Biology subjects

Mao, H.

Publications and source records attributed to Mao, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Super-resolution histology of paraffin-embedded samples via photonic chip-based microscopy

Fluorescence-based super-resolution optical microscopy (SRM) techniques allow the visualization of biological structures beyond the diffraction limit of conventional microscopes. Despite its successful adoption in cell biology, the integration of SRM into the field of histology has been deferred due to several obstacles. These include limited imaging throughput, high cost, and the need for complex sample preparation. Additionally, the refractive index heterogeneity and high labeling density of commonly available formalin-fixed paraffin-embedded (FFPE) tissue samples pose major challenges to applying existing super-resolution microscopy methods. Here, we demonstrate that photonic chip-based microscopy alleviates several of these challenges and opens avenues for super-resolution imaging of FFPE tissue sections. By illuminating samples through a high refractive-index waveguide material, the photonic chip-based platform enables ultra-thin optical sectioning via evanescent field excitation, which reduces signal scattering and enhances both the signal-to-noise ratio and the contrast. Furthermore, the photonic chip provides decoupled illumination and collection light paths, allowing for total internal reflection fluorescence (TIRF) imaging over large and scalable fields of view. By exploiting the spatiotemporal signal emission via MUSICAL, a fluorescence fluctuation-based super-resolution microscopy (FF-SRM) algorithm, we demonstrate the versatility of this novel microscopy method in achieving superior contrast super-resolution images of diverse FFPE tissue sections derived from human colon, prostate, and placenta. The photonic chip is compatible with routine histological workflows and allows multimodal analysis such as correlative light-electron microscopy (CLEM), offering a promising tool for the adoption of super-resolution imaging of FFPE sections in both research and clinical settings.

pathology↗

bioRxiv2023

SEL1L-HRD1 ER-associated degradation is a new ataxia gene

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD); however, definitive evidence for the importance of SEL1L in HRD1 ERAD is lacking. Here we report that attenuation of the interaction between SEL1L and HRD1 impairs HRD1 ERAD function and has pathological consequences in mice. Our data show that SEL1L variant p.Ser658Pro (SEL1LS658P) previously identified in Finnish Hound suffering cerebellar ataxia is a recessive hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Mechanistically, SEL1LS658P variant attenuates the SEL1L-HRD1 interaction and causes HRD1 dysfunction by generating electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes revealed that the SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L recruits not only the lectins OS9 and ERLEC1, but the E2 UBE2J1 and retrotranslocon DERLIN, to HRD1. These data underscore the pathophysiological importance and disease relevance of the SEL1L-HRD1 complex, and identify a key step in organizing the HRD1 ERAD complex.

cell biology↗

bioRxiv2023

RawHash: Enabling Fast and Accurate Real-Time Analysis of Raw Nanopore Signals for Large Genomes

Nanopore sequencers generate electrical raw signals in real-time while sequencing long genomic strands. These raw signals can be analyzed as they are generated, providing an opportunity for real-time genome analysis. An important feature of nanopore sequencing, Read Until, can eject strands from sequencers without fully sequencing them, which provides opportunities to computationally reduce the sequencing time and cost. However, existing works utilizing Read Until either 1) require powerful computational resources that may not be available for portable sequencers or 2) lack scalability for large genomes, rendering them inaccurate or ineffective. We propose RawHash, the first mechanism that can accurately and efficiently perform real-time analysis of nanopore raw signals for large genomes using a hash-based similarity search. To enable this, RawHash ensures the signals corresponding to the same DNA content lead to the same hash value, regardless of the slight variations in these signals. RawHash achieves an accurate hash-based similarity search via an effective quantization of the raw signals such that signals corresponding to the same DNA content have the same quantized value and, subsequently, the same hash value. We evaluate RawHash on three applications: 1) read mapping, 2) relative abundance estimation, and 3) contamination analysis. Our evaluations show that RawHash is the only tool that can provide high accuracy and high throughput for analyzing large genomes in real-time. When compared to the state-of-the-art techniques, UNCALLED and Sigmap, RawHash provides 1) 25.8x and 3.4x better average throughput and 2) significantly better accuracy for large genomes, respectively. Source code is available at https://github.com/CMU-SAFARI/RawHash.

