Biology subjects

Wang, J.

Publications and source records attributed to Wang, J..

At least 1,153 records · Page 64Linked to original sources

bioRxiv2024

Structural basis for the inhibition of coronaviral main proteases by PF-00835231

The main protease (Mpro) of coronaviruses plays a key role in viral replication, thus serving as a hot target for drug design. It has been proven that PF-00835231 is promising inhibitor of SARS-CoV-2 Mpro. Here, we report the inhibition potency of PF-00835231 against SARS-CoV-2 Mpro and seven Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) from SARS-CoV-2 variants. The results confirm that PF-00835231 has broad-spectrum inhibition against various coronaviral Mpros. In addition, the crystal structures of SARS-CoV-2 Mpro, SARS-CoV Mpro, MERS-CoV Mpro, and seven SARS-CoV-2 Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) in complex with PF-00835231 are solved. A detailed analysis of these structures reveal key determinants essential for inhibition and elucidates the binding modes of different coronaviral Mpros. Given the importance of the main protease for the treatment of coronaviral infection, structural insights into the Mpro inhibition by PF-00835231 can accelerate the design of novel antivirals with broad-spectrum efficacy against different human coronaviruses.

molecular biology↗

bioRxiv2024

DNA glycosylases Ogg1 and Mutyh mediate gene expression of PRC2 target genes important for neuronal processes underlying memory formation

Base excision repair (BER) initiated by DNA glycosylases is known to preserve genomic integrity by removing damaged bases. Recently, several DNA glycosylases were identified as potential readers of epigenetic modifications and proteins involved in BER have been associated with active DNA demethylation. DNA glycosylases Ogg1 and Mutyh were shown to alter the hippocampal transcriptome associated with cognitive function and independent of global DNA damage accumulation. However, the mechanism of DNA glycosylases in regulating cognition and their role in epigenetic remodeling in the brain remains elusive. Here we report that the combined deficiency of Ogg1 and Mutyh impairs spatial but not associative long-term memory. We demonstrate that Ogg1 or Mutyh modulate DNA methylation at gene regulatory regions of polycomb repressive complex 2 (PRC2) target genes in the adult hippocampus. Moreover, we find that the distribution of the PRC2 complex and histone modifications associated with PRC2 activity changes in both hippocampal neurons and glia depend on Ogg1 and Mutyh. Epigenetic alterations correlated with cell-type specific gene expression changes which were associated with pathways important for neuronal function and cognition. Our results provide a novel role for Ogg1 and Mutyh beyond DNA repair in modulating the epigenome to control transcriptional responses in the brain important for memory formation.

neuroscience↗

bioRxiv2024

Wampee chromosome-level reference genome elucidates fruit sugar-acid metabolism

Wampee (Clausena lansium) is an economically significant subtropical fruit tree widely cultivated in Southern China. High-quality genomic resources are unavailable, but they are essential for functional genomics and germplasm enhancement of wampee. Here, we provide a chromosome-level genome sequence for the wampee cultivar JinFeng and a population genomic analysis of 266 accessions. The 297.1 Mb wampee genome, containing nine chromosomes with a scaffold N50 of 29.2 Mb and encoding 23,468 protein-coding genes, showed a significant improvement over the previous version. We dissected the wampee population structure and genetic differentiation in China using population genomic analysis, which detected 110 and 671 genes under a selective sweep associated with sour and sweet wampee evolution in domesticated clones, respectively. Homozygous non-synonymous single nucleotide polymorphisms are likely associated with fruit flavor differentiation. A genome-wide association study identified 220 remarkable marker-trait associations for total acid content, harboring 289 genes encoding transcription factors, transporters, and enzymes involved in sugar and acid metabolism, which are potentially useful for sour and sweet taste development in wampee fruit. Furthermore, the ethylene response factor family gene ClERF061 and the SWEET family gene ClSWEET7 were identified. Linkage assessment between the relative expression levels of ClERF061 or ClSWEET7 and the total acid/total sugar contents implied their potential involvement in sugar-acid metabolism in wampee fruits. High-quality genome resources are valuable for expediting wampee research and genome-assisted breeding.

