Biology subjects

Nguyen, T.

Publications and source records attributed to Nguyen, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Systematic functional dissection of germline noncoding risk variants impacting clonal hematopoiesis

Clonal hematopoiesis of indeterminate potential (CHIP) is a precursor condition characterized by the expansion of blood cell clones harboring somatic mutations originating in hematopoietic stem cells (HSCs). Since individuals with CHIP face a high risk of developing myeloid malignancies, targeting CHIP clones could provide a viable strategy for leukemia prevention. Despite its clinical significance, the mechanisms underlying CHIP predisposition and progression remain poorly understood. Recent genome wide association studies (GWAS) have identified several non-coding genetic loci that are strongly associated with CHIP; however, their underlying mechanisms still remain unknown. We hypothesize that risk variants in these non-coding loci modulate enhancer elements active in HSCs. To test this, we selected 1,374 non-coding variants from 51 loci associated for CHIP risk in the UK Biobank and screened them for regulatory activity using a Massively Parallel Reporter Assay (MPRA). We performed our lentiviral MPRA screen in MUTZ-3 cells, a human hematopoietic cell line relevant to HSCs, which express CD34 surface marker and are dependent on HSC-specific transcription factors. Using a MPRA library of [~]73,000 constructs in CD34+ fraction of MUTZ-3 cells, we identified 87 variants representing 32 GWAS loci. We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MPRA variants that affect the transcription of NKD2, FLT3, and MSI2. Our functional studies on MSI2 indicate that presence of higher levels of MSI2 mediated by CHIP risk allele enhances the clonal expansion of TET2 knockout hematopoietic stem and progenitor cells, providing a mechanistic link whereby non-coding genetic variants can influence the expansion of mutant CHIP clones.

genetics↗

bioRxiv2026

Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. We also showed that while fork-MMEJ is promoted by Pol{theta}, it operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, which is distinct from canonical MMEJ (cMMEJ) defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. Combined inactivation of ATR and Pol{theta} synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.

molecular biology↗

bioRxiv2026

Mutant ASXL1 Drives Transcriptional Activation and Repression in Human Hematopoiesis

Mutations in the epigenetic regulator ASXL1 are common in myeloid malignancies and portend a near-universally poor prognosis. While multiple mechanisms for mutant ASXL1-dependent oncogenesis have been proposed, none have been functionally validated in the context of the human hematopoietic stem cell, where these mutations almost certainly arise. Here, we extensively characterized a CRISPR-engineered human hematopoietic stem and progenitor cell model of ASXL1 mutations. In this context, mutant ASXL1 expression decreases differentiation, increases clonogenicity in serial replating experiments, and improves engraftment in immunodeficient mice. We also show that endogenous truncating mutations in ASXL1 drive protein stabilization and confirm that mutant ASXL1 is resistant to proteasomal degradation. At the transcriptional level, these phenotypes are driven by significant repression of the stress-response genes and by increased expression of bromodomain and extra-terminal family protein targets. Using protein-interaction screens, genomic and functional approaches, we link the positive transcriptional changes in ASXL1-mutant cells to BRD4-dependent RNA polymerase II pause release and identify a mechanism for transcriptional repression via a previously uncharacterized interaction with the transcription factor MECOM. Finally, we demonstrate that ASXL1-mutant AML exhibits increased MECOM activity consistent with our gene-editing models. Collectively, these studies highlight a highly reproducible model of mutant ASXL1 in the appropriate cell context. Further, they are the first to functionally describe the mutant ASXL1 interactome in the context of the human HSC, identifying new dependencies with therapeutic potential.

cancer biology↗

bioRxiv2026

MYST acetyltransferases are a targetable therapeutic vulnerability in SETBP1-mutant leukemia