bioinformatics↗

bioRxiv2023

Effect of caffeine and other xanthines on liver sinusoidal endothelial cell ultrastructure

Xanthines such as caffeine and theobromine are among the most consumed psychoactive stimulants in the world, either as natural components of coffee, tea and chocolate, or as food additives. The present study assessed if xanthines affect liver sinusoidal endothelial cells (LSEC). Cultured primary rat LSEC were challenged with xanthines at concentrations typically obtained from normal consumption of xanthine-containing beverages, food or medicines; and at higher concentrations below the in vitro toxic limit. The fenestrated morphology of LSEC were examined with scanning electron and structured illumination microscopy. All xanthine challenges had no toxic effects on LSEC ultrastructure as judged by LSEC fenestration morphology, or function as determined by endocytosis studies. All xanthines in high concentrations (150 g/mL) increased fenestration frequency but at physiologically relevant concentrations, only theobromine (8 g/mL) showed an effect. LSEC porosity was influenced only by high caffeine doses which also shifted the fenestration distribution towards smaller pores. Moreover, a dose-dependent increase in fenestration number was observed after caffeine treatment. If these compounds induce similar changes in vivo, age-related reduction of LSEC porosity can be reversed by oral treatment with theobromine or with other xanthines using targeted delivery.

cell biology↗

bioRxiv2022

Enhancing detectable fluorescence fluctuation for high-throughput and four-dimensional live-cell super-resolution imaging

Super-resolution (SR) imaging with high-throughput is invaluable to fast and high-precision profiling in a wide range of biomedical applications. However, prevalent SR methods require sophisticated acquisition devices and specific imaging control, and may cost a fairly long time on a single field-of-view. These essentially increase the construction difficulty, including challenges in imaging throughput, system establishment, and automation. Using the natural photophysics of fluorescence, fluctuation-based microscopy techniques can routinely break the diffraction limit with no need for additional optical components, but its long acquisition time still poses a challenge for high-throughput imaging or visualizing transient organelle dynamics. Here, we propose an SR method based on the Auto-Correlation with two-step Deconvolution (SACD) that reduces the number of frames required by maximizing the detectable fluorescence fluctuation behavior in each measurement, with further removal of tunable parameters by a Fourier ring correlation analysis. It only needs 20 frames for twofold lateral and axial resolution improvements, while the SR optical fluctuation imaging (SOFI) needs more than 1000 frames. By capturing raw images for [~]10 minutes, we record an SR image with [~]128 nm resolution that contains 2.4 gigapixels covering an area of [~]2.0 mm x 1.4 mm, including more than 2,000 cells. Beyond that, by applying continuity and sparsity joint constraint, the Sparse deconvolution-assisted SACD enables 4D live-cell SR imaging of events such as mitochondrial fission and fusion. Overall, as an open-sourced module, we anticipate SACD can offer direct access to SR, which may facilitate the biology studies of cells and organisms with high-throughput and low-cost.

biophysics↗

bioRxiv2022

Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation

In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions, which is especially intractable in emerging deep-learning ones. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) framework to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Beyond that, we provide a strategy for learning-based restorations, allowing a direct detection of both data and model uncertainties, and expect the representative cases can inspire further advances in this rapidly developing field.

biophysics↗

bioRxiv2022

Towards Hierarchical Causal Representation Learning for Nonstationary Multi-Omics Data

Though open chromatin may promote active transcription, gene expression responses may not be directly coordinated with changes in chromatin accessibility. Most existing methods for single-cell multi-omics data focus only on learning stationary and shared information among these modalities, overlooking modality-specific information delineating cellular states and dynamics resulting from causal relations among modalities. To account for this, the epigenome and transcriptome relationship can be characterized in relation to time as "coupled" (changing dependently) or "decoupled" (changing independently). We propose the framework HALO, which adopts a causal approach to model these temporal causal relations on two levels. On the representation level, HALO factorizes these two modalities into both coupled and decoupled latent representations, identifying the dynamic interplay between chromatin accessibility and transcription through temporal modulations in the latent space. On the individual gene level, HALO matches gene-peak pairs and characterizes changing dynamics between gene expression and local peaks with time. HALO reveals bipotency in a subset of AT2 cells that exhibit different decisions in lineage specification between systemic sclerosis (SSc) and normal conditions. We demonstrate that using coupled and decoupled information, HALO discovers analogous biological functions between modalities, distinguishes epigenetic factors for lineage specification, and identifies temporal cis-regulation interactions relevant to cellular differentiation and complex human diseases.

