Biology subjects

Ouyang, M.

Publications and source records attributed to Ouyang, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Specification of Origins of DNA Replication Within Distinct Chromatin States in Human Cells

A comprehensive map of binding sites for the Origin Recognition Complex (ORC) in the human genome reveals relationships among replication licensing, chromatin state, three-dimensional genome organization, location of origins of DNA replication and DNA replication timing. Defining these relationships is essential for understanding how replication licensing is integrated with the broader organization and duplication of the human genome. Using 30 high-quality ORC1 CUT&RUN datasets, an ORC1 three-dimensional binding matrix (ORC1 3DM) was constructed that captures genomic position, binding intensity, and reproducibility across replicates. This analysis reveals considerable heterogeneity in ORC1 binding, and integration with chromatin-state maps shows that ORC1-binding sites separate into distinct epigenomic groups, with high-confidence sites enriched in H2A{middle dot}Z1- and H3K4me3- associated open chromatin. Hi-C analysis demonstrates preferential localization of ORC1 binding to sub-regions of genomic A compartments and frequent association with loop anchors, supported by a direct interaction between ORC1 and CTCF. Replication-timing analysis shows that ORC1 binding sites are enriched in initiation zones and exhibit subgroup-specific temporal patterns. An integrated analysis combining multiple replication initiation assays and chromatin features further places ORC1 3DM within a unified replication-chromatin framework. Together, these results support a hierarchical, probabilistic model of replication licensing that is embedded within chromatin architecture.

biochemistry↗

bioRxiv2025

CaRPOOL: A Pooled Calcium-Recording CRISPR Screening Platform Identifies CCR7 as a Modulator of Cellular Osmomechanosensing

Cells must continuously sense and respond to environmental changes by translating physical and chemical cues into intracellular signals. However, systematic discovery of genes governing these sensory processes has been limited by the transient nature of signaling events and the low throughput of measurement assays. Here, we present CaRPOOL, a pooled, high-throughput genetic screening platform that integrates the calcium-activity recorder CaMPARI2 with CRISPR interference (CRISPRi), enabling stable capture of transient calcium signals for genome-scale functional screening. Using osmomechanical stimulation as a model, we demonstrate that CaMPARI2 photoconversion faithfully reports stimulus-dependent calcium responses and supports pooled fluorescence-activated cell sorting (FACS)-based screening. A CRISPRi library targeting membrane-associated genes identified both known and previously uncharacterized regulators of mechanotransduction, including the chemokine receptor CCR7. Mechanistic analyses revealed that CCR7 promotes osmomechanical calcium signaling through a PIEZO1-dependent Gs-cAMP-PKA pathway, establishing it as a mechanically responsive GPCR. Notably, osmotic stress upregulated CCR7 expression in immune cell lines and enhanced mechanical responsiveness, suggesting a role in immunomechanical adaptation. Together, these findings introduce a broadly applicable platform for high-throughput discovery of genes controlling dynamic signaling responses and reveal a GPCR-ion channel crosstalk mechanism in mechanotransduction with potential implications for immune cell mechanoadaptation.

cell biology↗

bioRxiv2024

Tunable picoliter-scale dropicle formation using amphiphilic microparticles with patterned hydrophilic patches

Microparticle-templated droplets or dropicles have recently gained interest in the fields of diagnostic immunoassays, single-cell analysis, and digital molecular biology. Amphiphilic particles have been shown to spontaneously capture aqueous droplets within their cavities upon mixing with an immiscible oil phase, where each particle templates a single droplet. Here, we fabricated an amphiphilic microparticle with four discrete hydrophilic patches embedded at the inner corners of a square-shaped hydrophobic outer ring of the particle (4C particle). 3D computational fluid dynamics simulations predicted droplet formation dynamics and differing equilibrium conditions depending on the patterning configuration. Experiments recapitulated equilibrium conditions enabling tunable dropicle configurations with reproducible volumes down to [~]200pL templated by the amphiphilic particles. The dropicle configurations depended predominantly on the size of the hydrophilic patches of the 4C particles. This validated modeling approach can inform the design of dropicles with varying volumes and numbers per particle, which can be harnessed in new amplified bioassays for greater sensitivity, dynamic range, and statistical confidence.

