Biology subjects

Zhang, R.

Publications and source records attributed to Zhang, R..

At least 271 records · Page 15Linked to original sources

bioRxiv2024

Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing

5-Methylcytosine (m5C) is one of the post-transcriptional modifications in mRNA and is involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m5C mapping still needs improvement. Here, we develop a detection method named DRAM (deaminase and reader protein assisted RNA methylation analysis), in which deaminases (APOBEC1 and TadA-8e) are fused with m5C reader proteins (ALYREF and YBX1) to identify the m5C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provides transcriptome-wide editing regions which are highly overlapped with the publicly available BS-seq datasets and allows for a more stable and comprehensive identification of the m5C loci. In addition, DRAM system even supports ultra-low input RNA (10ng). We anticipate that the DRAM system could pave the way for uncovering further biological functions of m5C modifications.

biochemistry↗

bioRxiv2024

Identify compound-protein interaction with knowledge graph embedding of perturbation transcriptomics

The emergence of perturbation transcriptomics provides a new perspective and opportunity for drug discovery, but existing analysis methods suffer from inadequate performance and limited applicability. In this work, we present PertKGE, a method designed to improve compound-protein interaction with knowledge graph embedding of perturbation transcriptomics. PertKGE incorporates diverse regulatory elements and accounts for multi-level regulatory events within biological systems, leading to significant improvements compared to existing baselines in two critical "cold-start" settings: inferring binding targets for new compounds and conducting virtual ligand screening for new targets. We further demonstrate the pivotal role of incorporating multi- level regulatory events in alleviating dataset bias. Notably, it enables the identification of ectonucleotide pyrophosphatase/phosphodiesterase-1 as the target responsible for the unique anti- tumor immunotherapy effect of tankyrase inhibitor K-756, and the discovery of five novel hits targeting the emerging cancer therapeutic target, aldehyde dehydrogenase 1B1, with a remarkable hit rate of 10.2%. These findings highlight the potential of PertKGE to accelerate drug discovery by elucidating mechanisms of action and identifying novel therapeutic compounds.

bioinformatics↗

bioRxiv2024

A multiplexed transcriptomic analysis of a plant embryonic hourglass

Zoologists have adduced morphological convergence among embryonic stages of closely related taxa, which has been called the phylotypic stage of embryogenesis. Transcriptomic analyses reveal a phylotypic hourglass pattern of gene expression during plant as well as animal embryogenesis, characterized by the accumulation of evolutionarily older and conserved transcripts during mid-embryogenesis, whereas younger less-conserved transcripts predominate at earlier and later embryonic stages. However, widespread comparisons of embryonic gene expression across animal phyla describe an inverse hourglass pattern, where gene expression is correlated during early and late stages but not during mid-development. Here, multiplexed spatial-transcriptomic analyses is used to investigate embryo ontogeny and homology in maize, which has novel, grass-specific morphology. An embryonic-organ genetic network is identified, replete for ancient/conserved genes manifesting a phylotypic hourglass during mid-embryogenesis. Transcriptomic comparisons of grass embryo ontogeny with that of a moss Physcomitrium patens identify a conserved, inverse hourglass pattern across plant phyla, as in animals. The data indicate that the plant phylotypic stage and that of animals is characterized by ancient gene network expression during early histo- and morphogenesis and polarized embryonic development. The data reveal an ancient, convergent mechanism for the evolution of morphological novelty.

plant biology↗

bioRxiv2024

Engineering probiotic Escherichia coli Nissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system

