Biology subjects

Nguyen, M.

Publications and source records attributed to Nguyen, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Injectable butyrate-prodrug micelles induce long-acting immune modulation and suppress autoimmune arthritis in mice

Dysbiosis is linked to autoimmune diseases such as rheumatoid arthritis (RA), where microbial metabolites, such as short chain fatty acids (SCFAs), mediate the so-called gut-joint axis. The therapeutic potential of SCFAs is limited due to the frequent and high oral dosage requirements. RA is characterized by aberrant activation of peripheral T cells and myeloid cells. We aim to deliver butyrate, an SCFA, directly to the lymphatics using a polymeric micelle as a butyrate prodrug, creating a depot for inducing long-lasting immunomodulatory effects. Notably, negatively charged micelles (Neg-ButM) demonstrate superior efficacy in targeting the lymphatics post-subcutaneous administration, and were retained in the draining lymph nodes, spleen, and liver for over a month. In a mouse RA model, we found that Neg-ButM substantially mitigated arthritis symptoms and promoted tolerogenic phenotypes in T cells and myeloid cells, both locally and systemically. These findings suggest potential applications of this approach in treating inflammatory autoimmune diseases.

bioengineering↗

bioRxiv2023

Atheroma plaque microenvironment stimulates kynurenine production by macrophages to induce endothelial adhesion molecules in the context of atherogenesis

Cardiovascular diseases, including atherosclerosis, are major causes of morbidity and mortality worldwide. Here, we investigate the role of the kynurenine pathway (KP) in macrophages in the context of atheroma plaque microenvironment and its impact on atherogenesis. Using an in vitro model of primary human macrophages, we observed that exposure to plaque homogenates induces a marked increase in the early steps of the KP which impacts on kynurenine production. This was confirmed by immunostaining on human plaque of carotid arteries. Further investigation into the underlying molecular mechanisms revealed that LXR signaling contributes to this plaque microenvironment-induced KP activation. We showed that kynurenine released from macrophages affected endothelial cells, leading to increased expression of ICAM-1 and VCAM-1 in an AhR-dependent manner. Consistently with the proatherogenic effects, in a cohort of atherosclerotic patients, we observed higher levels of plasma kynurenine in patients with lower extremity arterial disease. In line with the results of in vitro investigations, the plasma kynurenine levels were associated plaque oxysterol content. Using a multiple logistic regression model, we showed that plasma kynurenine was independently associated with lower extremity arterial disease in atherosclerotic patients. Altogether, our data indicate that the activation of KP in macrophages in the context of atheroma plaque is partially mediated by LXR axis and leads to the release of kynurenine. This, in turn, contributes to the exacerbation of both local and peripheral atherosclerosis particularly through the activation of endothelial cells.

immunology↗

bioRxiv2023

A paradigm for ethanol consumption in head-fixed mice during prefrontal cortical two-photon calcium imaging

The prefrontal cortex (PFC) is a hub for higher-level cognitive behaviors and is a key target for neuroadaptations in alcohol use disorders. Preclinical models of ethanol consumption are instrumental for understanding how acute and repeated drinking affects PFC structure and function. Recent advances in genetically encoded sensors of neuronal activity and neuromodulator release combined with functional microscopy (multiphoton and one-photon widefield imaging) allow multimodal in-vivo PFC recordings at subcellular and cellular scales. While these methods could enable a deeper understanding of the relationship between alcohol and PFC function/dysfunction, they require animals to be head-fixed. Here, we present a method in mice for binge-like ethanol consumption during head-fixation. Male and female mice were first acclimated to ethanol by providing home cage access to 20% ethanol (v/v) for 4 or 8 days. After home cage drinking, mice consumed ethanol from a lick spout during head-fixation. We used two-photon calcium imaging during the head-fixed drinking paradigm to record from a large population of PFC neurons (>1000) to explore how acute ethanol affects their activity. Drinking modulated activity rates in a subset of neurons on slow (minutes) and fast (seconds) time scales but the majority of neurons were unaffected. Moreover, ethanol intake did not significantly affect network level interactions in the PFC as assessed through inter-neuronal pairwise correlations. By establishing a method for binge-like drinking in head-fixed mice, we lay the groundwork for leveraging advanced microscopy technologies to study alcohol-induced neuroadaptations in PFC and other brain circuits. HighlightsO_LIC57BL/6J mice voluntarily consume ethanol to binge-like levels during head-fixation, with females consuming more ethanol than males. C_LIO_LIMice show differences in frontloading and licking behavior for head-fixed ethanol and sucrose consumption. C_LIO_LIHead-fixed paradigm enables two-photon calcium imaging in the anterior cingulate cortex subdivision of the prefrontal cortex. C_LIO_LIAcute ethanol increases and decreases single neuron activity at fast (seconds) and slow (minutes) time scales but does not alter pairwise correlations between neurons. C_LI