genomics↗

bioRxiv2024

A generalized framework to identify SARS-CoV-2 broadly neutralizing antibodies

Monoclonal antibodies (mAbs) targeting the SARS-CoV-2 receptor-binding domain (RBD) are used to treat and prevent COVID-19. However, the rapid evolution of SARS-CoV-2 drives continuous escape from therapeutic mAbs. Therefore, the ability to identify broadly neutralizing antibodies (bnAbs) against future variants is needed. Here, we use deep mutational scanning (DMS) to predict viral RBD evolution and to select for mAbs neutralizing both existing and prospective variants. A retrospective analysis of 1,103 SARS-CoV-2 wildtype-elicited mAbs shows that this method can increase the probability of identifying effective bnAbs against the XBB.1.5 strain from 1% to 40% in an early pandemic setup. Among these bnAbs, BD55-1205 exhibited potent activity against all tested variants. Cryo-EM structural analyses revealed the receptor mimicry of BD55-1205, explaining its broad reactivity. Delivery of mRNA-LNPs encoding BD55-1205-IgG in mice resulted in ~5,000 serum NT50 against XBB.1.5, HK.3.1, and JN.1 variants. Combining bnAb identification using viral evolution prediction with the versatility of mRNA delivery technology can enable rapid development of next-generation antibody-based countermeasures against SARS-CoV-2 and potentially other pathogens with pandemic potential.

immunology↗

bioRxiv2024

Polycystins recruit cargo to distinct ciliary extracellular vesicle subtypes

Therapeutic use of tiny extracellular vesicles (EVs) requires understanding cargo loading mechanisms. Here, we used a modular proximity label approach to identify EV cargo associated with the transient potential channel (TRP) polycystin PKD-2 of C. elegans. Polycystins are conserved receptor-TRP channel proteins affecting cilium function; dysfunction causes polycystic kidney disease in humans and mating deficits in C. elegans. Polycystin-2 EV localization is conserved from algae to humans, hinting at an ancient and unknown function. We discovered that polycystins associate with and direct specific cargo to EVs: channel-like PACL-1, dorsal and ventral membrane C-type lectins PAMLs, and conserved tumor necrosis-associated factor (TRAF) signaling adaptors TRF-1 and TRF-2. Loading of these components relied on polycystin-1 LOV-1. Our modular EV-TurboID approach can be applied in both cell- and tissue-specific manners to define the composition of distinct EV subtypes, addressing a major challenge of the EV field.

cell biology↗

bioRxiv2024

Complementing Muscle Regeneration: Fibro-Adipogenic Progenitor and Macrophage-Mediated Repair of Elderly Human Skeletal Muscle

The capacity to regenerate skeletal muscle function after injury requires a complex and well-coordinated cellular response. Here, we unravel the intricate dynamics of human skeletal muscle regeneration by combining spatial, temporal, and single cell transcriptomics. Using spatial RNA sequencing, we profiled the expression of human protein-coding genes in elderly human skeletal muscle biopsies before as well as 2-, 8-, and 30-days post injury. Single Cell-Spatial deconvolution analysis highlights monocytes/macrophages and fibro-adipogenic progenitors (FAPs) as pivotal players in human muscle regeneration. By utilizing flow cytometry and cell sorting we confirmed increased cellular content and activity during regeneration. Spatial correlation analysis unveils FAPs and monocytes/macrophages co-localization and intercellular communication, mediated by complement factor C3. Immunostaining confirms C3 expression in FAPs and FAP secretion of C3, suggesting a role in phagocytosis. Functional assays demonstrate C3s impact on monocyte metabolism, survival and phagocytosis, unveiling its involvement in skeletal muscle regeneration. These insights elucidate FAP-macrophage interplay with perspectives for future therapeutic interventions.