Mutations in SET binding protein 1 (SETBP1) are associated with an adverse prognosis in myeloid malignancies. These mutations stabilize SETBP1 protein, driving increased expression of a progenitor-associated gene expression program through incompletely described mechanisms. A proteomic screen revealed interactions between SETBP1 and members of the MYST acetyltransferase complexes, including the catalytic subunits--KAT6A and KAT7. Mutant SETBP1 increases the localization of MYST complexes at known SETBP1 target genes, including the Hoxa cluster, where it drives increased histone acetylation and gene expression. Treatment of SETBP1D868N-expressing myeloid progenitors with MYST inhibitors reduced target gene expression. To establish the efficacy of MYST inhibition in vivo, we treated mice harboring a syngeneic SETBP1-mutant leukemia with the clinical-grade MYST inhibitor--PF-9363. This resulted in complete hematologic control and increased survival. MYST inhibition was also highly effective against a SETBP1-mutant PDX model. These studies identify MYST acetyltransferases as promising therapeutic targets in SETBP1-mutant malignancies. Statement of SignificanceSETBP1 mutations are markers of high-risk myeloid malignancies, but we lack any targeted therapies to improve outcomes. In this study, we identify MYST acetyltransferases as key drivers of mutant SETBP1-driven transcription. MYST inhibitors are highly effective against SETBP1-mutant leukemia and represent a promising avenue for clinical translation.

cancer biology↗

bioRxiv2025

Myelin Supports Cortical Circuit Function Underlying Skilled Movement

Primary motor cortex (M1) is among the most heavily myelinated cortical regions and generates tightly coordinated neuronal activity patterns that drive skilled movement. Activity-dependent myelination is required for motor skill acquisition, and myelin loss in demyelinating diseases such as multiple sclerosis leads to motor impairment. Yet how myelination influences neuronal activity underlying skilled behavior remains unclear. By combining in vivo imaging of oligodendrocytes with high density Neuropixels recordings during dexterous reaching, we demonstrate that cuprizone-induced demyelination impairs movement efficiency, and alters cell-type-specific neuronal activity and synchrony in a manner that predicts motor output. Using a computational model constrained by these data, we identify inhibitory axonal propagation failures as a mechanistic link between myelin loss and altered circuit function. Partial remyelination normalizes cortical network-level metrics and reach consistency but leaves smooth movement impaired, revealing a selective vulnerability in inhibitory circuits. These findings close a critical gap between cellular models of demyelination and clinical motor impairment by demonstrating how myelin supports cortical circuit dynamics driving skilled behavior.

neuroscience↗

bioRxiv2025

Combinatorial Protein Language Model-Guided Engineering of TEV Protease for Enhanced Stability and Production

Industrial enzyme engineering focuses on improvement of enzyme production yield, stability, catalytic activity, and substrate specificity, but often suffers from low efficiency with time-consuming and labor-intensive design and screening processes of massive libraries. Recent advances in AI and machine learning created protein language models trained by numerous datasets and shed new lights to speed up the enzyme engineering processes with high accuracy structural prediction. Here, we developed a highly efficient enzyme engineering strategy combining three protein language models (xTrimoMPNN-Thermo, ESM-IF, and MPNNsol) and use it to generate TEV protease variants with improved expression, stability, and function. The results indicated that a small number of TEV protease designs (<50 designs) were sufficient to develop variants with desired properties, demonstrating its high efficiency. Our strategy could be broadly applied to accelerate designing and engineering various industrial enzymes.

bioengineering↗

bioRxiv2025

Multi-omics analysis of TNBC organoids identifies phosphorylation of the membrane trafficking machinery as a key event associated with FER-mediated invasion