Many multidrug-resistant (MDR) bacteria evolved through accumulation of antibiotic-resistance genes (ARGs). Although the potential risk of probiotics as reservoirs of ARGs has been recognized, strategies for blocking transfer of ARGs while using probiotics have rarely been explored. The probiotic Escherichia coli Nissle 1917 (EcN) has long been used for treating intestinal diseases. Here, we showed frequent transfer of ARGs into EcN both in vitro and in vivo, raising its potential risk of accumulating antibiotic resistance. Given that no CRISPR-Cas system is found in natural EcN, we integrated the endogenous type I-E CRISPR-Cas system derived from E. coli BW25113 into EcN, and showed that the engineered EcN was able to efficiently cleave multiple ARGs (i.e., mcr-1, blaNDM-1 and tet(X)). By co-incubation of EcN expressing Cas3-Cascade and that expressing Cas9, we showed that the growth of the former strain outcompeted the latter strain, demonstrating better clinical application prospect of EcN expressing the type I-E CRISPR-Cas system. Finally, the engineered EcN exhibited immunity against transfer of targeted ARGs in the intestine of a model animal (i.e. zebrafish). Our work provides a new strategy for restricting transfer of ARGs in EcN, paving the way for safe use of this probiotic and development of probiotics as living therapeutics.

microbiology↗

bioRxiv2024

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 {micro}m.1 Within the past decade, reporter genes for US have been introduced2,3 and engineered4,5 to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs).6 Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semi-automated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologs of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

bioengineering↗

bioRxiv2024

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

cell biology↗

bioRxiv2024

Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous levels

ZNRF3, a negative regulator of {beta}-catenin signaling, removes Wnt receptors from the membrane. Currently, it is unknown which tumor-associated variants can be considered driver mutations and through which mechanisms they contribute to cancer. Here we show that all truncating mutations analyzed at endogenous levels exhibit loss-of-function, with longer variants retaining partial activity. Regarding missense mutations, we show that 27/82 ZNRF3 variants in the RING and R-Spondin domain structures, lead to (partial) loss-of-function/hyperactivation. Mechanistically, defective R-spondin domain variants appear to undergo endoplasmic-reticulum-associated degradation due to protein misfolding. They show reduced stability and fail to reach the membrane correctly, which can be partially restored for several variants by culturing cells at 27{degrees}C. Although RING and R-spondin domain mutations in RNF43/ZNRF3 are often considered to possess dominant-negative oncogene-like activity in cancers, our findings challenge this notion. When representative variants are heterozygously introduced into endogenous ZNRF3, their impact on {beta}-catenin signaling mirrors that of heterozygous knockout, suggesting that the supposed dominant-negative effect is non-existent. In other words, so-called "hyperactivating" ZNRF3/RNF43 mutations behave as classical loss-of-function mutations at endogenous levels. Taken together, our findings provide valuable information on ZNRF3 mutation impact in tumorigenesis and clarify their mechanism of action.

cancer biology↗

bioRxiv2024

Spatial integration of sensory input and motor output in Pseudomonas aeruginosa chemotaxis through colocalized distribution

The opportunistic pathogen Pseudomonas aeruginosa serves as a model organism for studying multiple signal transduction pathways. The chemoreceptor cluster, a core component of the chemotaxis pathway, is assembled from hundreds of proteins. The unipolar distribution of receptor clusters has long been recognized, yet the precise mechanism governing their assembly remains elusive. Here, we directly observed the relative positions of the flagellar motor and chemoreceptor cluster using flagellar filament labeling and gene editing techniques. Surprisingly, we found that both are located at the same cell pole, with the distribution pattern controlled by the polar anchor protein FlhF. Additionally, the efficient assembly of the chemoreceptor cluster is partially dependent on the integrity of the motor structure. Furthermore, we discovered that overexpression of the chemotaxis regulatory protein CheY leads to high intracellular levels of the second messenger c-di-GMP, triggering cell aggregation. Therefore, the colocalization of the chemoreceptor cluster and flagellum in P. aeruginosa serves to avoid cross-pathway signaling interference, enabling cells to conduct various physiological activities in an orderly manner.

microbiology↗

bioRxiv2024

An octopamine-specific GRAB sensor reveals a monoamine relay circuitry that boosts aversive learning