neuroscience↗

bioRxiv2023

Strategy of selection and optimization of single domain antibodies targeting the PHF6 linear peptide within the Tau intrinsically disordered protein

The use of VHHs (Variable domain of the Heavy-chain of the Heavy-chain-only antibodies) as disease-modifying biomolecules in neurodegenerative disorders holds promises including to target aggregation-sensitive proteins. Exploitation of their clinical values dependents however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the Tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using nuclear magnetic resonance spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures of three complexes consisting of an optimized VHH and a peptide containing the Tau epitope. Interestingly, the ability of the VHH variants to inhibit Tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in our capacity to propose two VHH variants derived from the parent Z70 for their next development steps.

biochemistry↗

bioRxiv2023

Human Th17- and IgG3-associated autoimmunity induced by a translocating gut pathobiont

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFN{gamma}+ Th17 differentiation and IgG3 subclass switch of anti-E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence SummaryTranslocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

immunology↗

bioRxiv2023

Seryl-butyrate: a prodrug that enhances butyrate's oral bioavailability and suppresses autoimmune arthritis and experimental autoimmune encephalomyelitis

Butyrate, a metabolite produced by commensal bacteria, has been intensively studied for its immunomodulatory effects on various immune cells, including T regulatory cells, macrophages, and dendritic cells. Butyrates development as a drug has been limited by its poor oral bioavailability due to its rapid metabolism in the gut, its low potency and thus high dosing, and its foul smell and taste. By simply esterifying butyrate to serine (O-butyryl-L-serine, SerBut), a design based on the concept of utilizing amino acid transporters to escape the gut and enhance systemic uptake thus increasing bioavailability, we developed an odorless and tasteless compound for oral administration. In the collagen antibody-induced arthritis (CAIA) and experimental autoimmune encephalomyelitis (EAE) murine models of rheumatoid arthritis and multiple sclerosis, we demonstrated that SerBut significantly ameliorated disease severity, modulated key immune cell populations both systemically and in disease-associated tissues, and reduced inflammatory responses without compromising global immune response to vaccination. Our findings highlight SerBut as a promising next-generation therapeutic agent for autoimmune and inflammatory diseases.

bioengineering↗

bioRxiv2023

Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients, which reduces efficacy and increases adverse reactions. Our laboratory has previously shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer (p(Man)). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We found that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by Tregs. We identify increased TCR signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

bioengineering↗

bioRxiv2023

Glycopolymer-conjugated antigens as an inverse vaccine platform prevent anaphylaxis in a pre-clinical model of food allergy

The only FDA-approved oral immunotherapy for a food allergy provides protection against accidental exposure to peanuts. However, this therapy often causes discomfort or side effects and requires long-term commitment. Better preventive and therapeutic solutions are urgently needed. We have developed a tolerance-inducing vaccine technology that utilizes glycosylation-modified antigens to induce antigen-specific non-responsiveness. The glycosylation-modified antigens were administered intravenously (i.v.) or subcutaneously (s.c.) and were found to traffic to the liver or lymph nodes, respectively, leading to preferential internalization by antigen-presenting cells, educating the immune system to respond in an innocuous way. In a mouse model of cows milk allergy, treatment with glycosylation-modified {beta}- lactoglobulin (BLG) was effective in preventing the onset of allergy. In addition, s.c. administration of glycosylation-modified BLG showed superior safety and potential in treating existing allergies in combination with an anti-CD20 co-therapy. This platform may provide an antigen-specific immunomodulatory strategy to prevent and treat food allergies.