Triple Negative Breast Cancer (TNBC) is characterised by unfavourable outcome due to the combination of its metastatic propensity, chemo-refractory behaviour and the lack of effective targeted interventions. Expression of the feline sarcoma-related (FER) kinase constitutes an independent prognostic factor that correlates with poor patient survival. FER promotes invasive behaviour in TNBC cells by regulating endosomal sorting and recycling (ESR) of adhesion proteins. Yet, the ESR machinery supporting invasion in TNBC, particularly within 3D environments, remains poorly understood. Here, we used FER-expressing TNBC patient-derived xenograft organoids (PDXOs) and MDA-MB-231 cells to identify the membrane trafficking machinery promoting invasion. Using a combination of proteomics, phospho-proteomics, and single cell RNA-sequencing, we show that the invasion of FER-expressing PDXO cells in collagen-I is mainly associated with the differential phosphorylation of membrane trafficking regulators, including SEC16A and a marked increase in Rab4-positive tubules. SEC16A depletion impairs cell invasion and reduces the number of focal adhesions and Rab4-positive tubules. Importantly, FER regulates SEC16A levels and localization, specifically in TNBC. Altogether, we identified SEC16A as a key player in FER-driven TNBC invasion, highlighting the membrane trafficking machinery as a promising target for the development of new therapeutic strategies.

cancer biology↗

bioRxiv2025

Salmonids reveal principles of regulatory evolution following autotetraploidization

Early vertebrate autotetraploidization events may have enabled major innovations by expanding the genetic material for functional diversification, yet their ancient timing obscures how genome doubling reshaped gene regulatory evolution. Salmonids provide a unique window to these mechanisms, because they experienced a comparatively recent autotetraploidization and are earlier in the rediploidization process - which creates new genes and regulatory elements during evolution. Here, using large-scale multiomics spanning embryonic and adult tissues in two salmonids, we investigate gene regulatory evolution following genome doubling and rediploidization, which we show is governed by developmental and tissue-specific context, with a period of maximal constraint at advanced stages of embryogenesis. This work advances understanding of vertebrate genome evolution, while providing an open resource supporting salmonid aquaculture and conservation.

evolutionary biology↗

bioRxiv2025

Conserved phenotype and function of human brain border-associated macrophages in iPSC-derived models

Border-associated macrophages (BAMs) are increasingly implicated in protective brain functions including the removal of pathogenic material such as amyloid-beta (A{beta}). However, there is a lack of available and deeply characterized human BAM models. Here, we report that postnatal transplantation of induced pluripotent stem (iPS) cell-derived hematopoietic progenitors to the murine brain sufficiently chimerizes the brain border immune compartment to allow functional interrogations. Human xenotransplanted BAMs (xBAMs) line the leptomeninges and brain vasculature beyond the glia limitans. Via single-cell RNA sequencing, we show that a conserved transcriptional signature distinguishes BAMs from microglia across species origin, age, and genetic background. Both xBAMs and murine BAMs are defined by a hyper-endocytic phenotype and function, surpass other brain macrophages in acute A{beta} scavenging, and exhibit compartment-restricted sampling of parenchymal material. Using a modified differentiation protocol, we find that we can generate iPS-derived BAM-like cells (iBAMs) in vitro, which are also characterized by a hyper-endocytic phenotype and enhanced engulfment capacity relative to iPS-derived microglia-like cells (iMGLs). Together, these data define a conserved hyper-endocytic BAM phenotype and provide a toolbox for studying human BAMs both in vivo and in vitro.

neuroscience↗

bioRxiv2025

High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB

Genome editing has revolutionized plant biology research. However, efficient and straightforward delivery of editing reagents remains a major challenge. Viral delivery systems can address these issues, but CRISPR-Cas nucleases are often too large for viral vectors. Recently, smaller editors like TnpBs have been identified, but wild-type TnpBs are significantly less active than commonly used Cas9 nucleases. Here, we optimized a tobacco rattle virus (TRV)-based system to deliver newly discovered, highly active engineered ISDra2 TnpB variants. Our results demonstrate that the eTnpBc variant delivered via TRV enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci, significantly higher than that of wild-type ISDra2 TnpB. Additionally, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here are expected to accelerate broader adoption of eTnpBc for transformation- and transgene-free genome editing in plants.

