Biology subjects

Hao, S.

Publications and source records attributed to Hao, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Primate-specific microRNA-1202 regulates dopaminergic neurogenesis by targeting APC2 and modulates WNT/β-catenin signaling pathway in midbrain organoid

MicroRNAs (miRNAs) are generally evolutionarily conserved, but a small number of them are found exclusively in primates. MiR-1202 is a primate-specific miRNA that was previously revealed as being involved in major depression disorder (MDD). Moreover, the genomic locus where miR-1202 locates (6q25.3) is strongly associated with recurrent early-onset MDD and neurodevelopmental disorder. We hypothesize that miR-1202 plays a unique role in the brain in fine-tuning the transcriptional network that governs neurogenesis. Here, we reported that microdeletion of miR-1202 resulted in retarded organoid growth but enhanced dopaminergic (DA) neuron differentiation in midbrain organoids. Integrated analysis of miRNA-interacting targets and transcriptional changes in miR-1202 knockout cells revealed that APC2, a negative regulator of canonical WNT/{beta}-catenin pathway, was a downstream target of miR-1202. Knockdown of APC2 resulted in attenuated DA neurogenesis, in contrast to the enhanced DA neurogenesis in miR-1202 knockout cells. Treatments of the brain organoids with antidepressants increased miR-1202 expression and simultaneously inhibited APC2 expression. Consistently, APC2 expression was found to increase in MDD patients. Our results suggest that miR-1202 regulates dopamine neurogenesis by inhibiting the APC2/WNT signaling pathway, which plays a distinct role in the midbrain-hindbrain regional patterning during the early development of the central nervous system.

cell biology↗

bioRxiv2024

Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors

We conducted a genome-wide CRISPR/Cas9 screen in suspension 293-F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screens included the previously reported KIAA039L, TM9SF2, and RNF121, along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 (SLC35A1), a Golgi apparatus-localized cytidine 5-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that SLC35A1 knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in KIAA0319L or TM9SF2 KO cells. Although SLC35A1 KO drastically reduced the expression of 2,6-linked SIA on the cell surface, the expression of 2,3-linked SIA, as well as the cell binding and internalization of rAAV5, were only moderately affected. Moreover, SLC35A1 KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3 which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that SLC35A1 KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion ({Delta}C Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression, but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization. IMPORTANCErAAV is an essential tool for gene delivery in the treatment of genetic disorders, yet the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293-F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV.

microbiology↗

bioRxiv2024

Deciphering gene regulatory programs in mouse embryonic skin through single-cell multiomics analysis

BackgroundCell type-specific transcriptional heterogeneity in embryonic mouse skin is well-documented, but few studies have investigated the regulatory mechanisms. ResultsHere, we present high throughput single-cell chromatin accessibility and transcriptome sequencing (HT-scCAT-seq), a method that simultaneously profiles transcriptome and chromatin accessibility. We utilized HT-scCAT-seq to dissect the gene regulatory mechanism governing epidermal stratification, periderm terminal differentiation, and fibroblast specification. ConclusionsBy linking chromatin accessibility to gene expression, we identified candidate cis-regulatory elements (cCREs) and target genes crucial for dermal and epidermal development. We described cells with similar gene expression profiles that exhibit distinct chromatin accessibility statuses during periderm terminal differentiation. Finally, we characterized the underlying lineage-determining transcription factors (TFs), and demonstrated that ALX4 and RUNX2 were candidate TF regulators of the dermal papilla lineage development through in silico perturbation analysis.

developmental biology↗

bioRxiv2024

Predicting the Impact of Dialyzer Choice and Binder Dialysate Flow Rate on Bilirubin Removal