bioengineering↗

bioRxiv2023

Targeted and selective knockout of the TLQP-21 neuropeptide unmasks its unique role in energy homeostasis

Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed. Here, we developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide ({Delta}TLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R21[->]A). We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. {Delta}TLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by a temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue.

physiology↗

bioRxiv2023

Depletion of LONP2 unmasks differential requirements for peroxisomal function between cell types and in cholesterol metabolism.

Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways is not yet understood. Herein, we report the cellular responses to acute peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered accumulation of its substrates, alterations in peroxisome size and numbers, and luminal protein import failure. Gene expression changes and lipidomic analysis revealed striking cell specific differences in the response to siLONP2. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway, and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight for our understanding of the tissue-specific responses seen in peroxisomal disorders.

cell biology↗

bioRxiv2023

A Flexible Transgene Integration Landing-Pad Toolkit in Human Induced Pluripotent Stem Cells Enables Facile Cellular Engineering, Gene Zygosity Control, and Parallel Transgene Integration

Development of a repeatable method for delivering transgene payloads to human induced pluripotent stem cells (hiPSCs) without risking unintended off-target effects is not fully realized. Yet, such methods are indispensable to fully unlocking the potential for applying synthetic biological approaches to regenerative medicine, delivering quantum impacts to cell-based therapeutics development. Here we present a toolkit for engineering hiPSCs centred on the development of two core landing-pad cell-lines, facilitating rapid high-efficiency delivery of transgenes to the AAVS1 safe-harbour locus using the Bxb1 large-serine recombinase. We developed two landing-pad cell lines expressing green and red fluorescent reporters respectively, both retaining stemness whilst fully capable of differentiation into all three germ layers. A fully selected hiPSC population can be isolated within 1-2 weeks after landing-pad recombinase-mediated cassette exchange. We demonstrate the capability for investigator-controlled homozygous or heterozygous transgene configurations in these cells. As such, the toolkit of vectors and protocols associated with this landing-pad hiPSC system has the potential to accelerate engineering workflows for researchers in a variety of disciplines.

synthetic biology↗

bioRxiv2023

Predicting Protein-encoding Gene Content in Escherichia coli Genomes

In this study, we built machine learning classifiers for predicting the presence or absence of the variable genes occurring in 10-90% of all publicly available high-quality Escherichia coli genomes. The BV-BRC genus-specific protein families were used to define orthologs across the set of genomes, and a single binary classifier was built for predicting the presence or absence of each family in each genome. Each model was built using the nucleotide k-mers from a set of 100 conserved genes as features. The resulting set of 3,259 XGBoost classifiers had a per-genome average macro F1 score of 0.944 [0.943-0.945, 95% CI]. We show that the F1 scores are stable across MLSTs, and that the trend can be recapitulated through sampling with a smaller number of core genes or diverse input genomes. Surprisingly, the presence or absence of poorly annotated proteins, including "hypothetical proteins", were easily predicted (F1 = 0.902 [0.898-0.906, 95% CI]). Models for proteins with horizontal gene transfer-related functions, including transposition- (F1 = 0.895 [0.882-0.907, 95% CI]), phage- (F1 = 0.872 [0.868-0.876, 95% CI]), and plasmid-related (F1 = 0.824 [0.814-0.834, 95% CI]) functions had slightly lower F1 scores, but were still accurate. Finally, we applied the models to a holdout set of 419 diverse E. coli genomes that were isolated from freshwater environmental sources and observed an average per-genome F1 score of 0.880 [0.876-0.883, 95% CI], demonstrating the extensibility of the models. Overall, this study provides a framework for predicting variable gene content using a limited amount of input sequence data. ImportanceHaving the ability to predict the protein-encoding gene content of a genome is important for a variety of bioinformatic tasks, including assessing genome quality, binning genomes from shotgun metagenomic assemblies, and assessing risk due to the presence of antimicrobial resistance (AMR) and other virulence genes. In this study, we built a series of binary classifiers for predicting the presence or absence of variable genes occurring in 10-90% of all publicly available E. coli genomes. Overall, the results show that a large portion of the E. coli variable gene content can be predicted with high accuracy, including genes with functions relating to horizontal gene transfer.