plant biology↗

bioRxiv2025

Glucose and Oxygen Metabolism Coordinate Human Cortical Developmental Decisions

Defining metabolic regulation of neurodevelopmental programs is essential to approach developmental disorders and injuries driven by alterations in metabolism. In vitro cultures are the only available method to temporally perturb and study living human brain cells throughout neurogenesis, however most culture systems use supraphysiologic conditions of essential nutrients, glucose and oxygen. We probed how environmental exposure to endogenous-like concentrations impact metabolic state and developmental progression of cortical cell types using organoids. Nutrient accessibility globally impacted metabolic state, yet developmental responses to metabolic changes were cell type-specific. Metabolomic and transcriptomic datasets reveal increased TCA metabolites and amino acids and oxidative phosphorylation (OXPHOS) genes, under physiologic glucose conditions. Oxygen level had a modest, yet specific, molecular impact on deep layer excitatory neurons. We assessed consequences of metabolic changes on fate and observed that physiologic glucose expanded the human-enriched population of cortical stem cells, outer radial glia, and their progeny, upper layer excitatory neurons. Alterations in oxygen, instead, affected production of neurogenic progenitors and neuronal differentiation, with higher oxygen availability supporting shifts toward mitochondrial metabolism necessary for maturing cell types. We functionally tested this transition by inhibiting glycolysis; total inhibition promoted neuronal differentiation, whereas inhibition of anaerobic glycolysis/lactate production led to oRG expansion. Lactate signaling was sufficient to suppress oRG development and promote self-renewal of neurogenic progenitors. These data suggest that refined metabolic switches and decreased reliance on glucose are required for transition from stem cell self-renewal to more mature, diversified progenitor subtypes, where switch from anaerobic to aerobic metabolism discretely impacts progenitor diversification.

neuroscience↗

bioRxiv2025

De Novo Computational Design of VHH Nanobodies Against LGR5

VHH discovery traditionally relies on animal immunization or large-scale library screening, methods that are slow, costly, and often ineffective for challenging targets such as GPCRs. We present a fully de novo computational pipeline for epitope-directed VHH design, integrating generative backbone modeling, deep learning-based sequence optimization, and iterative experimental feedback. Using LGR5 as a model, we progressed from in silico design to functional binders without structural templates. Across three design-test-learn cycles, millions of candidates were reduced to epitope-specific binders with nanomolar affinity and high thermal stability (melting temperature [Tm] > 65 {degrees}C). Cryogenic electron microscopy (cryo-EM) confirmed atomic-level agreement (RMSD {approx} 2.2 [A]). This structure-validated approach accelerates timelines, reduces cost, and is broadly applicable to GPCRs and other membrane proteins, enabling "on-demand" therapeutic antibody generation.

bioengineering↗

bioRxiv2025

GPR34 regulation of disease-associated microglial states and responses to physiological stimuli

Expression of the G protein coupled receptor GPR34 is highly enriched in microglia and has been reported to be downregulated in several brain disease contexts, including Alzheimers disease (AD) and multiple sclerosis (MS). GPR34 function is poorly understood, as is its role in regulation of microglial states. Using RNA-sequencing, we find that microglia from Gpr34 knockout (KO) mouse brains exhibited a transcriptomic shift toward disease-associated microglia (DAM) and inflammatory profiles, partially resembling the microglial phenotype seen in 5xFAD AD model mice. Moreover, when Gpr34 KO mice were crossed with 5xFAD mice, the DAM transcriptional profile of microglia and glial pathology were further enhanced beyond the already robust DAM signature driven by 5xFAD alone. This occurred without affecting amyloid plaque burden. Human stem cell-derived microglia (iMGLs) lacking GPR34 showed reduced calcium (Ca{superscript 2}) and phosphorylated ERK (pERK) signaling in response to stimulation with known GPR34 agonists (lyso-phosphatidylserine (lysoPS) and myelin), as well as transcriptomic changes in immune regulation and cell proliferation related pathways. Interestingly, GPR34 KO iMGLs were selectively impaired in phagocytosis of myelin but not amyloid-{beta} (A{beta}) or E. coli, and showed a diminished transcriptional response elicited by myelin. Together, these findings suggest that GPR34 is important for maintaining microglia in a homeostatic state, promotes phagocytosis of and transcriptional response to myelin, and limits microglial activation in neurodegenerative disease conditions.

