Biology subjects

Nguyen, T.

Publications and source records attributed to Nguyen, T..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Glucocerebrosidase Deficiency Dysregulates Human Astrocyte Lipid Metabolism

BackgroundDeficiency in the lysosomal enzyme, glucocerebrosidase (GCase), caused by mutations in the GBA1 gene, is the most common genetic risk factor for Parkinsons disease (PD). However, the consequence of reduced enzyme activity within neural cell sub-types remains ambiguous. Thus, the purpose of this study was to define the effect of GCase deficiency specifically in human astrocytes and test their non-cell autonomous influence upon dopaminergic neurons in a midbrain organoid model of PD. MethodsWild-type (GBA+/+), N370S mutant (GBA+/N370), and GBA1 knockout (GBA-/-) astrocytes were rapidly and directly induced from human pluripotent stem cells (hPSCs) via transcription factor-based differentiation. These astrocytes were extensively characterized for GCase-dependent phenotypes using immunocytochemistry, organoid coculture, enzymatic assays, lipid tracers, transcriptomics, and lipidomics. ResultshPSC lines were rapidly induced into astrocytes and enzymatic assays confirmed that GBA-/- astrocytes completely lacked GCase activity, while GBA+/N370 preserved partial activity. GBA-/-, but not GBA+/N370S, exhibited lysosomal alterations, with enlarged lysosomes and glucosylceramide (GlcCer) accumulation. GCase deficiency also exacerbated TNF-alpha-induced secretion of the inflammatory biomarker, CCL2. In midbrain organoids, GCase activity did not modulate the ability of astrocytes to support dopamine neuron production and survival. Lipidomics revealed a GBA-/--specific increase in sphingomyelin, and a decrease of triglycerides. Direct rescue of GCase activity with GBA1 mRNA treatment reduced GlcCer accumulation. Astrocytes exhibited a relatively high uptake and storage of fatty acid analogs as lipid droplets, in comparison to neurons, and this process was impaired in GBA-/- astrocytes. Lastly, GBA-/- astrocytes accumulate neuronal membrane-derived GlcCer. These findings highlight the critical role of astrocytic GCase in lipid metabolism and its neuronal influence. ConclusionGCase deficiency does not inhibit human astrocyte differentiation nor cause a non-cell autonomous neurotoxic effect upon dopaminergic neurons within midbrain organoids. However, it does elicit enhanced inflammatory reactivity, accumulation of GlcCer, and a distinct lipidomic profile, indicating impaired lipid metabolism in astrocytes that can dysregulate neuron-astrocyte intercellular signaling. Overall, these insights underscore dysfunctional astrocyte lipid metabolism as a high priority therapeutic target in Parkinsons disease and related neurodegenerative disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/632210v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1b1138forg.highwire.dtl.DTLVardef@7b26aborg.highwire.dtl.DTLVardef@127b950org.highwire.dtl.DTLVardef@f74a59_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

TSPO-PET Reveals Higher Inflammation in White Matter Disrupted by Paramagnetic Rim Lesions in Multiple Sclerosis

ObjectiveTo explore whether the inflammatory activity is higher in white matter (WM) tracts disrupted by paramagnetic rim lesions (PRLs) and if inflammation in PRL-disrupted WM tracts is associated with disability in people with multiple sclerosis (MS). MethodsForty-four MS patients and 16 healthy controls were included. 18 kDa-translocator protein positron emission tomography (TSPO-PET) with the 11C-PK11195 radioligand was used to measure the neuroinflammatory activity. The Network Modification Tool was used to identify WM tracts disrupted by PRLs and non-PRLs that were delineated on MRI. The Expanded Disability Status Scale was used to measure disability. ResultsMS patients had higher inflammatory activity in whole brain WM compared to healthy controls (p=0.001). Compared to patients without PRLs, patients with PRLs exhibited higher levels of inflammatory activity in the WM tracts disrupted by any type of lesions (p=0.02) or PRLs (p=0.004). In patients with at least one PRL, inflammatory activity was higher in WM tracts highly disrupted by PRLs compared to WM tracts highly disrupted by non-PRLs (p=0.009). Elevated inflammatory activity in highly disrupted WM tracts was associated with increased disability in patients with PRL (p=0.03), but not in patients without PRL (p=0.2). InterpretationThis study suggests that patients with PRLs may exhibit more diffuse WM inflammation in addition to higher inflammation along WM tracts disrupted by PRLs compared to non-PRLs, which could contribute to larger lesion volumes and faster disability progression. Imaging PRLs may serve to identify patients with both focal and diffuse inflammation, guiding therapeutic interventions aimed at reducing inflammation and preventing progressive disability in MS.