bioengineering↗

bioRxiv2024

Os-circANK negatively regulates resistance to bacterial blight in rice via Osa-miR398b/OsCSD1/OsCSD2 pathways

CircRNAs are widely present in plants, yet there have been no reports on the role of circRNA in the regulatory mechanisms of interactions between rice and pathogenic bacteria. As the sequence of circRNAs matches the parent gene except for the back splicing site, there is no feasible way to specifically knockdown circRNAs in plants. We identified a circRNA, named Os-circANK, derived from an ankyrin repeat protein, which was significantly downregulated post Xanthomonas oryzae pv. oryzae (Xoo) inoculation. We have created a CRISPR-Cas13d system to specifically knockdown a plant circular RNA, resulting in genetically edited rice with a targeted decrease in Os-circANK expression. Os-circANK was found to function as a sponge for Osa-miR398b, suppressing the cleavage of OsCSD1/OsCSD2 by Osa-miR398b, leading to a reduction in ROS levels following Xoo infection and a negative regulation of resistance to bacterial blight in rice. Our findings demonstrate that Os-circANK inhibits the resistance to bacterial blight in rice via the Osa-miR398b/OsCSD1/OsCSD2 pathways.

microbiology↗

bioRxiv2024

Spatiotemporal cerebral blood flow dynamics underlies emergence of the limbic-sensorimotor-association cortical gradient in human infancy

Infant cerebral blood flow (CBF) delivers nutrients and oxygen to fulfill brain energy consumption requirements for the fastest period of postnatal brain development across lifespan. However, organizing principle of whole-brain CBF dynamics during infancy remains obscure. Leveraging a unique cohort of 100+ infants with high-resolution arterial spin labeled MRI, we found the emergence of the cortical hierarchy revealed by highest-resolution infant CBF maps available to date. Infant CBF across cortical regions increased in a biphasic pattern with initial rapid and sequentially slower rate, with break-point ages increasing along the limbic-sensorimotor-association cortical gradient. Increases in CBF in sensorimotor cortices were associated with enhanced language and motor skills, and frontoparietal association cortices for cognitive skills. The study discovered emergence of the hierarchical limbic-sensorimotor-association cortical gradient in infancy, and offers standardized reference of infant brain CBF and insight into the physiological basis of cortical specialization and real-world infant developmental functioning.

neuroscience↗

bioRxiv2023

Development of Sensorimotor-Visual Connectome Gradient at Birth Predicts Cognitive Outcomes at 2 Years of Age

Functional connectome gradients represent fundamental brain organizing principles. Here, we investigate the maturation of the macroscopic connectome gradients in a task-free functional MRI dataset consisting of 39 preterm and term babies aged 31-42 postmenstrual weeks and its connection with postnatal cognitive growth. We show that the principal sensorimotor-to-visual gradient is present in baby connectomes around 35.6 postmenstrual weeks and continuously develops towards a more term-like pattern. The gradients global topographical structure undergoes progressive maturation with age, characterized by increases in explanation ratio, gradient range and gradient variation. The development of focal gradients is mostly localized in primary regions serving sensory, motor and visual functions. Machine learning approaches revealed that the connectome gradient at birth significantly predicts individual cognitive outcomes at 2-y follow-up. These findings highlight the early emerging rules of functional connectome gradients and have implications for understanding how the connectome gradients at birth underlies cognitive growth in later life. HighlightsO_LISensorimotor-to-visual gradient is present around 35.6 postmenstrual weeks C_LIO_LIGlobal gradient structure undergoes substantial changes during the third trimester C_LIO_LIRegional gradient develops mostly in primary sensory, motor, and visual regions C_LIO_LISensorimotor-to-visual gradient at birth predicts cognitive outcomes at age 2 C_LI

neuroscience↗

bioRxiv2023

Development of connectome dynamics at birth predicts neurocognitive outcomes at age 2

