiv2025

Avoidance of fratricide and improved efficacy of CD33-MSLN CAR-iNK cells in treating acute myeloid leukemia

Acute myeloid leukemia (AML) patients are often older, which brings challenges of endurance and persistent efficacy of autologous CAR-T cell therapies. Allogenic CAR-NK cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. In this study, we designed a novel CD33-mesothelin loop CAR (Loop CAR) and evaluated its anti-tumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. The Loop CAR exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established a hPSC-derived cell line via knockout of CD33 gene (CD33KO) and engineered Loop CAR. Rather than enforced expression of exogenous CD16, we generated abundant mature CD33KO-Loop CAR-iNK cells highly expressing endogenous CD16 via an organoid induction approach. This innovative strategy effectively mitigated NK cell fratricide and significantly enhanced CD33 and mesothelin-mediated specific cytotoxicity. Moreover, the CD33KO-Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mice and significantly prolonged survival. hPSC-derived CD33KO-Loop CAR-iNK cells possess unique advantages and translational potential for treating AML.

cancer biology↗

bioRxiv2025

Engineered CRO-CD7 CAR-NK cells derived from pluripotent stem cells avoid fratricide and efficiently suppress human T-cell malignancies

BackgroundT cell malignancies are highly aggressive hematological tumors with limited effective treatment options. CAR-NK cell therapy targeting CD7 has emerged as a promising approach for treating T-cell malignancies. However, conventional CAR-NK cell therapy faces the challenges of cell fratricide due to CD7 expression on both malignant cells and normal NK cells. Additionally, engineering CARs into human tissue-derived NK cells demonstrates heterogeneity, low transduction efficiency, and high manufacturing costs. MethodsThe human pluripotent stem cells (hPSCs) were genetically modified by knocking out the CD7 gene and introducing the CD7 CAR expression cassette to generate CD7 KO-CD7 CAR-hPSCs. These modified hPSCs were subsequently differentiated into CD7 KO-CD7 CAR-iNK cells using an efficient organoid induction method. The cytotoxicity of CD7 KO-CD7 CAR-iNK cells against CD7+ tumor cells was evaluated. Furthermore, we overexpressed the CXCR4 gene in CD7 KO-CD7 CAR-hPSCs and derived CXCR4-expressing CD7 KO-CD7 CAR-iNK (CRO-CD7 CAR-iNK) cells. The dynamics of CRO-CD7 CAR-iNK cells in vivo were tracked, and their therapeutic efficacy was assessed using human T-cell acute lymphoblastic leukemia (T-ALL) xenograft models. ResultsThe CD7 KO-CD7 CAR-iNK cells derived from CD7 KO-CD7 CAR-hPSCs effectively avoided fratricide, demonstrated normal expansion, and exhibited potent and specific anti-tumor activity against CD7+ T-cell tumor cell lines and primary T-ALL cells. CXCR4 overexpression in CRO-CD7 CAR-iNK cells improved their homing capacity and extended their persistence in vivo. The CRO-CD7 CAR iNK cells significantly suppressed tumor growth and prolonged the survival of T-ALL tumor-bearing mice. ConclusionsOur study provides a reliable strategy for the large-scale generation of fratricide-resistant CD7 CAR-iNK cells with robust anti-tumor effects from hPSCs, offering a promising cell product to treat T-cell malignancy.

cancer biology↗

bioRxiv2025

A cytoskeletal scaffold promotes motile cilia assembly by regulating transition-zone integrity