neuroscience↗

bioRxiv2024

Design and Validation of a Wearable Imaging System for Automated Wound Monitoring in Porcine Model

Effective wound monitoring has the potential to guide treatment regiments and improve healing outcomes, yet current clinical assessment methods remain largely subjective and labor-intensive. To address this challenge, we present a high-resolution wearable imaging system designed for continuous wound monitoring. The system integrates a 64 MP camera with a plano-convex lens housed in an enclosure measuring 73 mm in diameter and 36.1 mm in height, and features a custom printed circuit board (PCB) for programmable LED illumination. The 3D-printed device enclosure is designed to accommodate a silicone bioelectronic device and can be securely attached using a commercially available ostomy skin barrier. In porcine model validation studies, the system successfully captured daily wound progression over periods up to 7 days. The captured images were wirelessly transmitted to a processing unit where DeepMapper, a machine learning algorithm, processed z-stacked images and performed multi-level feature extraction to predict wound healing stages and indicate potential complications such as infection. This imaging system enables automated analysis of wound progression and supports the development of smart wound care platforms for personalized treatment strategies. The integrated design approach demonstrates the feasibility of creating compact, high-resolution imaging systems suitable for clinical wound monitoring applications.

bioengineering↗

bioRxiv2024

Remote-Controlled Wireless Bioelectronics for Fluoxetine Therapy to Promote Wound Healing in a Porcine Model

Wound healing presents a significant challenge in biomedical science, requiring precise therapeutic delivery and real-time monitoring. Bioelectronic systems offer a promising solution but remain largely unexplored for wound care, particularly in large animal models that reflect human healing dynamics. This study introduces a remote controlled wireless bioelectronic platform equipped with an iontophoretic pump to deliver fluoxetine, a selective serotonin reuptake inhibitor that promotes wound repair. In vitro and ex-vivo testing validated efficient on demand fluoxetine delivery. In vivo experiments in a porcine wound model demonstrated clear therapeutic efficacy over 3-day and 7-day periods. The system enhanced healing outcomes, increasing re-epithelialization by 37% (H&E staining), reducing the M1/M2 macrophage ratio by 33%, and stimulating neuronal growth at the wound site. This bioelectronic platform delivers fluoxetine in a controlled, remotely-controlled manner while allowing for wound direct wound imaging that can be used to monitor wound healing progress. Additionally, it allows precise dose and temporal delivery of treatment to enhance the outcome of future large animal wound healing studies.

bioengineering↗

bioRxiv2024

Wireless bioelectronic device for wound healing

Wireless bioelectronic actuators have been developed to deliver targeted treatments over multiple days while continuously monitoring delivery, thereby improving wound healing. Specifically, these devices can deliver charged biomolecules such as fluoxetine cations (Flx+) and electric field (EF) in freely moving pigs. Treatments can be controlled and monitored in real time via WiFi, with options for both user-specified delivery rates and durations, as well as automated closed-loop (CL) control. The devices are engineered to handle various failure scenarios that may arise in dynamic, real-world experiments--such as communication or power interruptions--ensuring that valuable experimental data is collected with minimal disruption. The ion pump features eight drug reservoirs and channels arranged around a 20 mm diameter-wound, with a central ground electrode (0 V). When voltages above 0 V are applied to the outer channels, currents flow from the reservoirs and channels into the wound, delivering Flx+ and/or EF depending on the reservoir solution. The device records applied voltages and currents locally to a microSD card at a high sampling rate, while simultaneously transmitting real-time measurements via a local WiFi network to a wound healing algorithm running on a nearby laptop. CL control of current/delivery rate is performed by an onboard microcontroller unit (MCU) and current-source microchips, based on instructions received from the wound healing algorithm. A graphical user interface (GUI) provides intuitive user control and real-time data visualization, with support for multiple devices. In vivo studies over seven days showed that Flx+-treated wounds had a 20% lower M1/M2 macrophage ratio and 41.67% greater re-epithelialization compared to controls (standard-of-care), demonstrating the actuators potential to enhance wound healing.