The functional connectome of the human brain comprises time-varying network structure that facilitates efficient inter-module communication and support flexible cognitive functions. However, little is known about how the connectome dynamics of the brain emerges and develops at very early stages of human life and whether this dynamics is predictive of neurocognitive outcomes later in life. Here, we employed resting-state functional MRI data from 39 infants (31 to 42 postmenstrual weeks) and a multilayer network model to characterize the development of connectome dynamics during the third trimester and its critical role in predicting future neurocognitive outcomes at 2 years of age. We observed that the modular architecture of baby functional connectomes spontaneously reconfigures over time, with lower network module switching across time primarily in the primary regions and higher module switching mainly in the association areas. With development, the dynamic switching between the brain modules was significantly decreased, primarily located in the lateral precentral gyrus, medial temporal lobe, and subcortical areas. The clustering analysis further revealed that the primary areas displayed a higher developmental rate than the higher-order systems. Using the support vector regression approach, we found that brain connectome dynamics at birth significantly predicted cognitive and language performance at 2 years of age. Our findings highlight the emergence and spatially inhomogeneous maturation of the neonate connectome dynamics, laying a critical neural foundation for the development of cognitive and language skills later in life.

neuroscience↗

bioRxiv2023

Amphiphilic particle-stabilized nanoliter droplet reactors with a multi-modal portable reader for distributive biomarker quantification

Compartmentalization, leveraging microfluidics, enables highly sensitive assays; but the requirement for significant infrastructure for their design, build, and operation limits access. Newer multi-material particle-based technologies thermodynamically stabilize monodisperse droplets as individual reaction compartments with simple liquid handling steps, precluding the need for expensive microfluidic equipment. Here, we further improve the accessibility of this lab on a particle technology to resource-limited settings by combining this assay system with a portable multi-modal reader, thus enabling nanoliter droplet assays in an accessible platform. We show the utility of this platform in measuring N-terminal propeptide B-type natriuretic peptide (NT-proBNP), a heart failure biomarker, in complex medium and patient samples. We report a limit of detection of [~]0.05 ng/ml and a linear response between 0.2 - 2 ng/ml in spiked plasma samples. We also show that, owing to the plurality of measurements per sample, "swarm" sensing acquires better statistical quantitation with a portable reader. Monte Carlo simulations show the increasing capability of this platform to differentiate between negative and positive samples, i.e. below or above the clinical cut-off for acute heart failure ([~]0.1ng/ml), as a function of the number of particles measured. Our platform measurements correlate with gold standard ELISA measurement in cardiac patient samples, and achieve lower variation in measurement across samples compared to the standard well plate-based ELISA. Thus, we show the capabilities of a cost-effective droplet-reader system in accurately measuring biomarkers in nanoliter droplets for diseases that disproportionally affect underserved communities in resource-limited settings.

bioengineering↗

bioRxiv2022

Cell remote mechanics regulates large-spatial matrix modeling with dynamic simulations

Tissues are often isotropic and heterogeneous organizations, which developmental processes are coordinated by cells and extracellular matrix modeling. Cells have the capability of modeling matrix in distance, however, the biophysical mechanism is largely unknown. We investigated underlying mechanism of large collagen I (COL) fibrillary modeling by cell mechanics with designed arrays of cell clusters. By incorporating dynamic contractions, Molecular Dynamics simulations yielded highly matching isotropic outcomes with observed COL clustering in experiments from variable geometrical arrays without spatial limitation. Further designed single polygons from triangles to hexagons resulted in predicted structural assembly which showed maintained spatial balance. Cell cytoskeletal integrity (actin filaments, microtubules), actomyosin contractions, and endoplasmic reticulum calcium channels were essential for remote fiber inductions, while membrane mechanosensitive integrin and Piezo showed coordinative role in regulating the fiber assembly. The study provides new insights on cell mechanics-induced isotropic matrix modeling with dynamic large-spatial scales and the associated cellular mechanism. The assembled biomechanical scaffolds with pre-designs may lead to applications in micro-tissue engineering. This work implicates heterogeneous tissue structures maybe partially derived from isotropic cell mechanics.

biophysics↗

bioRxiv2022

Mechanical communication-induced cell directional migration and branching connections mediated by calcium channels, integrin β1 and N-cadherin