bioinformatics↗

bioRxiv2021

Escape Steering by Cholecystokinin Peptidergic Signaling

Escape is an evolutionarily conserved and essential avoidance response. Considered to be innate, most studies on escape responses focused on hard-wired circuits. We report here that peptidergic signaling is an integral and necessary component of the Caenorhabditis elegans escape circuit. Combining genetic screening, electrophysiology and calcium imaging, we reveal that a neuropeptide NLP-18 and its cholecystokinin receptor CKR-1 enable the escape circuit to execute a full omega ({Omega}) turn, the last motor step where the animal robustly steers away from its original trajectory. We demonstrate in vivo and in vitro that CKR-1 is a Gq protein coupled receptor for NLP-18. in vivo, NLP-18 is mainly secreted by the gustatory sensory neuron (ASI) to activate CKR-1 in the head motor neuron (SMD) and the turn-initiating interneuron (AIB). Removal of NLP-18, removal of CKR-1, or specific knockdown of CKR-1 in SMD or AIB neurons lead to shallower turns hence less robust escape steering. Consistently, elevation of head motor neuron (SMD)s Ca2+ transients during escape steering is attenuated upon the removal of NLP-18 or CKR-1. in vitro, synthetic NLP-18 directly evokes CKR-1-dependent currents in oocytes and CKR-1-dependent Ca2+ transients in SMD. Thus, cholecystokinin signaling modulates an escape circuit to generate robust escape steering.

neuroscience↗

bioRxiv2021

RNA-seq and differential expression analysis of the duck transcriptome: The effect of short-term cage-rearing

Ducks are an important source of meat and egg products for human beings. In China, duck breeding has gradually changed from the traditional floor-water combination system to multilayer cage breeding. Therefore, the present study collected the hypothalamus and pituitary of 113-day-old ducks after being caged for 3 days, in order to investigate the effect of cage-rearing on the birds. In addition, the same tissues (hypothalamus and pituitary) were collected from ducks raised in the floor-water combination system, for comparison. Thereafter, the transcriptomes were sequenced and the expression level of genes were compared. The results of sequencing analysis showed that a total of 506 and 342 genes were differentially expressed in the hypothalamus and pituitary, respectively. Additionally, the differentially expressed genes were mainly enriched in signaling pathways involved in processing environmental information, including ECM-receptor interaction, neuroactive ligand-receptor interaction and focal adhesion. The findings also showed that there was a change in the alternative splicing of genes when ducks were transferred into the cage rearing system. However, there was no difference in the expression of some genes although there was a change in the expression of the isoforms of these genes. The findings herein can therefore help in understanding the mechanisms underlying the effect of caging on waterfowl. The results also highlight the gene regulatory networks involved in animal responses to acute stress.

genetics↗

bioRxiv2021

Structured illumination microscopy artifacts caused by illumination scattering

Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artifacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index (RI) from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artifacts caused by scattering in thick samples.

biophysics↗

bioRxiv2020

Systematic evaluation of chromosome conformation capture assays

Chromosome conformation capture (3C)-based assays are used to map chromatin interactions genome-wide. Quantitative analyses of chromatin interaction maps can lead to insights into the spatial organization of chromosomes and the mechanisms by which they fold. A number of protocols such as in situ Hi-C and Micro-C are now widely used and these differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of experimental parameters of 3C-based protocols. We find that different protocols capture different 3D genome features with different efficiencies. First, the use of cross-linkers such as DSG in addition to formaldehyde improves signal-to-noise allowing detection of thousands of additional loops and strengthens the compartment signal. Second, fragmenting chromatin to the level of nucleosomes using MNase allows detection of more loops. On the other hand, protocols that generate larger multi-kb fragments produce stronger compartmentalization signals. We confirmed our results for multiple cell types and cell cycle stages. We find that cell type-specific quantitative differences in chromosome folding are not detected or underestimated by some protocols. Based on these insights we developed Hi-C 3.0, a single protocol that can be used to both efficiently detect chromatin loops and to quantify compartmentalization. Finally, this study produced ultra-deeply sequenced reference interaction maps using conventional Hi-C, Micro-C and Hi-C 3.0 for commonly used cell lines in the 4D Nucleome Project.