physiology↗

bioRxiv2024

Aldehydic load as an objective imaging biomarker of mild traumatic brain injury

Concussion is a mild traumatic brain injury (mTBI) defined as complex neurological impairment induced by biomechanical forces without structural brain damage. There does not yet exist an objective diagnostic tool for concussion. Downstream injury from mTBI stems from oxidative damage leading to the production of neurotoxic aldehydes. A collagen-based 3D corticomimetic scaffold was developed affording an in vitro model of concussion, which confirmed increased aldehyde production in live neurons following impact. To evaluate total aldehyde levels in vivo following mTBI, a novel CEST-MRI contrast agent, ProxyNA3, has been implemented in a new model of closed-head, awake, single-impact concussion developed in aged and young mice with aldehyde dehydrogenase 2 (ALDH2) deficiency. Behavioural tests confirm deficits immediately after injury. ProxyNA3-MRI was performed before impact, and on days two- and seven- post-impact. MRI signal enhancement significantly increased at two days post-injury and decreased to baseline seven days post-injury in all mice. An increase in astrocyte activation at seven days post-injury confirms the onset of a neuroinflammatory response following aldehyde production in the brain. The data suggest that advanced age and ALDH2 deficiency contribute to increased aldehydic load following mTBI. Overall, ProxyNA3 was capable of mapping concussion-associated aldehydes, supporting its application as an objective diagnostic tool for concussion.

biochemistry↗

bioRxiv2024

Confinement in fibrous environments positions and orients mitotic spindles

Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.

biophysics↗

bioRxiv2024

Mechanical stress through growth on stiffer substrates impacts animal health and longevity in C. elegans.

Mechanical stress is a measure of internal resistance exhibited by a body or material when external forces, such as compression, tension, bending, etc. are applied. The study of mechanical stress on health and aging is a continuously growing field, as major changes to the extracellular matrix and cell-to-cell adhesions can result in dramatic changes to tissue stiffness during aging and diseased conditions. For example, during normal aging, many tissues including the ovaries, skin, blood vessels, and heart exhibit increased stiffness, which can result in a significant reduction in function of that organ. As such, numerous model systems have recently emerged to study the impact of mechanical and physical stress on cell and tissue health, including cell-culture conditions with matrigels and other surfaces that alter substrate stiffness and ex vivo tissue models that can apply stress directly to organs like muscle or tendons. Here, we sought to develop a novel method in an in vivo, model organism setting to study the impact of mechanical stress on aging, by increasing substrate stiffness in solid agar medium of C. elegans. To our surprise, we found shockingly limited impact of growth of C. elegans on stiffer substrates, including limited effects on cellular health, gene expression, organismal health, stress resilience, and longevity. Overall, our studies reveal that altering substrate stiffness of growth medium for C. elegans have only mild impact on animal health and longevity; however, these impacts were not nominal and open up important considerations for C. elegans biologists in standardizing agar medium choice for experimental assays.

cell biology↗

bioRxiv2024

In vivo evaluation of thermally drawn biodegradable optical fibers as brain neural interfaces

Optical fiber technology has a critical role in modern neuroscience towards understanding the complex neuronal dynamics within the nervous system. In this study, we manufactured amorphous thermally drawn poly D, L-lactic acid (PDLLA) optical fibers in different diameters. These fibers were then implanted into the lateral posterior (LP) region of the mouse brain for 4 months, allowing us to assess their degradation characteristics. The gradual dissolution of the implanted PDLLA optical fibers in the brain was confirmed by optical microscope and scanning electron microscopy (SEM), molecular weight measurements, and light transmission spectroscopy. The results indicate that the degradation rate was mainly pronounced during the first week. Degradation after 4 months resulted in the formation of micropores on the surface of the implanted fiber within the gray matter region of the brain. Moreover, the current PDLLA optical fiber implant offers efficient light transmission in the short-wavelength near-infrared (SW-NIR) range. SW-NIR enables deeper tissue penetration and reduced light scattering, making it ideal for optogenetics and functional imaging with therapeutic potential in neurological disorders. We believe that the provided PDLLA optical fiber in this study constitutes a promising candidate for the development of next-generation biocompatible, soft, and biodegradable bi-directional neural interfaces

bioengineering↗

bioRxiv2024

Soft-lithographically defined template for arbitrarily patterned acoustic bioassembly