Liver failure is the 12th leading cause of death worldwide. Protein bound toxins such as bilirubin are responsible for many complications of the disease. Binder dialysis systems use albumin dialysate and detoxifying sorbent columns to remove these toxins. Systems like the Molecular Adsorbent Recirculating System (MARS) and BioLogic-DT have existed since the 1990s, but survival benefit in randomized controlled trials have not been consistent. Thus, a new generation of binder dialysis systems, including Open Albumin Dialysis (OPAL) and the Advanced Multi-Organ Replacement System (AMOR) are being developed. Optimal conditions for binder dialysis have not been established. We developed and validated a computational model of bound solute dialysis using established thermodynamic theories. Our objective is to improve AMOR therapy. We confirmed our models validity by predicting the impact of changing between two benchtop dialysis setups using different polysulfone dialyzers (F3 and F6HPS). We then applied it to predict the impact of varying dialysate flow rate on toxin removal. We found that bilirubin removal is independent of dialysate flow rate within the clinically relevant range (20 mL/min - 800 mL/min), matching our models predictions. At very low dialysate flow rates (2 mL/min), bilirubin removal declines, deviating from the thermodynamic model. This model may be useful to achieving optimal clinical outcomes by setting optimal dialyzer and flow rate conditions. Further improvement is possible by accounting for toxin adsorption onto the dialyzer membrane.

bioengineering↗

bioRxiv2024

Generators of the frequency-following response in the subthalamic nucleus: implications for non-invasive deep brain stimulation

While Deep Brain Stimulation (DBS) is effective treatment for several movement disorders, non-invasive stimulation modes have major clinical relevance. We report on a novel method holding potential for non-invasive subthalamic nucleus (STN) stimulation. We used an auditory frequency-following response task (FFR), a popular tool for studying the auditory brainstem as the neural response in the cortical and midbrain generator, as it precisely reflects the ongoing dynamics of a speech or non-speech sound. We recorded EEG and DBS electrodes from 5 patients, in 4 from the STN, and one from the anterior thalamus and a number of cortical and subcortical areas located in the hippocampus and frontal regions, during an FFR at a frequency higher than the upper limit of phase-locking in the cortex (333Hz). Our results revealed a neural response local to the STN, but not other structures. This finding is novel. Auditory perception in the basal ganglia is rather unexplored, and the STN generator of the FFR has likely gone unseen due to the limitations of our tools and research focus. The potential clinical implications are far-reaching. Future research should investigate whether auditory stimuli at common electrical stimulation frequencies and waveforms of electrical DBS stimulation can induce clinical improvement.

neuroscience↗

bioRxiv2024

pH-Sensitive Nanodroplets for Controlled Delivery of Berberine Chloride

BackgroundThe development of nanocarriers with precise control over drug release is crucial for targeted therapy. This study focuses on the design and optimization of pH-sensitive gelatin/perfluorohexane (PFH) nanodroplets loaded with berberine chloride, a model drug relevant to traditional Chinese medicine. Subjects and MethodsNanodroplets were prepared using an emulsion technique, with optimization of parameters including homogenization rate, polymer concentration, surfactant, drug, and perfluorocarbon conte nt. ResultsThe optimized formulation resulted in nanodroplets with a mean particle size of 281.7 nm and a drug encapsulation efficiency of 66.8 {+/-} 1.7%. Characterization studies confirmed successful encapsulation and pH-responsive behavior. Ultrasound stimulation significantly enhanced drug release, with 150 kHz frequency proving more effective than 1 MHz. Stability studies demonstrated prolonged stability over one month at 4{degrees}C. Following 10 minutes of ultrasound irradiation, the nanodroplets exhibited 89.4% cumulative drug release. ConclusionsIn conclusion, these pH-sensitive nanodroplets show potential for delivering berberine chloride in a controlled manner, connecting traditional Chinese medicine with contemporary drug delivery methods.

biophysics↗

bioRxiv2024

Fertilization-induced synergid cell death by RALF12-triggered ROS production and ethylene signaling

