Biology subjects

Do, H.

Publications and source records attributed to Do, H..

18 recordsLinked to original sources

bioRxiv2026

Sequential HIV-1 fusion protein gates control the initiation of host cell entry

HIV-1 fusion with host cells is initiated upon engagement of the viral envelope (Env) protein by its receptors and co-receptors. Receptor-induced conformational changes in the Env ectodomain expose the fusion machinery and position the HIV-1 fusion peptide for release and transit to the host membrane. However, no allosteric rearrangements in the envelope receptor/co-receptor binding domain that could induce fusion peptide release have been identified, and the trigger for the structural cascade that brings the viral and host membranes together remains unknown. Here, we identified two sequential conformational gates that control fusion peptide release, thereby facilitating its transition to the host membrane. We show that destabilizing a clasp holding the fusion peptide proximal motif enhances fusion peptide release, and that a coupled downstream gate controls the release of the receptor-binding gp120 subunit. Single-particle cryo-EM structures, biased and unbiased atomistic simulations, and functional experiments reveal that this process is controlled by shifts in the angular orientation of the gp120 subunits, induced by the geometries required for receptor and co-receptor binding. These results show how HIV-1 senses contact with the host cell to initiate fusion, thereby permitting viral entry.

molecular biology↗

bioRxiv2025

Modeling σE biochemical network reveals context-dependent feedback control and kinetic constraints in the envelope stress response

The bacterial cell envelope is essential for mechanical stability, barrier function, defining cell size and shape, and supporting cellular processes. Because external or internal stressors can challenge its integrity, bacteria have evolved stress response systems to maintain envelope homeostasis. For example, under stress, E. coli activates {sigma}E to induce a regulon that restores envelope homeostasis. The molecular mechanisms underlying the transcriptional and post-translational regulation of {sigma}E and its anti-sigma factor RseA have been well-mapped. However, how these regulatory layers function at the systems level to regulate {sigma}E activity remains unclear. Here, we combine mathematical modeling with quantitative gene expression measurements to determine how the interplay of transcription and post-translational regulation affects the dynamics of the {sigma}E response. The results suggest that the {sigma}E activity is determined by the balance between the release from RseA, resulting from its degradation, and slow binding of free {sigma}E to the intact RseA. Notably, the combined action of transcriptional and post-translational regulation reveals that autoregulation, traditionally assumed to be a positive feedback, transitions from negative to positive feedback under extreme stress, enabling a finely tuned response that prevents premature activation while ensuring a robust response under severe envelope stress. In summary, our findings elucidate the mechanisms of {sigma}E regulation, thereby advancing our understanding of how alternative sigma factor bacterial networks control stress-response pathways.

systems biology↗

bioRxiv2025

AGO1 in neural progenitor cells orchestrates brain development and sociability via LIN28A-REELIN axis

AGO1, an essential RNA binding protein (RBP) in RNA interference, is associated with autism spectrum disorder (ASD). However, the precise functions of AGO1 in brain development and related disorders remain largely unexplored. Here, we report the critical roles of AGO1 in neural progenitor cells (NPCs) in promoting sociability and shaping the structure of the developing brain. Ago1 knockout (KO) in the mouse brain leads to hyposociability, a characteristic symptom of ASD. In human forebrain organoids, AGO1 KO disrupts the formation of ventricle-like structures and delays cortical layer development. We discovered that AGO1 KO results in a loss of polarity in NPCs, which subsequently reduces neuronal development. These AGO1 KO phenotypes originate from the failure to suppress LIN28A in NPCs. AGO1 is primarily localized in the nucleus within NPCs and binds to the LIN28A promoter region, thereby inhibiting LIN28A transcription. We found that increased LIN28A in AGO1 KO NPCs reduces REELIN expression, a key regulator of brain development, by binding directly to the REELIN mRNA. The aberrant polarity phenotype in AGO1 KO NPCs was successfully ameliorated by either LIN28A knockdown or recombinant REELIN treatment. Collectively, our findings elucidate the intricate molecular mechanisms orchestrated by nuclear AGO1 in NPCs and underscores its pivotal roles in brain development, providing significant insights into ASD.