neuroscience↗

bioRxiv2025

Simulating Tandem Mass Spectra for Small Molecules using a General-Purpose Large-Language Model

We show a practical application of the Google Gemini large-language-model for simulating tandem mass spectra for compounds from the Blood Exposome Database. This approach bypasses the need for domain-specific model training, suggesting that the chemical fragmentation knowledge could be latently encoded within the Gemini model. General-purpose LLMs represent a useful and accessible tool for expanding in-silico spectral libraries and may accelerate the compound annotation in mass spectrometry-based metabolomics and exposomics.

bioinformatics↗

bioRxiv2025

Chronic Electronic Cigarette Exposure Promotes Atherosclerosis and Chondrogenic Modulation of Smooth Muscle Cells

BackgroundElectronic cigarette (E-cig) use has reached epidemic proportions worldwide, yet its cardiovascular consequences remain poorly defined. While several lines of evidences in human epidemiological and animal studies suggest chronic aerosol exposure accelerates atherosclerosis; the cellular and molecular mechanisms underlying this pathological remain unknown. MethodsWe exposed hyperlipidemic mice to chronic e-cigarette aerosol inhalation and characterized the plaque cellular landscape by coupling SMC lineage tracing with single-cell transcriptomic/epigenomic profiling and histologic phenotyping. We subsequently leveraged human coronary artery smooth muscle cells (HCASMCs) to validate in vivo discovery and identified E-cig specific pathological signaling pathways relevant to human vascular disease risk. ResultsChronic E-cig aerosol exposure accelerated atherosclerosis, increasing both SMC phenotypic modulation and plaque macrophage burden in a lipid-independent manner. Transcriptomically, SMCs are particularly more sensitive to E-cig than other vascular cell types. E-cig exposure reprogrammed SMCs toward a pro-calcifying, chondrogenic phenotype, thereby enhancing vascular ossification in vivo and in HCASMCs in vitro. Mechanistically, E-cig mediated SMC fate alteration occurs through activation of a glutamatergic/NMDAR signaling program, that increased NMDAR-dependent Ca2+ influx in a GRIN2A dependent manner. Notably, inhibition of GRIN2A mediated signaling reversed E-cig-induced pathological shifts in SMC phenotype. ConclusionsSMC chondrogenic reprogramming and subsequent vascular calcification are central to the detrimental cardiovascular consequences of E-cig exposure. Our findings implicate a GRIN2A-dependent glutamatergic/NMDAR signaling axis in SMC as a primary driver of this calcifying remodeling program. These findings define a SMC-specific vulnerability to E-cig aerosols and establish the GRIN2A/NMDAR pathway as potential therapeutic targets for mitigating E-cig-associated cardiovascular disease.

genomics↗

bioRxiv2025

A phenotypic brain organoid atlas for neurodevelopmental disorders

Thousands of genes are associated with neurodevelopmental disorders (NDDs), yet mechanisms and targeted treatments remain elusive. To fill these gaps, we present a CIRM-initiated NDD biobank of 352 publicly-available genetically-diverse patient-derived iPSCs, along with clinical details, brain imaging and genomic data, representing four major categories of disease: microcephaly (MIC), polymicrogyria (PMG), epilepsy (EPI), and intellectual disability (ID). From 35 representative patients, we studied over 6000 brain organoids for histology and single cell transcriptomics. Compared with an organoid library from ten neurotypicals, patients showed distinct cellular defects linked to underlying clinical disease categories. MIC showed defects in cell survival and excessive TTR+ cells, PMG showed intermediate progenitor cell junction defects, EPI showed excessive astrogliosis, and ID showed excessive generation of TTR+ cells. Our organoid atlas demonstrates both conserved and divergent NDD category-specific phenotypes, bridging genotype and phenotype. This NDD iPSC biobank can support future disease modeling and therapeutic approaches. HIGHLIGHTSO_LIResource of 352 CIRM-funded genetically-diverse IPSC lines from patients with neurodevelopmental disorders (NDDs). C_LIO_LIGenome/exome and brain images available for these genetically-diverse IPSC lines. C_LIO_LIDerived human brain organoids (hBOs) show disease-specific histological and cellular phenotypes. C_LIO_LIhBO phenotypes show unanticipated differentiation towards non-neuronal cell fates in NDDs. C_LI