Fertilization-dependent elimination of the persistent synergid cell is essential to block supernumerary pollen tubes and thus to avoid polyspermy in flowering plants. Little is known about the molecular mechanisms ensuring timely induction and execution of synergid cell death. We analysed manually isolated maize synergid cells along their degeneration and show that they are gland cells expressing batteries of genes encoding small secreted proteins under control of the MYB98 transcription factor. This network is down-regulated after fertilization, while genes involved in reactive oxygen species (ROS) production, ethylene biosynthesis and response, senescence, and oxidative stress regulation are induced before synergid elimination and its ultimate fusion with the endosperm. We further show that fertilization-induced RALF12 peptide specifically triggers mitochondrial ROS and apoptosis, while ethylene promotes synergid degeneration. In conclusion, this study sheds light on developmental programmed cell death (dPCD) in plants and provides a unique resource to discover novel PCD regulators.

cell biology↗

bioRxiv2024

Label free, capillary-scale blood flow mapping in vivo reveals that low intensity focused ultrasound evokes persistent dilation in cortical microvasculature

Non-invasive, low intensity focused ultrasound (FUS) is an emerging neuromodulation technique that offers the potential for precision, personalized therapy. An increasing body of research has identified mechanosensitive ion channels that can be modulated by FUS and support acute electrical activity in neurons. However, neuromodulatory effects that persist from hours to days have also been reported. The brains ability to provide targeted blood flow to electrically active regions involve a multitude of non-neuronal cell types and signaling pathways in the cerebral vasculature; an open question is whether persistent effects can be attributed, at least partly, to vascular mechanisms. Using a novel in vivo optical approach, we found that microvascular responses, unlike larger vessels which prior investigations have explored, exhibit persistent dilation following sonication without the use of microbubbles. This finding and approach offers a heretofore unseen aspect of the effects of FUS in vivo and indicate that concurrent changes in neurovascular function may partially underly persistent neuromodulatory effects.

neuroscience↗

bioRxiv2023

Identification of Host Restriction Factors Critical for Recombinant AAV Transduction of Polarized Human Airway Epithelium

Recombinant (r)AAV2.5T was selected from the directed evolution of an AAV capsid library in human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequence of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome wide CRISPR gRNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screen identified several genes that are critical for rAAV2.5T transduction in HeLa S3 cells, including previously reported genes KIAA0319L, TM9SF2, VPS51, and VPS54, as well as a novel gene WDR63. We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed WDR63 is an important player in rAAV2.5T transduction of HAE, while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T. SignificanceThe essential steps of a successful gene delivery by rAAV include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

microbiology↗

bioRxiv2023

Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5' untranslated region

One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as C30 and C34, which bind to a four-way RNA helix called SL5 in the 5 UTR of the SARS-CoV-2 RNA genome. To locate the binding site, we developed a novel sequencing-based method namely cgSHAPE-seq, in which the acylating chemical probe was directed to crosslink with the 2-OH groups of ribose at the ligand binding site. This crosslinked RNA could then create read-through mutations during reverse transcription (i.e., primer extension) at single-nucleotide resolution to uncover the acylation locations. cgSHAPE-seq unambiguously determined that a bulged G in SL5 was the primary binding site of C30 in the SARS-CoV-2 5 UTR, which was validated through mutagenesis and in vitro binding experiments. C30 was further used as a warhead in RNA-degrading chimeras to reduce viral RNA expression levels. We demonstrated that replacing the acylating moiety in the cgSHAPE probe with ribonuclease L recruiter (RLR) moieties yielded RNA degraders active in the in vitro RNase L degradation assay and SARS-CoV-2 5 UTR expressing cells. We further explored another RLR conjugation site on the E ring of C30/C34 and discovered improved RNA degradation activities in vitro and in cells. The optimized RNA-degrading chimera C64 inhibited live virus replication in lung epithelial carcinoma cells.

biochemistry↗

bioRxiv2022

Spateo: multidimensional spatiotemporal modeling of single-cell spatial transcriptomics