neuroscience↗

bioRxiv2025

Streamlining Spatial Transcriptomics for Human Kidney Tissue

Single-cell spatial technologies have emerged in recent years, enabling tissue architecture and organization characterization at unprecedented resolution. However, computational analysis of spatial transcriptomics data is often a bottleneck for scientific discoveries in the absence of a dedicated bioinformatician. Here, we describes a workflow to annotate cell types from a dataset generated using NanoStrings CosMx single-cell resolution spatial transcriptomic technology, enabling a comparison between healthy kidney biopsies and diseased tissue. We validated our pipelines accuracy with both gene expression analysis and pathological changes associated with biopsy-proven diabetic kidney disease (DKD). Through precise cell type annotation, we observed significant changes in the proportions of podocytes and immune cells in DKD, with DKD tissue showing regional enrichment of immune cells and differential gene expression. Notably, injured proximal tubules had the expected increased expression of HAVCR1 and VCAM1 and genes associated with diabetes, including IL18, ITGA3 and ITGB1. The entire workflow, now fully integrated into the BioTuring SpatialX (Lens V2.0), is available as a platform designed for users with no formal bioinformatics training.

bioinformatics↗

bioRxiv2025

MEG3 Enhances Survival of Developing Human Neurons with CLCN4-Linked Autophagy Impairment

Genetic variations in CLCN4, encoding the H+/Cl- exchanger CLC-4, are associated with human neurodevelopmental disorders with highly variable phenotypes. A lack of physiologically relevant models has hampered molecular understanding of pathogenic mechanisms. We now establish engineered brain organoid and neuronal cell systems to examine impacts of patient-relevant CLCN4 genetic variations. We find that CLCN4 variants reduced excitatory neuron numbers due to early-stage cell death, accompanied by altered endo-lysosomal dynamics and disrupted autophagic flux. Transcriptomic profiling showed significant downregulation of long non-coding RNA MEG3 in CLCN4-variant neurons. Restoring MEG3 expression is sufficient to rescue cellular defects and improve survival of CLCN4-variant neurons. These findings link CLCN4 dysfunction with impaired autophagy and neuronal cell death, highlighting MEG3 as a potential therapeutic target for neurodevelopmental disorders involving autophagic dysfunction.

neuroscience↗

bioRxiv2025

Hypoosmolarity inhibits archaeal ammonia oxidation

Salinity strongly influences the physiology and distribution of nitrifying microorganisms, yet the effects of low salinity on them remain understudied. This study investigates the impact of hypoosmolarity on different groups of ammonia oxidizers in soil and lake environments, as well as in pure culture isolates. In soil microcosms amended with ammonium, at low salinity levels ([~]120 S/cm), comparable to values commonly found in pristine terrestrial and aquatic environments, the abundance of ammonia-oxidizing bacteria (AOB), dominated by Nitrosomonas oligotropha, significantly increased. In contrast, the growth of ammonia-oxidizing archaea (AOA), dominated by "Ca. Nitrosotenuis" of the Nitrosopumilaceae family, was stimulated by high salinity ([~]760 S/cm). In ammonium-fed lake microcosms, the abundance of AOB, dominated by N. oligotropha, significantly increased under both low ([~]170 S/cm) and high salinity ([~]850 S/cm) conditions. In the presence of allylthiourea, a bacterial nitrification inhibitor, AOA were sensitive to low salinity in both soil and lake microcosms. Consistently, pure culture studies revealed marked growth inhibition of AOA, especially members of Nitrosopumilaceae, under hypoosmolarity, unlike AOB and complete ammonia oxidizer (comammox) strains. Comparative genomic analyses with AOB and comammox, along with transcriptomic studies, suggested that the sensitivity of AOA to hypoosmolarity stress was possibly due to a lack of sophisticated osmoregulatory transport systems and their S-layer cell wall structure. Overall, this study highlights hypoosmolarity as a key factor shaping the ecological niches and distribution of ammonia oxidizers, as well as nitrification activities, in terrestrial and aquatic environments that are increasingly affected by intensified water cycles due to climate change.