Octopamine (OA), analogous to norepinephrine in vertebrates, is an essential monoamine neurotransmitter in invertebrates that plays a significant role in various biological functions, including olfactory associative learning. However, the spatial and temporal dynamics of OA in vivo remain poorly understood due to limitations associated with the currently available methods used to detect it. To overcome these limitations, we developed a genetically encoded GPCR activation-based (GRAB) OA sensor called GRABOA1.0. This sensor is highly selective for OA and exhibits a robust and rapid increase in fluorescence in response to extracellular OA. Using GRABOA1.0, we monitored OA release in the Drosophila mushroom body (MB), the flys learning center, and found that OA is released in response to both odor and shock stimuli in an aversive learning model. This OA release requires acetylcholine (ACh) released from Kenyon cells, signaling via nicotinic ACh receptors. Finally, we discovered that OA amplifies aversive learning behavior by augmenting dopamine-mediated punishment signals via Oct{beta}1R in dopaminergic neurons, leading to alterations in synaptic plasticity within the MB. Thus, our new GRABOA1.0 sensor can be used to monitor OA release in real-time under physiological conditions, providing valuable insights into the cellular and circuit mechanisms that underlie OA signaling.

neuroscience↗

bioRxiv2024

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility

Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1s high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only [~]20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 mutation, R256H. We previously identified this mutation in multiple family members with dilated cardiomyopathy (DCM), who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent functional effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using Cryo-EM, and we see alterations in actins structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1R256H/+ human induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric disorganization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy. Significance StatementSkeletal muscle actin mutations are well-known to cause skeletal myopathies, but their role in cardiomyopathies have been controversial as skeletal muscle actin is only expressed at modest levels in the heart. Here, we demonstrate that a skeletal muscle actin mutation potently causes multiple defects in actin function at the atomic and molecular scales, and it functions in a dominant fashion, leading to cardiomyocyte contractile defects. Our results establish how skeletal muscle actin mutations may cause cardiomyocyte dysfunction and lay the foundation for future studies of the role of skeletal muscle actin in cardiomyopathy.

biophysics↗

bioRxiv2024

Spatiotemporal regulation of Pseudorabies Virus Thymidine kinase UL23 by post-translational modification

Post-translational modification plays a significant role in the interaction between viruses and their hosts. The pseudorabies virus (PRV) is a highly contagious herpesvirus that affects the central nervous system and respiratory tract of swine. However, the role of post-translational modifications, including Ubiquitination and SUMOylation, in host and PRV interplays is poorly understood. Here we examined the SUMO modification of PRV proteins and revealed that the PRV thymidine kinase UL23 can undergo SUMO modification. Bioinformatic analysis suggested four potential modification sites for UL23. Site-directed mutagenesis indicates that SUMO modification occurs at sites K242 and K267 as the wild type localizes in the nucleus while the mutants localize in the cytoplasm. Subsequently, polyclonal antibodies against mouse derived UL23 were employed to reveal that wild type UL23 was mainly located in the nucleus during PRV infection. Co-expression of UL23 with SUMO deconjugating enzymes showed that SENP1/2 inhibited the nuclear import of UL23. Whereas SUMO modification significantly impacted the localization of UL23, it did not detectably affect its stability.

cell biology↗

bioRxiv2024

Multi-condition and multi-modal temporal profile inference during mouse embryonic development

The emergence of single-cell time-series datasets enables modeling of changes in various types of cellular profiles over time. However, due to the disruptive nature of single-cell measurements, it is impossible to capture the full temporal trajectory of a particular cell. Furthermore, single-cell profiles can be collected at mismatched time points across different conditions (e.g., sex, batch, disease) and data modalities (e.g., scRNA-seq, scATAC-seq), which makes modeling challenging. Here we propose a joint modeling framework, Sunbear, for integrating multi-condition and multi-modal single-cell profiles across time. Sunbear can be used to impute single-cell temporal profile changes, align multi-dataset and multi-modal profiles across time, and extrapolate single-cell profiles in a missing modality. We applied Sunbear to reveal sex-biased transcription during mouse embryonic development and predict dynamic relationships between epigenetic priming and transcription for cells in which multi-modal profiles are unavailable. Sunbear thus enables the projection of single-cell time-series snapshots to multi-modal and multi-condition views of cellular trajectories.