biochemistry↗

bioRxiv2024

Impact of wildfire smoke and diesel exhaust on inflammatory response in aging human microglia

BackgroundAir pollution, particularly from Diesel Exhaust Particles (DEP) and Wildfire Smoke (WFS), is increasingly recognised as a significant driver of neuroinflammation linked to brain diseases. However, the role of microglia in mediating these neuroinflammatory responses remains poorly understood. This study aimed to investigate the effects of air pollution on monocyte-derived microglia-like cells (MDMi) from both young (< 40 years of age), and (older > 60 years of age) healthy individuals, focusing on immune response, cytokine secretion, nitrosative stress, and phagocytic activity. ResultsOur study demonstrated that DEP and WFS extract (WFSE) significantly upregulated expression of the oxidative stress marker, heme-oxygenase-1 (HO-1) in MDMi after 24 hr, with levels normalising by 96 hr, indicating a transient oxidative stress response. Both DEP and WFSE elicited distinct inflammatory cytokine profiles. DEP induced a rapid response, increasing TNF-, IL-6, IL-23, and IL-33 within 2 hr in young MDMi and 24 hr in aged MDMi. In contrast, WFSE triggered a delayed but sustained inflammatory response, with TNF-, IFN-{gamma}, IL-23, and IL-33 levels persisting at 96 hr in aged MDMi, highlighting an age-related vulnerability to air pollutant-induced inflammation. Both pollutants activated p38, ERK, and NF-{kappa}B pathways, with p38 activity resolving by 96 hours and ERK activation persisting, reflecting their distinct roles in cellular stress and inflammation. NF-{kappa}B p65 nuclear translocation, observed at 24 hours, highlighted its critical role in cytokine release and inflammation following exposure to DEP and WFSE. This is the first report of NF-{kappa}B activation in human microglia exposed to air pollutants. ConclusionsThese results highlight the distinct and potentially harmful effects of DEP and WFSE on immune and inflammatory responses in MDMi, particularly in ageing populations, with significant implications for brain health. DEP triggers acute oxidative stress and inflammatory responses, while WFSE induces more prolonged effects, especially in aged microglia. Both pollutants activate the MAPK and NF-{kappa}B pathways and exhibit unique cytokine profiles, underscoring their overlapping yet distinct mechanisms of action. These findings advance our understanding of air pollutant-induced neuroinflammation and its contribution to neurodegeneration, providing a foundation for developing targeted interventions to mitigate the neurotoxic effects of air pollution.

cell biology↗

bioRxiv2024

Imaging the dancing brain: Decoding sensory, motor and social processes during dyadic dance

Real-world social cognition requires processing and adapting to multiple dynamic information streams. Interpreting neural activity in such ecological conditions remains a key challenge for neuroscience. This study leverages advancements in de-noising techniques and multivariate modeling to extract interpretable EEG signals from pairs of participants (male-male, female-female, and male-female) engaged in spontaneous dyadic dance. Using multivariate temporal response functions (mTRFs), we investigated how music acoustics, self-generated kinematics, other-generated kinematics, and social coordination uniquely contributed to EEG activity. Electromyogram recordings from ocular, face, and neck muscles were also modeled to control for artifacts. The mTRFs effectively disentangled neural signals associated with four processes: (I) auditory tracking of music, (II) control of self-generated movements, (III) visual monitoring of partner movements, and (IV) visual tracking of social coordination. We show that the first three neural signals are driven by event-related potentials: the P50-N100-P200 triggered by acoustic events, the central lateralized movement-related cortical potentials triggered by movement initiation, and the occipital N170 triggered by movement observation. Notably, the (previously unknown) neural marker of social coordination encodes the spatiotemporal alignment between dancers, surpassing the encoding of self-or partner-related kinematics taken alone. This marker emerges when partners can see each other, exhibits a topographical distribution over occipital areas, and is specifically driven by movement observation rather than initiation. Using data-driven kinematic decomposition, we further show that vertical bounce movements best drive observers EEG activity. These findings highlight the potential of real-world neuroimaging, combined with multivariate modeling, to uncover the mechanisms underlying complex yet natural social behaviors. Significance statementReal-world brain function involves integrating multiple information streams simultaneously. However, due to a shortfall of computational methods, laboratory-based neuroscience often examines neural processes in isolation. Using multivariate modeling of EEG data from pairs of participants freely dancing to music, we demonstrate that it is possible to tease apart physiologically established neural processes associated with music perception, motor control, and observation of a partners movement. Crucially, we identify a previously unknown neural marker of social coordination that encodes the spatiotemporal alignment between dancers, beyond self- or partner-related kinematics alone. These findings highlight the potential of computational neuroscience to uncover the biological mechanisms underlying real-world social and motor behaviors, advancing our understanding of how the brain supports dynamic and interactive activities.