neuroscience↗

bioRxiv2025

Computational Design of Soluble CCR8 Analogues with Preserved Antibody Binding

G protein-coupled receptors (GPCRs) represent the largest class of drug targets, yet their membrane-embedded nature poses significant challenges for structural studies and therapeutic development. Here, we report the successful computational design and experimental validation of soluble CCR8 analogues that maintain native antibody binding properties. Using an integrated pipeline combining ProteinMPNN-sol sequence design, and structure-based filtering, we generated 13 CCR8 analogues from 272 initial designs across three N-terminal truncation strategies. Experimental validation confirmed 62% success rate (8/13 designs) with protein yields of 1.19-73.72 mg/L in aqueous buffer, representing a significant improvement over traditional membrane protein production method. Surface plasmon resonance analysis demonstrated that all analogues retained mAb1 binding with dissociation constants ranging from 77-857 nM, comparable to wild-type CCR8 (KD = 190 nM). Despite extensive sequence divergence (10-13% identity with wild-type CCR8), structural integrity was preserved as evidenced by binding affinity maintenance and computational structural validation. This work demonstrates the feasibility of computationally designing functional soluble analogues of challenging membrane proteins, with implications for accelerating drug discovery, antibody development, and structural biology studies. Our approach addresses critical limitations in membrane protein accessibility while preserving native epitope presentation, opening new avenues for therapeutic target characterization and binder discovery.

bioengineering↗

bioRxiv2025

Cellular and Molecular Architecture of Renin-Angiotensin System Signaling in the PVN Under Cardiometabolic Stress

The hypothalamic paraventricular nucleus (PVN) integrates neuroendocrine and autonomic signals that regulate blood pressure and metabolism. Although the renin- angiotensin system (RAS) is implicated in neurogenic hypertension and obesity, cell-type- specific expression and regulation of its components within the PVN remain poorly understood. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to profile the transcriptomic landscape of the PVN in male mice under baseline conditions and in models of DOCA-salt-induced hypertension and high-fat diet (HFD)-induced obesity. We identified major PVN cell types, including neurons, astrocytes, precursor oligodendrocytes, oligodendrocytes, microglia and endothelial cells, and further resolved eight transcriptionally distinct neuronal subtypes. Expression of RAS-related genes was highly cell-type specific: Agt (angiotensinogen) was enriched in astrocytes, whereas Ace (angiotensin-converting enzyme), Atp6ap2 (also known as the (pro)renin receptor [PRR]), Agtr1a (angiotensin II type 1a receptor, aka AT1aR), Lnpep (leucyl/cystinyl aminopeptidase, aka angiotensin 4 receptor [AT4R]), and the Mas1 proto-oncogene were predominantly expressed in neurons. DOCA-salt treatment increased the proportion of GABAergic and vasopressin neurons and enhanced neuronal Agt and Atp6ap2 expression, while reducing astrocytic Agt, suggesting activation of a vasoconstrictive RAS axis. HFD exposure increased excitatory and stress-responsive neuronal subtypes (glutamatergic, vasopressin, corticotropin-releasing hormone) and upregulated Atp6ap2, Agtr1b, Lnpep, and Mas1 in vasopressin neurons, while downregulating multiple RAS genes in GABAergic neurons. These findings reveal dynamic, cell-type-specific remodeling of RAS signaling in the PVN in response to hypertensive and metabolic stress, providing a transcriptomic atlas of RAS expression in the PVN and identifying potential cellular targets for therapeutic strategies addressing cardiometabolic disorders.

genomics↗