bioinformatics↗

bioRxiv2024

MarmoPose: A Deep Learning-Based System for Real-time Multi-Marmoset 3D Pose Trackingp

The ability to track positions and poses (body parts) of multiple monkeys in a 3D space in real time is highly desired by non-human primate (NHP) researchers in behavioral and systems neuroscience because it allows both analyzing social behaviors among multiple NHPs and performing close-loop experiments (e.g., delivering sensory or optogenetics stimulation during a particular behavior). While a number of animal pose tracking systems have been reported, nearly all published work lacks the real-time analysis capacity. Existing methods for tracking freely moving animals have been developed primarily for rodents which typically move on a 2D space. In contrast, NHPs roam in a 3D space and move at a much faster speed than rodents. We have designed a real-time 3D pose tracking system (MarmoPose) based on deep learning to capture and quantify social behaviors in natural environment of a highly social NHP species, the common marmosets (Callithrix jacchus) which has risen to be an important NHP model in neuroscience research in recent years. This system has minimum hardware requirement and can accurately track the 3D poses (16 body locations) of multiple marmosets freely roaming in their homecage. It employs a marmoset skeleton model to optimize the 3D poses and estimate invisible body locations. Furthermore, it achieves high inference speed and provides an online processing module for real-time closed-loop experimental control based on the 3D poses of marmosets. While this system is optimized for marmosets, it can also be adapted for other large animal species in a typical housing environment with minimal modifications.

animal behavior and cognition↗

bioRxiv2024

Degradation of IKAROS prevents epigenetic progression of T cell exhaustion in a novel antigen-specific assay

In cancer, chronic antigen stimulation drives effector T cells to exhaustion, limiting the efficacy of T cell therapies. Recent studies have demonstrated that epigenetic rewiring governs the transition of T cells from effector to exhausted states and makes a subset of exhausted T cells non-responsive to PD1 checkpoint blockade. Here, we describe an antigen-specific assay for T cell exhaustion that generates T cells that are phenotypically and transcriptionally similar to those found in human tumors. We performed a screen of human epigenetic regulators, identifying and validating IKAROS as a driver of T cell exhaustion. We found that the IKAROS degrader iberdomide prevents exhaustion by blocking chromatin remodeling at T cell effector enhancers and preserving binding of AP-1, NF-{kappa}B, and NFAT. Thus, our study uncovered a role for IKAROS as a driver of T cell exhaustion through epigenetic modulation, providing a rationale for the potential use of iberdomide in solid tumors to prevent T cell exhaustion. HighlightsO_LINovel in vitro assay generates antigen-specific exhausted T cells from human T cells C_LIO_LIIKAROS (IKZF1) is a key driver of T cell exhaustion C_LIO_LIIberdomide (IKZF1/3 degrader) prevents the progression of exhaustion C_LIO_LITF footprinting reveals IKAROS silences effector genes by inhibiting AP-1, NF-{kappa}B and NFAT binding C_LI

genomics↗

bioRxiv2024

A Systematically Optimized Miniaturized Mesoscope (SOMM) for large-scale calcium imaging in freely moving mice

Understanding how neuronal dynamics gives rise to ethologically relevant behavior requires recording of neuronal population activity via technologies that are compatible with unconstrained animal behavior. However, realizations of cellular resolution head-mounted microscopes for mice have been based on conventional microscope designs that feature various forms of ad-hoc miniaturization and weight reduction measures necessary for compatibility with the weight-limits for free animal behavior. As a result, they have typically remained limited to a small field of view (FOV) or low resolution, a shallow depth range and often remain susceptible to motion-induced artifacts. Here, we present a systematically optimized miniaturized mesoscope (SOMM), a widefield, head-mounted fluorescent mesoscope based on a principled optimization approach that allows for mesoscale, cellular resolution imaging of neuroactivity while offering robustness against motion-induced artifacts. This is achieved by co-optimization of a compact diffractive optical element and the associated computational algorithm under form-factor and weight constraints while maximizing the obtainable FOV, depth of field (DOF), and resolution. SOMM enables recordings of neuronal population activity at up to 16 Hz within a FOV of 3.6 x 3.6 mm2 in the cortex of freely moving mice while featuring 4-{micro}m resolution, a DOF of 300 {micro}m at a weight of less than 2.5 g. We show SOMMs performance of recording large-scale neuronal population activity during social interactions, during conditioning-type experiments and by investigating neurovascular coupling using dual-color imaging.