neuroscience↗

bioRxiv2024

Galectin-1 Modulates Hepatocellular Carcinoma Response to Thermal Ablation Through Regulating Glycolysis

I.Background & AimsThermal ablation is the standard of care treatment modality with curative intent for early-stage hepatocellular carcinoma (HCC), but its efficacy remains moderately limited-- with up to 40% of HCC patients experiencing local recurrence post-treatment. This study aimed to evaluate the prognostic value of galectin-1 (Gal-1) in predicting thermal-ablation responsiveness. We then evaluated the therapeutic potential of targeting Gal-1 in inhibition of glycolysis and subsequently enhancing thermal-ablation efficacy. MethodsLiquid-Chromatography Mass-Spectrometry (LC-MS) was employed to analyze proteomic profiles of retrospectively collected pre-ablation FFPE samples of known thermal-ablation responders and nonresponders. An in-vitro thermal peri-ablation model was established using a heated water bath. Gal-1 inhibition via OTX008 or knockdown was utilized to investigate hyperthermic sensitivity. Hyperthermia-resistant SNU449 cells were used to establish an orthotopic murine model to evaluate the combination therapy of OTX008 and thermal ablation. Harvested tumors were analyzed by LC-MS to determine their metabolic profiles. ResultsThis study revealed that responders had significantly longer tumor progression-free survival compared to nonresponders (57.0{+/-}1.6 (median not reached) versus 8.3{+/-}0.5 months (median: 13.6 months), p<0.001). Moreover, responders were found to have significant downregulation of Gal-1 expression compared to that of nonresponders. Gal-1 inhibition or knockdown markedly increased hyperthermic sensitivity in hyperthermia-resistant HCC SNU449 cells. Targeting Gal-1 by OTX008 in combination with thermal ablation significantly reduced SNU449-derived tumor growth compared to the thermal-ablation alone group in vivo. Metabolomic analysis revealed decreased glycolytic metabolites, fructose 1,6-bisphosphate, 3-phosphoglycerate and phosphoenolpyruvate, while western blot analysis showed decreased Gal-1 expression in the combined treatment group compared to monotherapy thermal ablation or OTX008 treatment. ConclusionsGal-1 overexpression correlates with thermal-ablation nonresponsiveness, and targeting Gal-1 enhances thermal-ablation efficacy by inhibiting glycolysis. Impact and ImplicationsDespite being a standard-of-care treatment for early-stage HCC, thermal ablation has a high local recurrence rate of approximately 40%. While thermal ablation can lead to cellular death in the central-treatment zone, its metabolic impact on cells in the peri-ablational region remains unclear. This study shows the direct association between Gal-1 overexpression and thermal-ablation nonresponsiveness. Moreover, it found that Gal-1 inhibition or knockdown increased hyperthermia sensitivity in vitro. Targeting Gal-1 in combination with thermal-ablation significantly reduced hyperthermia-resistant SNU449 tumor growth by inhibiting glycolysis in vivo. These findings suggest that the efficacy of thermal ablation in HCC can be enhanced by pharmacologically inhibiting Gal-1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/628238v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18788ccorg.highwire.dtl.DTLVardef@1261839org.highwire.dtl.DTLVardef@5d57org.highwire.dtl.DTLVardef@1f00b2a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIProfiling pre-ablation HCC biopsies reveals Galectin-1 as a key prognostic biomarker for response prediction in thermal ablation C_LIO_LITargeting Galectin-1 with a selective inhibitor (OTX008) enhances the efficacy of thermal ablation in HCC C_LIO_LIGalectin-1 modulates thermal-ablation response via regulating glycolysis in HCC C_LI