Cells do not live in a vacuum, but in a milieu defined by cell-cell communication that can be measured via emerging high-resolution spatial transcriptomics approaches. However, analytical tools that fully leverage such data for kinetic modeling remain lacking. Here we present Spateo (aristoteleo/spateo-release), a general framework for quantitative spatiotemporal modeling of single-cell resolution spatial transcriptomics. Spateo delivers novel methods for digitizing spatial layers/columns to identify spatially-polar genes, and develops a comprehensive framework of cell-cell interaction to reveal spatial effects of niche factors and cell type-specific ligand-receptor interactions. Furthermore, Spateo reconstructs 3D models of whole embryos, and performs 3D morphometric analyses. Lastly, Spateo introduces the concept of "morphometric vector field" of cell migrations, and integrates spatial differential geometry to unveil regulatory programs underlying various organogenesis patterns of Drosophila. Thus, Spateo enables the study of the ecology of organs at a molecular level in 3D space, beyond isolated single cells.

systems biology↗

bioRxiv2022

YAP condensates are highly organized hubs for YAP/TEAD transcription

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. The transcription factor TEAD1 actively stabilizes YAP condensates, which also recruit BRD4, a coactivator that is enriched at active enhancers. Using single molecule tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

cell biology↗

bioRxiv2022

Nanobody-tethered transposition allows for multifactorial chromatin profiling at single-cell resolution

Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility, and other factors. However, most current single-cell-resolution methods are unable to measure more than one aspect of chromatin state in a single experiment, limiting our ability to accurately measure chromatin states. Here, we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), a new assay capable of measuring the genome-wide presence of multiple histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq utilizes recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk- and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and in cells of the human immune system, demonstrating the high accuracy and sensitivity of the method. We further extend NTT-seq to enable simultaneous profiling of cell-surface protein expression alongside multimodal chromatin states to study cells of the immune system.

genomics↗

bioRxiv2022

Longitudinal Morphological and Functional Characterization of Human Heart Organoids Using Optical Coherence Tomography

Organoids play an increasingly important role as in vitro models for studying organ development, disease mechanisms, and drug discovery. Organoids are self-organizing, organ-like three-dimensional (3D) cell cultures developing organ-specific cell types and functions. Recently, three groups independently developed self-assembling human heart organoids (hHOs) from human pluripotent stem cells (hPSCs). In this study, we utilized a customized spectral-domain optical coherence tomography (SD-OCT) system to characterize the growth of hHOs. Development of chamber structures and beating patterns of the hHOs were observed via OCT and calcium imaging. We demonstrated the capability of OCT to produce 3D images in a fast, label-free, and non-destructive manner. The hHOs formed cavities of various sizes, and complex interconnections were observed as early as on day 4 of differentiation. The hHOs models and the OCT imaging system showed promising insights as an in vitro platform for investigating heart development and disease mechanisms.

bioengineering↗

bioRxiv2021

Antigen presentation by type 3 innate lymphoid cells instructs the differentiation of gut microbiota-specific regulatory T cells

The mutualistic relationship of gut-resident microbiota and cells of the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a poised, but largely non-aggressive, immune cell compartment1, 2. Consequences of disturbing this balance, by environmental or genetic factors, include proximal inflammatory conditions, like Crohns disease, and systemic illnesses, both metabolic and autoimmune. One of the means by which this equilibrium is achieved is through induction of both effector and suppressor or regulatory arms of the adaptive immune system. In mice, Helicobacter species induce regulatory (iTreg) and follicular helper (Tfh) T cells in the colon-draining mesenteric lymph nodes under homeostatic conditions, but can instead induce inflammatory Th17 cells when iTreg cells are compromised3, 4. How Helicobacter hepaticus and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here, we investigated which cells and molecular components are required to convey the microbial instruction for the iTreg differentiation program. We found that antigen presentation by cells expressing ROR{gamma}t, rather than by classical dendritic cells, was both required and sufficient for iTreg induction. These ROR{gamma}t+ cells, likely to be type 3 innate lymphoid cells (ILC3) and/or a recently-described population of Aire+ cells termed Janus cells5, require the MHC class II antigen presentation machinery, the chemokine receptor CCR7, and v integrin, which activates TGF-{beta}, for iTreg cell differentiation. In the absence of any of these, instead of iTreg cells there was expansion of microbiota-specific pathogenic Th17 cells, which were induced by other antigen presenting cells (APCs) that did not require CCR7. Thus, intestinal commensal microbes and their products target multiple APCs with pre-determined features suited to directing appropriate T cell differentiation programs, rather than a common APC that they endow with appropriate functions. Our results illustrate the ability of microbiota to exploit specialized functions of distinct innate immune system cells, targeting them to achieve the desired composition of equipoised T cells, thus maintaining tolerance.