microbiology↗

bioRxiv2024

Food intake enhances hippocampal sharp wave-ripples

Effective regulation of energy metabolism is critical for survival. Metabolic control involves various nuclei within the hypothalamus, which receive information about the bodys energy state and coordinate appropriate responses to maintain homeostasis, such as thermogenesis, pancreatic insulin secretion, and food-seeking behaviors. It has recently been found that the hippocampus, a brain region traditionally associated with memory and spatial navigation, is also involved in metabolic regulation. Specifically, hippocampal sharp wave ripples (SWRs), which are high-frequency neural oscillations supporting memory consolidation and foraging decisions, have been shown to reduce peripheral glucose levels. However, whether SWRs are enhanced by recent feeding- when the need for glucose metabolism increases, and if so, whether feeding-dependent modulation of SWRs is communicated to other brain regions involved in metabolic regulation, remains unknown. To address these gaps, we recorded SWRs from the dorsal CA1 region of the hippocampus of mice during sleep sessions before and after consumption of meals of varying caloric values. We found that SWRs occurring during sleep are significantly enhanced following food intake, with the magnitude of enhancement being dependent on the caloric content of the meal. This pattern occurred under both food-deprived and ad libitum feeding conditions. Moreover, we demonstrate that GABAergic neurons in the lateral hypothalamus, which are known to regulate food intake, exhibit a robust SWR-triggered increase in activity. These findings identify the satiety state as a factor modulating SWRs and suggest that hippocampal-lateral hypothalamic communication is a potential mechanism by which SWRs could modulate peripheral metabolism and food intake.

neuroscience↗

bioRxiv2024

Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

Novel antibodies have been created for targeted degradation of extracellular and membrane proteins in the lysosome. The mechanism of degradation of target proteins for these antibodies has involved either chemical conjugation of synthetic mannose 6-phosphate (M6P) or engineered bispecific antibodies. Currently, recombinant antibodies cannot be produced with naturally phosphorylated N-glycans. Here, we report the development of a novel platform technology for producing bifunctional therapeutic antibodies with high levels of M6P-bearing glycans directly from producing cells. The antibodies designated as phosphorylated N-glycosylated peptide chimeric antibodies (PNCA) maintain their affinity for antigens with concurrent high affinity binding to cell surface cation-independent mannose-6-phosphate receptors that facilitate internalization and delivery of antibody/antigen complexes to lysosomes for efficient degradation of both target extracellular soluble and membrane proteins. This PNCA approach provides a simple, scalable, and viable approach for producing naturally phosphorylated bifunctional antibodies from production cell lines for targeted protein degradation in lysosomes.

biochemistry↗

bioRxiv2023

Alternative Polyadenylation Determines the Functional Landscape of Inverted Alu Repeats

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' UTRs. Yet, the biological significance of 3' UTR IRAlus remains largely unknown. Here, we find that IRAlus induce the silencing of genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

molecular biology↗

bioRxiv2023

The sleep quality- and myopia-linked PDE11A-Y727C variant impacts neural physiology by reducing catalytic activity and altering subcellular compartmentalization of the enzyme

Recently, a Y727C variant in the dual-specific 3,5-cyclic nucleotide phosphodiesterase 11A (PDE11A-Y727C) was linked to increased sleep quality and reduced myopia risk in humans. Given the well-established role that the PDE11 substrates cAMP and cGMP play in eye physiology and sleep, we determined if 1) PDE11A protein is expressed in the retina or other eye segments in mouse, 2) PDE11A-Y7272C affects catalytic activity and/or subcellular compartmentalization more so than the nearby suicide-associated PDE11A-M878V variant, and 3) Pde11a deletion alters eye growth or sleep quality in male and female mice. Western blots show distinct protein expression of PDE11A4, but not PDE11A1-3, in eyes of Pde11a WT--but not KO mice--that vary by eye segment and age. In HT22 and COS-1 cells, PDE11A4-Y727C reduces PDE11A4 catalytic activity far more than PDE11A4-M878V, with both variants reducing PDE11A4-cAMP more so than PDE11A4-cGMP activity. Despite this, Pde11a deletion does not alter age-related changes in retinal or lens thickness, axial length, nor vitreous or anterior chamber depth. Further, Pde11a deletion only minimally changes refractive error and sleep quality. That said, both variants also dramatically alter the subcellular compartmentalization of human and mouse PDE11A4, an effect occurring independently of dephosphorylating PDE11A4-S117/S124 or phosphorylating PDE11A4-S162. Rather, re-compartmentalization of PDE11A4-Y727C is due to the loss of the tyrosine changing how PDE11A4 is packaged/repackaged via the trans-Golgi network. Therefore, the protective impact of the Y727C variant may reflect a gain-of-function (e.g., PDE11A4 displacing another PDE) that warrants further investigation in the context of reversing/preventing sleep disturbances or myopia.