Cell-cell mechanical communications at large spatial scale (above hundreds of micrometers) have been increasingly recognized in recent decade, which shows importance in tissue-level assembly and morphodynamics. The involved mechanosensing mechanism and resulted physiological functions are still to be fully understood. Recent work showed that traction force sensation in the matrix induces cell communications for self-assembly. Here, based on the experimental model of cell directional migration on Matrigel hydrogel containing 0.5 mg/ml type I collagen, we studied the mechano-responsive pathways for cell distant communications. Airway smooth muscle (ASM) cells assembled network structure on the hydrogel, whereas stayed isolated individually when cultured on glass without force transmission. Cell directional migration, or network assembly was significantly attenuated by inhibited actomyosin activity, or inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) calcium channel or SERCA pump on endoplasmic reticulum (ER) membrane, or L-type calcium channel on the plasma membrane. Inhibition of integrin {beta}1 with siRNA knockdown reduced cell directional migration and branching assembly, whereas inhibition of cell junctional N-cadherin with siRNA had little effect on distant attractions but blocked branching assembly. Our work demonstrated that the ER calcium channels and integrin are mechanosensing signals for cell mechanical communications regulated by actomyosin activity, while N-cadherin is responsible for traction force-induced cell stable connections in the assembly.

biophysics↗

bioRxiv2022

Transcription factor determines the sources of extracellular vesicle DNA

Extracellular vesicle DNAs (evDNAs) possess the important diagnostic value for multiple diseases and play roles for horizontally transferring genetic materials among cells. In this study, we have found that transcription factor FOXM1 can mediate the loading of certain chromatin genes or DNA fragments (named FOXM1-chDNAs) to extracellular vesicles (EVs). FOXM1 interacts with LC3 in nucleus and FOXM1-chDNAs (such as DUX4 gene and Telomere DNA) are specified by FOXM1 and translocated to cytoplasm. These DNAs are released to EVs through the process of an LC3-involved autophagosome-multivesicular body (MVB) transport. The roles of FOXM1 on loading FOXM1-chDNAs to EVs are further confirmed by DNA-FISH experiments, tracing the translocation of selected chromatin loci with the TetO/TetR-GFP method, and PCR analysis of the DNA samples from MVBs and EVs. Furthermore, disrupting the expression of FOXM1 or the process of autophagosome-MVB transport impairs the loading of FOXM1-chDNAs to EVs. This discovery suggests that transcription factor FOXM1 contributes the constitution of evDNAs from nuclear chromatin, providing the first example to explain how chromatin DNA fragments are specified and loaded to EVs. It also provide a foundation to further explore the roles of evDNAs in biological processes such as the horizontal gene transfer.

cell biology↗

bioRxiv2021

Transient late gestation prenatal hypoxic insult results in functional deficits but not gross neuroanatomic deficits in mice

Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain.

animal behavior and cognition↗

bioRxiv2021

Physiological underpinning of default-mode network emergence in the human infant brain

Human infancy is characterized by most rapid cerebral blood flow (rCBF) increases across lifespan and emergence of a fundamental brain system default-mode network (DMN). However, how infant rCBF change spatiotemporally across the brain and how the rCBF dynamics support emergence of vital functional networks such as DMN remains unknown. Here, by acquiring cutting-edge multi-modal MRI including pseudo-continuous arterial-spin-labeled perfusion MRI and resting-state functional MRI of infants aged 0 to 24 months, we elucidated unprecedented 4D spatiotemporal infant rCBF framework and region-specific physiology-function coupling across infancy. We found faster rCBF increases in the DMN than other regions. We also found strongly coupled increases of rCBF and network strength specifically in the DMN, suggesting faster local blood flow increase to meet extra neuronal metabolic demands in the DMN maturation. These results offer insights into physiological mechanism of brain functional network emergence and have important implications in altered network maturation in brain disorders.

neuroscience↗

bioRxiv2020

Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures

In vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood. Here, using in vitro assay with engineered MDCK cells expressing H2B-mCherry (nucleus) and gp135/Podocalyxin-GFP (apical marker), we show in multi-dimensions that such coordination for epithelial morphogenesis can be determined by cell-soluble ECM interaction in the fluidic phase. The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM) and type I collagen (COL) fibers: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time-lapse two-photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135-GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi-lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment. Our results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell-soluble ECM interaction and self-assembly.

biophysics↗

bioRxiv2020

Diffusion-MRI-based regional cortical microstructure at birth for predicting neurodevelopmental outcomes at 2 years of age