cancer biology↗

bioRxiv2024

THE FAM53C/DYRK1A axis regulates the G1/S transition of the cell cycle

A growing number of therapies are being developed to target the cell cycle machinery for the treatment of cancer and other human diseases. Consequently, a greater understanding of the factors regulating cell cycle progression becomes essential to help enhance the response to these new therapies. Here, using data from the Cancer Dependency Map, we identified FAM53C as a new regulator of cell cycle progression. We found that FAM53C is critical for this cell cycle transition and that it acts upstream of the CyclinD-CDK4/6-RB axis and of p53 in the regulation of the G1/S transition. By mass spectrometry, biochemical, and cellular assays, we identified and validated DYRK1A as a cell cycle kinase that is inhibited by and directly interacts with FAM53C. Consistent with the role for FAM53C identified in cells in culture, FAM53C knockout human cortical organoids display increased cell cycle arrest and growth defects. Fam53C knockout mice show minor behavioral phenotypes. Because DYRK1A dysregulation contributes to developmental disorders such as Down syndrome as well as tumorigenesis, future strategies aiming at regulating FAM53C activity may benefit a broad range of patients.

cell biology↗

bioRxiv2024

Potent antitumor activity of a designed interleukin-21 mimic

Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a broad range of affinities while dampening suppressive regulatory T (Treg) cell responses, but current approaches achieve these goals with limited success. Here, we report a de novo IL-21 mimic, 21h10, designed to have augmented stability and high signaling potency in both humans and mice. In multiple animal models and in ex vivo human melanoma patient derived organotypic tumor spheroids (PDOTS), 21h10 showed robust antitumor activity. 21h10 generates significantly prolonged STAT signaling in vivo compared with native IL-21, and has considerably stronger anti-tumor activity. Toxicities associated with systemic administration of 21h10 could be mitigated by TNF blockade without compromising antitumor efficacy. In the tumor microenvironment, 21h10 induced highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells and driving high expression of interferon- (IFN-) and granzyme B compared to native IL-21, while increasing the frequency of IFN-+ Th1 cells and reducing that of Foxp3+ Tregs. As 21h10 has full human/mouse cross-reactivity, high stability and potency, and potentiates low-affinity antitumor responses, it has considerable translational potential.

biochemistry↗

bioRxiv2024

Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of a chemical moiety to DNA to expand current modalities for precision gene editing.

synthetic biology↗

bioRxiv2024

PYR1 biosensor-driven genome-wide CRISPR screens for improved monoterpenoid production in Kluyveromyces marxianus

Monoterpenoids are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microbial biosynthesis offers sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpenoid production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor--a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket--we screened 24 monoterpenoids and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve titer of a desired metabolite.

synthetic biology↗

bioRxiv2024

Polymorphic tandem repeats shape single-cell gene expression across the immune landscape

Tandem repeats (TRs) - highly polymorphic, repetitive sequences across the human genome - are important regulators of gene expression but remain underexplored due to challenges in accurate genotyping and analysis1. Here, we generate new whole genome and single-cell RNA sequencing from >5.4 million blood-derived cells across 1,925 individuals in two cohorts [Cuomo et al., accompanying manuscript], and perform meta-analysis to characterize the impact of variation in >1.7 million TR loci on immune cell type-specific gene expression. We identify >69,000 single-cell expression TR loci (sc-eTRs), 30.7% of which are specific to one of 28 immune cell types, and reveal dynamic regulatory effects using cell-state inference. Matched single-cell ATAC sequencing profiles from >3.4 million nuclei in 922 individuals [Xue et al., accompanying manuscript]. uncover chromatin accessibility QTLs for nearly one-third of expression-associated TRs, supporting coordinated effects on cis-regulatory architecture. Fine-mapping implicates 1,490 TRs as candidate causal drivers of gene expression in 6.1% of tested genes, and colocalization analyses highlight >200 genes in which TRs likely mediate genetic associations with immune and hematological traits. Together, these results provide a genome-wide, multiomic view of TR-mediated regulation in the human immune system, establishing TRs as key contributors to cell type-specific regulatory variation and complex trait architecture.

genomics↗

bioRxiv2024

Evolutionary history and rhizosphere microbial community composition in domesticated hops (Humulus lupulus L.)