neuroscience↗

bioRxiv2023

Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Alzheimers disease is characterized pathologically by cerebral deposition of 42-residue amyloid {beta}-peptide (A{beta}42), proteolytically produced from amyloid precursor protein (APP) by {beta}- and {gamma}-secretases.1 Although mutations in APP and presenilin, the catalytic component of {gamma}-secretase, cause familial Alzheimers disease (FAD), a role for A{beta}42 as the primary disease driver has not been clearly established and remains controversial.2,3 Here we show through comprehensive analysis of the multi-step proteolysis of APP substrate C99 by {gamma}-secretase that FAD mutations are consistently deficient in early proteolytic events, not later events that produce secreted A{beta} peptides. Cryo-electron microscopy revealed that a substrate mimetic traps {gamma}-secretase at the transition state for intramembrane proteolysis, and this structure closely aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and fluorescence lifetime imaging microscopy in cultured cells support stabilization by FAD mutations of enzyme-substrate and/or enzyme-intermediate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorabditis elegans led to age-dependent synaptic loss only when one of the transgenes carried an FAD mutation. Designed mutations that stabilize the enzyme-substrate complex and block proteolysis likewise led to synaptic loss. Collectively, these findings implicate the stalled process--not the released products--of {gamma}-secretase cleavage of substrates in FAD pathogenesis.

neuroscience↗

bioRxiv2023

Changes in coding and efficiency through modular modifications to a One Pot PURE system for in vitro transcription & translation

The incorporation of unnatural amino acids are attractive methods for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. In this work, we further adapted and modified the One Pot PURE for a robust and modular system of enzymatic single site-specific of unnatural amino acid. We demonstrated the flexibility of this system through the introduction of two orthogonal aminoacyl tRNA synthetases and the suppression of two distinctive stop codons.

synthetic biology↗

bioRxiv2023

Synthetic microbial sensing and biosynthesis of amaryllidaceae alkaloids

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimers medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from low-yielding plants, including the wild daffodil Narcissus pseudonarcissus. Engineered biocatalytic methods have the potential to stabilize the supply chain of amaryllidaceae alkaloids. Here, we propose a highly efficient biosensor-AI technology stack for biocatalyst development, which we apply to engineer amaryllidaceae alkaloid production in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50= 20 uM) and specific biosensor for the key amaryllidaceae alkaloid branchpoint 4-OMethylnorbelladine. A machine learning model (MutComputeX) was subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which were rapidly screened with the biosensor. Functional enzyme variants were identified that yielded a 60% improvement in product titer, 17-fold reduced remnant substrate, and 3-fold lower off-product regioisomer formation.

synthetic biology↗

bioRxiv2021

Unique Features of Different Classes of G-Protein-Coupled Receptors Revealed from Sequence Coevolutionary and Structural Analysis