Brain cerebral cortical architecture encoding regionally differential dendritic arborization and synaptic formation at birth underlies human behavior emergence at 2 years of age. Brain changes in 0-2 years are most dynamic across lifespan. Effective prediction of future behavior with brain microstructure at birth will reveal structural basis of behavioral emergence in typical development, and identify biomarkers for early detection and tailored intervention in atypical development. Here, we aimed to evaluate the neonate whole-brain cortical microstructure quantified by diffusion MRI for predicting future behavior. We found that individual cognitive and language functions assessed at age of 2 years were robustly predicted by neonate cortical microstructure using support vector regression. Remarkably, cortical regions contributing heavily to the prediction models exhibited distinctive functional selectivity for cognition and language. These findings highlight regional cortical microstructure at birth as potential sensitive biomarker in predicting future neurodevelopmental outcomes and identifying individual risks of brain disorders.

neuroscience↗

bioRxiv2020

Monodisperse drops templated by 3D-structured microparticles

The ability to create uniform sub-nanoliter compartments using microfluidic control has enabled new approaches for analysis of single cells and molecules. However, specialized instruments or expertise have been required, slowing the adoption of these cutting-edge applications. Here, we show that 3D-structured microparticles with sculpted surface chemistries template uniformly-sized aqueous drops when simply mixed with two immiscible fluid phases. In contrast to traditional emulsions, particle-templated drops of a controlled volume occupy a minimum in the interfacial energy of the system, such that a stable monodisperse state results with simple and reproducible formation conditions. We describe techniques to manufacture microscale drop-carrier particles and show that emulsions created with these particles prevent molecular exchange, concentrating reactions within the drops, laying a foundation for sensitive compartmentalized assays with minimal instrumentation.

bioengineering↗

bioRxiv2020

Formation of uniform reaction volumes using concentric amphiphilic microparticles

Reactions performed in uniform microscale volumes have enabled numerous applications in the analysis of rare entities (e.g. cells and molecules), however, sophisticated instruments are usually required to form large numbers of uniform compartments. Here, uniform aqueous droplets are formed by simply mixing microscale multi-material particles, consisting of concentric hydrophobic outer and hydrophilic inner layers, with oil and water. The particles are manufactured in batch using a 3D printed device to co-flow four concentric streams of polymer precursors which are polymerized with UV light. The size of the particles is readily controlled by adjusting the fluid flow rate ratios and mask design; whereas the cross-sectional shapes are altered by microfluidic nozzle design in the 3D printed device. Once a particle encapsulates an aqueous volume, each "dropicle" provides uniform compartmentalization and customizable shape-coding for each sample volume to enable multiplexing of uniform reactions in a scalable manner. We implement an enzymatically-amplified affinity assay using the dropicle system, yielding a detection limit of <1 pM with a dynamic range of at least 3 orders of magnitude. Moreover, multiplexing using two types of shape-coded particles was demonstrated without cross talk, laying a foundation for democratized single-entity assays.

bioengineering↗

bioRxiv2019

Cell motion-coordinated fibrillar assembly of soluble collagen I to promote MDCK cell branching formation

Extracellular Matrix (ECM) assembly and remodeling are critical physiological events in vivo, and abnormal ECM assembly or remodeling is related to pathological conditions such as osteoarthritis, fibrosis, cancers, and genetic diseases. ECM assembly/remodeling driven by cells represents more physiological processes. Collagen I (COL) is very abundant in tissues, which assembly/remodeling is mediated by biochemical and mechanical factors. How cells regulate COL assembly biomechanically still remains to be well understood. Here we used fluorescent COL in the medium to study how cells assembled ECM which represents more physiological structures. The results showed that MDCK cells actively recruited COL from the medium and helped assemble the fibers, which in turn facilitated cell branching morphogenesis, both displaying high spatial associations and mutual dependency. Inhibition of cellular contraction force by ROCK and Myosin II inhibitors attenuated but did not block the COL fiber formation, while cell motion showed high consistency with the fiber assembly. Under ROCK or Myosin II inhibition, further analysis indicated high correlation between local cell movement and COL fiber strength as quantified from different regions of the same groups. Blocking cell motion by actin cytoskeleton disruption completely inhibited the fiber formation. These suggest that cell motion coordinated COL fiber assembly from the medium, possibly through generated strain on deposited COL to facilitate the fiber growth.

biophysics↗