immunology↗

bioRxiv2021

Single-cell Stereo-seq enables cell type-specific spatial transcriptome characterization in Arabidopsis leaves

Understanding the complex functions of plant leaves requires spatially resolved gene expression profiling with single-cell resolution. However, although in situ gene expression profiling technologies have been developed, this goal has not yet been achieved. Here, we present the first in situ single-cell transcriptome profiling in plant, scStereo-seq (single-cell SpaTial Enhanced REsolution Omics-sequencing), which enabled the bona fide single-cell spatial transcriptome of Arabidopsis leaves. We successfully characterized subtle but significant transcriptomic differences between upper and lower epidermal cells. Furthermore, with high-resolution location information, we discovered the cell type-specific spatial gene expression gradients from main vein to leaf edge. By reconstructing those spatial gradients, we show for the first time the distinct spatial developmental trajectories of vascular cells and guard cells. Our findings show the importance of incorporating spatial information for answering complex biological questions in plant, and scStereo-seq offers a powerful single cell spatially resolved transcriptomic strategy for plant biology.

plant biology↗

bioRxiv2021

Mapping the nuclear microenvironment of genes at agenome-wide scale

The nuclear folding of chromosomes relative to nuclear bodies is an integral part of gene function. Here, we demonstrate that population-based modeling--from ensemble Hi-C data--can provide a detailed description of the nuclear microenvironment of genes and its role on gene function. We define the microenvironment by the subnuclear positions of genomic regions with respect to nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single cell models on a genome-wide scale, thereby revealing the structural variability between cells. We demonstrate that the structural microenvironment of a genomic region is linked to its functional potential in gene transcription, replication and chromatin compartmentalization. Some chromatin regions are distinguished by their strong preferences to a single microenvironment, due to associations to specific nuclear bodies in most cells. Other chromatin shows high structural variability, which is a strong indicator of functional heterogeneity. Moreover, we identify specialized nuclear microenvironments, which distinguish chromatin in different functional states and reveal a key role of nuclear speckles in chromosome organization. We demonstrate that our method produces highly predictive 3-dimensional genome structures, which accurately reproduce data from TSA-seq, DamID, GPSeq and super-resolution imaging. Thus, our method considerably expands the range of Hi-C data analysis and is widely applicable.

genomics↗

bioRxiv2021

Discovery of target genes and pathways of blood trait loci using pooled CRISPR screens and single cell RNA sequencing

The majority of variants associated with complex traits and common diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown regulatory effects in cis and trans. By leveraging biobank-scale GWAS data, massively parallel CRISPR screens and single cell transcriptome sequencing, we discovered target genes of noncoding variants for blood trait loci. The closest gene was often the target gene, but this was not always the case. We also identified trans-effects networks of noncoding variants when cis target genes encoded transcription factors, such as GFI1B and NFE2. We observed that GFI1B trans-target genes were enriched for GFI1B binding sites and fine-mapped GWAS variants, and expressed in human bone marrow progenitor cells, suggesting that GFI1B acts as a master regulator of blood traits. This platform will enable massively parallel assays to catalog the target genes of human noncoding variants in both cis and trans.

genomics↗