bioinformatics↗

bioRxiv2022

Integration of event experiences to build relational knowledge in the human brain

We investigated how the human brain integrates experiences of specific events to build general knowledge about typical event structure. We examined an episodic area important for temporal relations, anterior-lateral entorhinal cortex (alEC), and a semantic area important for action concepts, middle temporal gyrus (MTG). Participants underwent fMRI while watching sequences of novel events and recalling their temporal relations over two sessions one week apart. Across different contexts, the roles of these events could either be consistent, or conflict; integrative representations should weaken in the conflicting condition. We found integrative representations that emerged quickly in alEC, and only subsequently in MTG, which showed a significant change across sessions. Thus, alEC is important in building integrative representations early in learning and may be a gateway to semantic knowledge in MTG. We suggest that both episodic and semantic systems participate in integrating new information when it is aligned with their content specializations.

neuroscience↗

bioRxiv2022

Dynamic hippocampal-cortical interactions during event boundaries support retention of complex narrative events

According to most memory theories, encoding involves continuous communication between the hippocampus and neocortex leaving the temporal dynamics of hippocampal-neocortical interactions often overlooked. Recent work has shown that we perceive complex events in our lives as dynamic, with relatively distinct starting and stopping points known as event boundaries. Event boundaries may be important for memory, as they are associated with increased activity in the hippocampus, and extended neocortical regions (the posterior cingulate cortex, lateral parietal cortex, and parahippocampal cortex). Our objective was to determine how functional connectivity between the hippocampus and neocortical regions during the encoding of naturalistic events (movies) related to subsequent retrieval and retention of those events. Participants encoded two 16-minute cartoon movies during fMRI scanning. After encoding, participants freely recalled one of the movies immediately, and the other after a 2-day delay. We quantified hippocampal-neocortical functional connectivity (FC) at time windows around each event onset, middle, and offset, and compared these FC measures with subsequent recall. These analyses revealed that higher FC between the hippocampus and the posterior medial network (PMN) at an events offset related to whether that event was subsequently recalled. In contrast, mid-event connectivity between the hippocampus and PMN was associated with poorer memory. Furthermore, hippocampal-PMN offset connectivity predicted not only whether events were retained in memory, but also the degree to which these events could be recalled in detail after a 2-day delay. These data demonstrate that the relationship between memory encoding and hippocampal-neocortical interaction is more dynamic than suggested by most memory theories, and they converge with recent modeling work suggesting that event offset is an optimal time for encoding.

neuroscience↗

bioRxiv2022

RTEL-1 and DNA polymerase theta promote subtelomeric DNA synthesis and telomere fusion in C. elegans

Interstitial telomere sequences (ITS) are degenerate telomere tracts scattered along chromosome arms whose functions are not well understood. We found that critically shortened telomeres of C. elegans telomerase mutants initiate DNA synthesis within ITS tracts that were close to or far from a telomere. Some ITS tracts were targeted recurrently. RTEL-1 dismantles T-loops and recombination intermediates, and DNA polymerase theta (POLQ-1) promotes end-joining using short segments of microhomology. In telomerase mutants, RTEL-1 and POLQ-1 promoted telomere fusion and DNA synthesis at subtelomeric ITS tracts. RTEL-1 is known to suppress homologous recombination, and we found that RTEL-1 similarly suppressed POLQ-1-mediated double-strand break repair. Mutation signatures characteristic of repair by POLQ-1 occurred during initiation of subtelomeric DNA synthesis and at subsequent template shifting events. We propose that RTEL-1 and POLQ-1 play distinct essential roles in subtelomeric DNA synthesis, a process that may contribute significantly to telomere fusion and tumor genome evolution.