genomics↗

bioRxiv2020

Developing Commotio Cordis Injury Metrics by Correlating Chest Force and Rib Deformation to Left Ventricle Strain and Pressure

Commotio cordis is a sudden death mechanism that occurs when the heart is impacted during the repolarization phase of the cardiac cycle. This study aimed to investigate commotio cordis injury metrics by correlating chest force and rib deformation to left ventricle (LV) strain and pressure. We simulated 128 chest impacts using a simulation matrix which included two initial velocities, 16 impact locations spread across the transverse and sagittal plane, and four baseball stiffness levels. Results showed that an initial velocity of 17.88 m/s and an impact location over the LV was the most damaging setting across all possible settings, causing the most considerable LV strain and pressure increases. The impact force metric did not correlate with LV strain and pressure, while rib deformations located over the LV were strongly correlated to LV strain. This leads us to the recommendation of exploring new injury metrics such as the rib deformations we have highlighted for future commotio cordis safety regulations.

bioengineering↗

bioRxiv2020

The NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

SummaryDouble-stranded DNA viruses use ATP-powered molecular motors to package their genomes. To do so, these motors must efficiently transition between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes dimerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Loss of Bone Morphogenetic Protein-binding Endothelial Regulator Causes Insulin Resistance

Accumulating evidence suggests chronic inflammation of metabolic tissues plays a causal role in obesity-induced insulin resistance. Yet, how specific endothelial factors exert impacts in metabolic tissues remains undefined. Bone morphogenetic protein (BMP)–binding endothelial regulator (BMPER) adapts endothelial cells to inflammatory stress in diverse organ microenvironments. Here we demonstrate BMPER is a driver of insulin sensitivity. Inducible knockout (iKO) of BMPER causes hyperinsulinemia, glucose intolerance and insulin resistance without increasing inflammation in metabolic tissues. Interestingly, BMPER can directly activate insulin signaling, which requires its internalization and interaction with Niemann-Pick C1 (NPC1), an integral membrane protein that transports intracellular cholesterol. These results suggest the endocrine function of the vascular endothelium maintains glucose homeostasis. Of potential clinical significance, the delivery of BMPER recombinant protein or its overexpression significantly alleviates insulin resistance and hyperglycemia in high-fat diet (HFD)-fed mice and Leprdb/db (db/db) diabetic mice. We conclude that BMPER exhibits therapeutic potential for the treatment of diabetes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pathology↗

bioRxiv2020

ITPK1-Dependent Inositol Polyphosphates Regulate Auxin Responses in Arabidopsis thaliana

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsP), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also the precursor of higher phosphorylated forms called inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recently, it was shown that Arabidopsis ITPK1 catalyzes the phosphorylation of InsP6 to the natural 5-InsP7 isomer in vitro. Here, we demonstrate that Arabidopsis ITPK1 contributes to the synthesis of InsP7 in planta. We further find a critical role of ITPK1 in auxin-related processes including primary root elongation, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity towards exogenously applied auxin. Notably, 5-InsP7 binds to recombinant auxin receptor complex, consisting of the F-Box protein TIR1, ASK1 and the transcriptional repressor IAA7, with high affinity. Furthermore, a specific increase in 5-InsP7 in a heterologous yeast expression system results in elevated interaction of the TIR1 homologs AFB1 and AFB2 with various AUX/IAA-type transcriptional repressors. We also identified a physical interaction between ITPK1 and TIR1, suggesting a dedicated channeling of an activating factor, such as 5-InsP7, to the auxin receptor complex. Our findings expand the mechanistic understanding of auxin perception and lay the biochemical and genetic basis to uncover physiological processes regulated by 5-InsP7.

plant biology↗

bioRxiv2020

Mask, the Drosophila Ankyrin Repeat and KH domain-containing protein, regulates microtubule dynamics