Humulus lupulus L., commonly known as hops, is a perennial crop grown worldwide and is well known for its pharmacological, commercial, and most importantly brewing applications. For hundreds of years, hops have undergone intense artificial selection with over 250 cultivated varieties being developed worldwide, all displaying differences in key characteristics such as bitter acid concentrations, flavor and aroma profiles, changes in photoperiod, growth, and pathogen/pest resistances. Previous studies have individually explored differences between cultivars, aiming to identify markers that can quickly and cost-effectively differentiate between cultivars. However, little is known about their evolutionary history and the variability in their associated rhizospheric microbial communities. Coupling phenotypic, genomic, and soil metagenomic data, our study aims to explore the global population structure and domestication history of 98 hops cultivars. Additionally, we assessed differences in growth rates, rates of viral infection, usage of dissolvable nitrogen, and soil microbial community compositions between US and non-US based cultivars. Contrary to previous studies, our study revealed that worldwide hop cultivars cluster into four primary subpopulations; Central European, English, and American ancestry as previously reported, and one new group, the Nobles, revealing further substructure amongst Central European cultivars. Modeling the evolutionary history of domesticated hops reveals an early divergence of the common ancestors of modern US cultivars around 2800 ybp, and more recent divergences with gene flow across English, Central European, and Noble cultivars, reconciled with key events in human history and migrations. Furthermore, cultivars of US origin were shown to overall outperform non-US cultivars in both growth rates and usage of dissolvable nitrogen and display novel microbial composition.

evolutionary biology↗

bioRxiv2024

Identification of the Regulatory Elements and Protein Substrates of Lysine Acetoacetylation

Short chain fatty acylations establish connections between cell metabolism and regulatory pathways. Lysine acetoacetylation (Kacac) was recently identified as a new histone mark. However, regulatory elements, substrate proteins, and epigenetic functions of Kacac are not yet fully understood, hindering further in-depth understanding of acetoacetate modulated (patho)physiological processes. Here, we created a chemo-immunological approach for reliable detection of Kacac, and demonstrated that acetoacetate serves as the primary precursor for histone Kacac. We report the enzymatic addition of the Kacac mark by the acyltransferases GCN5, p300, and PCAF, and its removal by deacetylase HDAC3. Furthermore, we establish acetoacetyl-CoA synthetase (AACS) as a key regulator of cellular Kacac levels. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveal the broad impacts of Kacac on multifaceted cellular processes. These findings unveil pivotal regulatory mechanisms for the acetoacetate-mediated Kacac pathway, opening a new avenue for further investigation into ketone body functions in various pathophysiological states.

biochemistry↗

bioRxiv2024

Inhibition of Ribosome Biogenesis in vivo Causes p53-Dependent Death and p53-Independent Dysfunction

Ribosomes are critical for cell function; their synthesis (known as ribosome biogenesis; "RiBi") is complex and energy-intensive. Surprisingly little is known about RiBi in differentiated cells in vivo in adult tissue. Here, we generated mice with conditional deletion of Nat10, an essential gene for RiBi and translation, to investigate effects of RiBi blockade in vivo. We focused on RiBi in a long-lived, ribosome-rich cell population, pancreatic acinar cells, during homeostasis and tumorigenesis. We observed a surprising latency of several weeks between Nat10 deletion and onset of structural and functional abnormalities and p53-dependent acinar cell death, which was associated with translocation of ribosomal proteins RPL5 and RPL11 into acinar cell nucleoplasm. Indeed, deletion of Trp53 could rescue acinar cells from apoptotic cell death; however, Nat10{Delta}/{Delta}; Trp53{Delta}/{Delta} acinar cells remained morphologically and functionally abnormal. Moreover, the deletion of Trp53 did not rescue the lethality of inducible, globally deleted Nat10 in adult mice nor did it rescue embryonic lethality of global Nat10 deletion, emphasizing p53-independent consequences of RiBi inhibition. Deletion of Nat10 in acinar cells blocked Kras-oncogene-driven pancreatic intraepithelial neoplasia and subsequent pancreatic ductal adenocarcinoma, regardless of Trp53 mutation status. Together, our results provide initial insights into how cells respond to defects in RiBi and translation in vivo.