Motile cilia are eukaryotic organelles with essential chemo- and mechano-sensing functions across evolution, from single cell organisms to humans. Motile cilia of the mammalian nervous, respiratory and reproductive systems are characterized by unique motility proteins to generate fluid flow essential for transporting metabolites and removing mucus. The molecular mechanism of motile cilia biogenesis remains unknown. Here, we use mouse genetics, single-molecule motility assays, proteomics, high-resolution imaging, and in situ cryo-tomography to identify mammalian KIF27, a motor protein of the Kinesin-4 family and homologue of the Hedgehog pathway regulator COS2/KIF7, as a key regulator of motile cilia assembly. We show that KIF27 promotes the integrity of the transition zone, a diffusion barrier situated at the cilium base. Loss of KIF27 causes specific and profound defects in axonemal structure and disrupts cilia beating, which collectively lead to organismal phenotypes that recapitulate primary ciliary dyskinesia. We show that the motile properties of KIF27 are dispensable for its function in motile cilia biogenesis. Instead, KIF27 acts as a microtubule scaffold to regulate the transition zone architecture and enable correct ciliary incorporation of motility-generating proteins. Given that KIF27 homologues exist in different evolutionarily lineages, we propose that the ancestral activities of KIF27/KIF7 kinesins were to form a microtubule-associated scaffold for protein-protein interactions pertinent to cilia formation and signaling. The transition-zone associated KIF27 activities may represent a general building principle for motile cilia assembly in diverse species and cell types.

developmental biology↗

bioRxiv2024

Priming versus propagating: distinct immune effects of an alpha- versus beta-particle emitting radiopharmaceutical when combined with immune checkpoint inhibition

Radiopharmaceutical therapy (RPT) enhances tumor response to immune checkpoint inhibitors (ICI) in preclinical models, but the effects of different radioisotopes have not been thoroughly compared. To evaluate mechanisms of response to RPT+ICI, we used NM600, an alkylphosphocholine selectively taken up by most tumors. Effects of 90Y-, 177Lu-, and 225Ac-NM600 + ICIs were compared in syngeneic murine models, B78 melanoma (poorly immunogenic) and MC38 colorectal cancer (immunogenic). 90Y-/177Lu-/or 225Ac-NM600 delivering 2 Gy mean tumor dose promoted tumor regression and improved survival when combined with ICIs in syngeneic mice bearing B78 or MC38 tumors. Regardless of the administered isotope, this combination was optimized with early ICI administration (days -3/0/3) relative to day 1 RPT. 90Y-NM600+ICI produced the greatest anti-tumor response for MC38, whereas high linear energy transfer (LET) alpha particle radiation from 225Ac-NM600+ICI was most effective against poorly immunogenic B78 tumors. Flow cytometry and single cell RNA and T cell receptor (TCR) sequencing illuminated distinct mechanisms of 90Y- or 177Lu-NM600 in promoting expansion of existing adaptive immunity and of 225Ac-NM600 in promoting immune priming when combined with ICI. Antitumor immune response can be achieved with appropriate application of - or {beta}- emitting RPT in combination with ICIs in diverse murine tumor models.

cancer biology↗

bioRxiv2024

Global and genetic regulation of gene expression in human endothelial and vascular smooth muscle cells

BackgroundThe understanding of genetic and epigenetic regulation of gene expression in endothelial and vascular smooth muscle cells remains fragmented with limited experimental validation. MethodsChromatin conformation (Micro-C), DNA methylation (RRBS), chromatin accessibility (ATAC-seq), and transcriptome profiles (RNA-seq) were mapped in human induced pluripotent stem cell (hiPSC)-derived, isogenic endothelial and vascular smooth muscle cells (iECs and iVSMCs). CTCF and RAD21 were depleted to assess the functional relevance of chromatin architecture, and genome editing was used to evaluate the allelic effect of a blood pressure-associated single nucleotide polymorphism (SNP). ResultsSignificant correlations were identified between gene expression levels and chromatin interactions, chromatin accessibility, and DNA methylation in iECs and iVSMCs, with chromatin interactions showing the strongest association. Chromatin contact regions displayed distinct epigenetic landscapes depending on the types of regulatory element interactions involved. Perturbation of CTCF and RAD21 revealed their differential regulatory effects, particularly on the expression of genes overlapping chromatin contacts, with RAD21 exhibiting a broader regulatory impact. SNPs associated with several vascular traits were enriched in chromatin loops or accessible regions in iECs or iVSMCs. Precise genome editing demonstrated allele-dependent effects of SNP rs9833313 on the expression of SHOX2 located 247.4 kbp away but within the same chromatin loops as the SNP. ConclusionThis study provides an extensive epigenetic landscape of vascular cells that may drive novel research on the role of genetic and epigenetic mechanisms of vascular function and disease as demonstrated by our targeted experiments.

genomics↗