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and represent the primary targets of about one third of currently marketed drugs. Despite the critical importance, experimental structures have been determined for only a limited portion of GPCRs and functional mechanisms of GPCRs remain poorly understood. Here, we have constructed novel sequence coevolutionary models of the A and B classes of GPCRs and compared them with residue contact frequency maps generated with available experimental structures. Significant portions of structural residue contacts were successfully detected in the sequence-based covariational models. "Exception" residue contacts predicted from sequence coevolutionary models but not available structures added missing links that were important for GPCR activation and allosteric modulation. Moreover, we identified distinct residue contacts involving different sets of functional motifs for GPCR activation, such as the Na+ pocket, CWxP, DRY, PIF and NPxxY motifs in the class A and the HETx and PxxG motifs in the class B. Finally, we systematically uncovered critical residue contacts tuned by allosteric modulation in the two classes of GPCRs, including those from the activation motifs and particularly the extracellular and intracellular loops in class A GPCRs. These findings provide a promising framework for rational design of ligands to regulate GPCR activation and allosteric modulation. SignificanceG-protein-coupled receptors (GPCRs) play key roles in cellular signaling and serve as the primary targets of [~]1/3 of currently marketed drugs. In this work, we have presented the first analysis of both residue sequence coevolution and structural contact maps in different classes of GPCRs. We have inferred pathways for GPCR signal transduction that could not be determined from structural analysis alone. Distinct residue contacts have been identified in the signaling pathways of class A and B GPCRs. Our combined sequence coevolutionary and structural contact analysis has thus revealed important insights into the mechanism of GPCR signal transduction, which is expected to facilitate rational drug design of the GPCRs.

bioinformatics↗

bioRxiv2020

Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila

Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells. Here, we used single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discovered novel hemocyte types, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identified the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. We unraveled similarities and differences between embryonically derived- and larval lymph gland hemocytes. Finally, the comparison of Drosophila lymph gland hemocytes and human immune cells highlights similarities between prohemocytes and hematopoietic stem cell, and between mature hemocytes and myeloid cells across species. Altogether, our study provides detailed insights on the development and evolution of hematopoiesis at single-cell resolution.

developmental biology↗

bioRxiv2019

Heterogeneity and targeted therapy-induced adaptations in lung cancer revealed by longitudinal single-cell RNA sequencing

Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNAseq) of metastatic lung cancer was performed using 44 tumor biopsies obtained longitudinally from 27 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNAseq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNAseq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes.

cancer biology↗

bioRxiv2018

Evaluating Named-Entity Recognition approaches in plant molecular biology

Text mining research is becoming an important topic in biology with the aim to extract biological entities from scientific papers in order to extend the biological knowledge. However, few thorough studies on text mining and applications are developed for plant molecular biology data, especially rice, thus resulting a lack of datasets available to train models able to detect entities such as genes, proteins and phenotypic traits. Since there is rare benchmarks for rice, we have to face various difficulties in exploiting advanced machine learning methods for accurate analysis of rice bibliography. In this article, we developed a new training datasets (Oryzabase) as the benchmark. Then, we evaluated the performance of several current approaches to find a methodology with the best results and assigned it as the state of the art method for our own technique in the future. We applied Name Entities Recognition (NER) tagger, which is built from a Long Short Term Memory (LSTM) model, and combined with Conditional Random Fields (CRFs) to extract information of rice genes and proteins. We analyzed the performance of LSTM-CRF when applying to the Oryzabase dataset and improved the results up to 86% in F1. We found that on average, the result from LSTM-CRF is more exploitable with the new benchmark.

bioinformatics↗

bioRxiv2017

GRIDSS: sensitive and specific genomic rearrangement detection using positional de Bruijn graph assembly

The identification of genomic rearrangements, particularly in cancers, with high sensitivity and specificity using massively parallel sequencing remains a major challenge. Here, we describe the Genome Rearrangement IDentification Software Suite (GRIDSS), a high-speed structural variant (SV) caller that performs efficient genome-wide break-end assembly prior to variant calling using a novel positional de Bruijn graph assembler. By combining assembly, split read and read pair evidence using a probabilistic scoring, GRIDSS achieves high sensitivity and specificity on simulated, cell line and patient tumour data, recently winning SV sub-challenge #5 of the ICGC-TCGA DREAM Somatic Mutation Calling Challenge. On human cell line data, GRIDSS halves the false discovery rate compared to other recent methods. GRIDSS identifies non-template sequence insertions, micro-homologies and large imperfect homologies, and supports multi-sample analysis. GRIDSS is freely available at https://github.com/PapenfussLab/gridss.

bioinformatics↗