cell biology↗

bioRxiv2024

Aryl-hydrocarbon receptor in smooth muscle cells protect against dioxin induced adverse remodeling of atherosclerosis

IntroductionEnvironmental exposure to dioxin has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity, however, the detailed mechanism linking dioxin exposure to adverse SMC modulation is not well understood. MethodsSingle-cell RNA and ATAC sequencing and histological analyses were performed on the aorta from mouse models of atherosclerosis exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or control. Primary human coronary artery SMC (HCASMC) treated in culture with TCDD were used to perform RNA-Seq, ATAC-Seq, and functional phenotypic assays. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21, two of known SMC modulating transcription factors. ResultsModulated SMC were the most transcriptionally responsive cell type to dioxin in the atherosclerotic aorta. Dioxin accelerated disease phenotype by promoting a modulated SMC phenotype early, resulting in increased lesion size, migration of SMC, and macrophage recruitment to the lesion. We found C3 expressing modulated SMCs to be likely contributing to the increased macrophage recruitment and inflammation. Analysis of the RNA-Seq data from HCASMC treated with TCDD showed differential enrichment of biological pathways related to cell migration, localization, and inflammation. Furthermore, ATAC-Seq data showed a significant activation for pathways regulating vascular development, cell migration, inflammation, and apoptosis. With TCDD treatment, there was also enrichment of AHR ChIP-Seq peaks, while the TCF21 enrichment decreased significantly. The SMC-specific Ahr knockout resulted in increased oxidative stress in SMC, increased lesion size and macrophage content, and loss of SMC lineage cells in the lesion cap when exposed to TCDD, consistent with a more vulnerable plaque phenotype. ConclusionDioxin adversely remodels atherosclerotic plaque by accelerating the SMC- phenotypic modulation, and increasing inflammation and oxidative stress resulting in increased macrophage recruitment and lesion size. Dioxin may adversely affect the SMC phenotype and disease state by affecting the TCF21 occupancy in the open chromatin regions. Furthermore, we observed that SMC-specific deletion of Ahr in mice resulted in worsening of dioxin mediated SMC modulation and atherosclerosis, suggesting that Ahr in SMC confers protection against dioxin by promoting a stable plaque phenotype and reducing dioxin induced oxidative stress. SummaryO_LIExposure to dioxin, an environmental pollutant present in tobacco smoke and air pollution, accelerates smooth muscle cell modulation, and atherosclerosis. C_LIO_LIDioxin exposure leads to inflammatory smooth muscle cell phenotype characterized by complement pathway activation and increased macrophage recruitment to plaque C_LIO_LIAryl-hydrocarbon receptor in SMC protects against oxidative stress, and promotes a stable plaque phenotype C_LI

molecular biology↗

bioRxiv2024

Broadly Reactive Anti-VHH Antibodies for Characterizing, Blocking, or Activating Nanobody-Based CAR-T Cells

Production of chimeric antigen receptor T cell (CAR-T) therapies is dependent on the use of antibody reagents to label, isolate, and/or expand T cell products. We sought to create antibody-based tools that directly target the variable domain of heavy-chain only antibodies (VHH or nanobody) used in some CAR molecules. Two murine antibodies were identified which bind to distinct epitopes in the conserved framework regions of llama-derived VHHs, and not to human VH domains. We produced a high-quality dual-clonal anti-VHH antibody product which reacts with over 98% of VHH proteins, regardless of their antigenic specificity. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus could be used for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies potently inhibited VHH-CAR function, blocking CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH antibodies could also be applied for activation and expansion of VHH CAR-T cells, inducing 730-fold mean expansion, >94% CAR purity, with retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen specific activity without functional exhaustion. Overall, these data identify a useful new tool for understanding and manipulating VHH-based CAR-T cells. Funding SourceThis work was funded by the National Research Council Canada Disruptive Technology Solutions Cell and Gene Therapy challenge program, and BioCanRx Declaration of interestsThe anti-VHH antibodies reported here are the subject of a provisional patent application by the National Research Council of Canada

immunology↗