genetics↗

bioRxiv2022

A phage weaponizes a satellite recombinase to subvert viral restriction

Bacteria can acquire mobile genetic elements (MGEs) to combat infection by viruses (phages). Satellite viruses, including the PLEs (phage-inducible chromosomal island-like elements) in epidemic Vibrio cholerae, are MGEs that restrict phage replication to the benefit of their host bacterium. PLEs parasitize the lytic phage ICP1, unleashing multiple mechanisms to restrict phage replication and promote their own spread. In the arms race against PLE, ICP1 uses nucleases, including CRISPR-Cas, to destroy PLEs genome during infection. However, through an unknown CRISPR-independent mechanism, specific ICP1 isolates subvert restriction by PLE. Here, we discover ICP1-encoded Adi that counteracts PLE by exploiting the PLEs large serine recombinase (LSR), which normally mobilizes PLE in response to ICP1 infection. Unlike previously characterized ICP1-encoded anti-PLE mechanisms, Adi is not a nuclease itself but instead appears to modulate the activity of the LSR to promote destructive nuclease activity of the LSRs specific attachment site, attP. The PLE LSR, its catalytic activity, and attP are additionally sufficient to sensitize a PLE encoding a resistant variant of the recombination module to Adi activity. This work highlights a unique type of adaptation arising from inter-genome conflicts, in which the intended activity of a protein can be weaponized to overcome the antagonizing genome.

microbiology↗

bioRxiv2022

SUMOylation of ABCD3 restricts bile acid synthesis and regulates metabolic homeostasis

Mitochondrial anchored protein ligase (MAPL) has been shown to function as both a SUMO and ubiquitin ligase with multiple roles in mitochondrial quality control, cell death pathways and inflammation. To examine the global function of MAPL we generated a knock-out mouse model and sought functional insight through unbiased BioID, transcriptomics and metabolic analysis. MAPL KO mice are lean and highly insulin sensitive, ultimately developing fully penetrant, spontaneous hepatocellular carcinoma after 18 months. BioID revealed the peroxisomal bile acid transporter ABCD3 as a primary MAPL interacting partner, which we show is SUMOylated in a MAPL-dependent manner. MAPL KO animals showed increased bile acid secretion in vivo and in isolated primary hepatocytes, along with robust compensatory changes in the expression of enzymes synthesizing and detoxifying bile acid. In addition, MAPL KO livers showed signs of ER stress and secreted high levels of Fgf21, the starvation hormone known to drive the reduction of white fat stores and promote insulin sensitivity. Lastly, during aging all MAPL KO mice developed hepatocellular carcinomas. These data reveal a major function for MAPL in the regulation of bile acid synthesis leading to profound changes in whole body metabolism and the ultimate generation of liver cancer when MAPL is lost.

cell biology↗

bioRxiv2021

Evolutionary sweeps of subviral parasites and their phage host bring unique parasite variants and disappearance of a phage CRISPR-Cas system

Vibrio cholerae is a significant threat to global public health in part due to its propensity for large-scale evolutionary sweeps where lineages emerge and are replaced. These sweeps may originate from the Bay of Bengal where bacteriophage predation and the evolution of anti-phage counter defenses is a recurring theme. The bacteriophage ICP1 is a key predator of epidemic V. cholerae and is notable for acquiring a CRISPR-Cas system to combat PLE, a defensive subviral parasite encoded by its V. cholerae host. Here we describe the discovery of five previously unknown PLE variants, including one found during recent surveillance of patient samples in Bangladesh. We also observed a lineage sweep of PLE negative V. cholerae occurring within a patient population in under a year which coincided with a loss of ICP1s CRISPR-Cas system. These findings reinforce the importance of surveillance to better understand the selective pressures that drive pandemic cholera.

microbiology↗