neuroscience↗

bioRxiv2024

PathEX: Make Good Choice for Whole Slide Image Extraction

BackgroundThe tile-based approach has been widely used for slide-level predictions in whole slide image (WSI) analysis. However, the irregular shapes and variable dimensions of tumor regions pose challenges for the process. To address this issue, we proposed PathEX, a framework that integrates intersection over tile (IoT) and background over tile (BoT) algorithms to extract tile images around boundaries of annotated regions while excluding the blank tile images within these regions. MethodsWe developed PathEX, which incorporated IoT and BoT into tile extraction, for training a classification model in CAM (239 WSIs) and PAIP (40 WSIs) datasets. By adjusting the IoT and BoT parameters, we generated eight training sets and corresponding models for each dataset. The performance of PathEX was assessed on the testing set comprising 13,076 tile images from 48 WSIs of CAM dataset and 6,391 tile images from 10 WSIs of PAIP dataset. ResultsPathEX could extract tile images around boundaries of annotated region differently by adjusting the IoT parameter, while exclusion of blank tile images within annotated regions achieved by setting the BoT parameter. As adjusting IoT from 0.1 to 1.0, and 1 - BoT from 0.0 to 0.5, we got 8 train sets. Experimentation revealed that set C demonstrates potential as the most optimal candidate. Nevertheless, a combination of IoT values ranging from 0.2 to 0.5 and 1-BoT values ranging from 0.2 to 0.5 also yielded favorable outcomes. ConclusionsIn this study, we proposed PathEX, a framework that integrates IoT and BoT algorithms for tile image extraction at the boundaries of annotated regions while excluding blank tiles within these regions. Researchers can conveniently set the thresholds for IoT and BoT to facilitate tile image extraction in their own studies. The insights gained from this research provide valuable guidance for tile image extraction in digital pathology applications.

pathology↗

bioRxiv2024

Unraveling the Role of Urea Hydrolysis in Salt Stress Response during Seed Germination and Seedling Growth in Arabidopsis thaliana

Urea is intensively utilized as a nitrogen fertilizer in agriculture, originating either from root uptake or from catabolism of arginine by arginase. Despite its extensive use, the underlying physiological mechanisms of urea, particularly its adverse effects on seed germination and seedling growth under salt stress remains unclear. In this study, we demonstrate that salt stress induces excessive hydrolysis of arginine-derived urea, leading to an increase in cytoplasmic pH within seed radical cells, which, in turn, triggers salt-induced inhibition of seed germination (SISG) and hampers seedling growth. Our findings challenge the long-held belief that ammonium accumulation and toxicity are the primary causes of SISG, offering a novel perspective on the mechanism underlying these processes. This study provides significant insights into the physiological impact of urea hydrolysis under salt stress, contributing to a better understanding of SISG.

plant biology↗

bioRxiv2024

Accumulation of Oncogenic Mutations During Progression from Healthy Tissue to Cancer

Cancers typically result from sequential accumulation of driver mutations in a previously healthy cell. Some of these mutations, such as inactivation of the first copy of a tumor suppressor gene, can be neutral, and some, like those resulting in activation of oncogenes, may provide cells with a selective growth advantage. We study a multi-type branching process that starts with healthy tissue in home-ostasis and models accumulation of neutral and advantageous mutations on the way to cancer. We provide results regarding the sizes of premalignant populations and the waiting times to the first cell with a particular combination of mutations, including the waiting time to malignancy. Finally, we apply our results to two specific biological settings: initiation of colorectal cancer and age incidence of chronic myeloid leukemia. Our model allows for any order of neutral and advantageous mutations and can be applied to other evolutionary settings.

cancer biology↗