Proper regulation of microtubule (MT) dynamics is vital for essential cellular processes and neuronal activities, including axonal transport as well as synaptic growth and remodeling. Here we demonstrate that Mask negatively regulates MT stability and maintains a balanced dynamics of MT length and architecture in both fly larval muscles and motor neurons. In larval muscles, loss of mask increases MT length, and genetically altering mask levels modifies the Tau-induced MT fragmentation. In motor neurons, loss of mask function reduces the number of End-Binding Protein 1 (EB1)-positive MT plus-end structures in the axons and also results in overexpansion of the presynaptic terminal at larval neuromuscular junctions (NMJs). mask shows strong genetic interaction with stathmin (stai), a neuronal modulator of MT dynamics, in regulation of axon transportation and synaptic terminal stability. Our structure/function analysis on Mask revealed that truncated Mask transgenes carrying only its N-terminal portion that contains the two Ankyrin repeats domains is able to rescue the MT-related mask loss-of-function defects in larval muscles and NMJs, suggesting an essential role of the Ankyrin repeats domains in mediating Masks MT stability-regulating function. Furthermore, we discovered that Mask negatively regulates the abundance of the microtubule-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss of function phenotypes at the larval NMJs. Together, our study identified Mask as a novel regulator for microtubule stability and dynamics. Author SummaryMicrotubules (MTs) are an essential part of the cellular cytoskeleton, providing the structural basis for critical cellular processes and functionality. A series of factors are required to orchestrate the assembly and disassembly of MTs. Here, we identified Mask as a novel regulator of MT dynamics in Drosophila. Mask shows prominent interplay with two important modulators of MT, Tau and Stathmin (Stai). These findings not only support the role of Mask as a novel microtubule regulator but also provide the foundation to explore future therapeutic strategies in mitigating deficits related to dysfunction of Tau and/or Stai, both of which are linked to human neurodegenerative disorders. Further analyses on Mask demonstrated that Jupiters localization to the MTs in the axons is negatively affected by Mask, and that reducing Jupiter level is able to partially suppress synaptic defects caused by mask mutant. Together, these data imply that Masks function in regulating MT dynamics requires Jupiter.

neuroscience↗

bioRxiv2020

Rapid development of an inactivated vaccine for SARS-CoV-2

The COVID-19 pandemic caused by SARS-CoV-2 has brought about an unprecedented crisis, taking a heavy toll on human health, lives as well as the global economy. There are no SARS-CoV-2-specific treatments or vaccines available due to the novelty of this virus. Hence, rapid development of effective vaccines against SARS-CoV-2 is urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies potently neutralized 10 representative SARS-CoV-2 strains, indicative of a possible broader neutralizing ability against SARS-CoV-2 strains circulating worldwide. Immunization with two different doses (3g or 6 g per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without any antibody-dependent enhancement of infection. Systematic evaluation of PiCoVacc via monitoring clinical signs, hematological and biochemical index, and histophathological analysis in macaques suggests that it is safe. These data support the rapid clinical development of SARS-CoV-2 vaccines for humans. One Sentence SummaryA purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc) confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology

microbiology↗

bioRxiv2019

Electrospun Ag-doped SnO2 hollow nanofibers with high antibacterial activity

With the continuous improvement in medical science in modern times, the spread of bacterial infection has become a matter of global concern. Therefore, the search for biological medical materials with antibacterial function has become a focus of intense research. In this work, pure SnO2 and Ag-doped SnO2 hollow nanofibers were fabricated by a combination of an electrospinning method and a calcination procedure, and the effects of the doped Ag on antibacterial activity were subsequently investigated. Through the process of high-temperature calcination, a high heating rate would lead to the formation of a hollow tubular structure in SnO2 fibers, and Ag2O would be reduced to Ag0 by a facile process with appropriate thermal treatment. Additionally, the existence of SnO2 as a tetragonal rutile structure was confirmed. On the basis of pure SnO2, doping with silver greatly improved the antibacterial activity of hollow nanofibers. The formation mechanism and the antibacterial mechanism of pure SnO2 and Ag-doped hollow nanofibers are also discussed. This study has broad application prospects for biological medicine.

biochemistry↗