Acoustic bioassembly is recently regarded as a highly efficient biofabrication tool to generate functional tissue mimics. Despite their capacity of directly patterning live cells with close intercellular proximity, most acoustic bioassembly techniques are currently limited to generate some specific simple types of periodic and symmetric patterns, which represents an urgent challenge to emulate geometrically complex cytoarchitecture in human tissue. To address this challenge, we herein demonstrate a soft-lithographically defined acoustic bioassembly (SLAB) technique that enables to assemble live cells into geometrically defined arbitrary multicellular structures. Particularly, we employed a widely accessible soft lithography technique to fabricate a PDMS construct that works as an amplitude modulation template to define the pressure distribution of near-field acoustic waves. We found that zero pressure areas of the near-field acoustic waves at the PDMS surface distribute above the air-filling regions of the PDMS construct when both the PDMS top layer and air layer are approximately one-tenth of the acoustic wavelength. Using this technique, bioparticles can be assembled into symmetrical or asymmetrical patterns. Specifically, we have demonstrated the SLAB of endothelial spheroids and hepatic cells into liver tissue mimics (LTMs). The functional analysis further indicates that the formed LTMs displayed liver-specific functions, including albumin secretion, urea synthesis, glucose metabolism, and lipid storage. We expect this SLAB technique will be broadly used to construct complex functional tissues for tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2024

Immune regulators EDS1 and PAD4 constrain ADR1-dependent disease resistance in rice

Withdrawal StatementThe authors have withdrawn this manuscript owing to finding inconsistencies in genotyping of kitaake rice eds1 pad4 double mutant lines and to establishing that combined loss of EDS1 and PAD4 does not underlie the stunted autoimmune-like phenotype reported in this preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors: Jane Parker, Thomas Kroj and Haitao Cui (co-corresponding)

plant biology↗

bioRxiv2024

Spatiotemporal transcriptomic profiling reveals the dynamic immunological landscape of alveolar echinococcosis

Alveolar echinococcosis (AE) is caused by the invasive growth of the metacestodes of Echinococcus multilocularis (E. multilocularis). The early diagnosis, management, and treatment of AE remains challenging. Herein, we integrated bulk RNA-seq, scRNA-seq, and ST technologies to reveal the immune characteristics both spatially and chronologically in E. multilocularis infected mouse liver. An unprecedented high-resolution spatial atlas of the E. multilocularis infection foci was obtained, revealing the pivotal role of neutrophils, Spp1+ monocyte-derived macrophages (MoMFs), and fibroblasts in AE progression. We observed continuous recruitment of neutrophils and macrophages into the infected tissues, indicating prolonged immune stimulation by the growing metacestodes. However, their spatial distribution patterns and functions changed during the infection cause. At the early infection stage, Il1bhi neutrophils aggregated in the lesion displayed the highest pattern-recognition receptor activity and may kill the metacestodes through NETosis, but their death in the infection foci might also trigger immune suppression. Meanwhile, Spp1+ MoMFs attached to the protoscoleces to clear the infection. At the late infection stage, the neutrophils failed to infiltrate into the lesion although their continuous recruitment into the infected liver. Even though the Spp1+ MoMFs enclosing the microcysts of E. multilocularis may retain their pathogen-killing ability, they may also promote fibrosis and angiogenesis through interaction with fibroblasts. The pro- and anti-inflammatory phenotype balance of these immune cells may be associated with the transition of parasite control strategy from "active killing" to "negative segregation" by the host. Our findings identify key molecular traits involved in AE development, which may benefit the treatment of echinococcosis.

immunology↗

bioRxiv2024

Structure and function of an intermediate GPCR-G protein complex

Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-G{beta}{gamma} structures, these snapshots primarily capture the fully activated end-state complex. Consequently, a comprehensive understanding of the conformational transitions during GPCR activation and the roles of intermediate GPCR-G protein complexes in signaling remain elusive. Guided by a conformational landscape profiled by 19F quantitative NMR (19F-qNMR) and Molecular Dynamics (MD) simulations, we resolved the structure of an unliganded GPCR-G protein intermediate complex by blocking its transition to the fully activated end-state complex. More importantly, we presented direct evidence that the intermediate GPCR-Gs{beta}{gamma} complex initiates a rate-limited nucleotide exchange without progressing to the fully activated end-state complex, thereby bridging a significant gap in our understanding the complexity of GPCR signaling. Understanding the roles of individual conformational states and their complexes in signaling efficacy and bias will help us to design drugs that discriminately target a disease-related conformation.

biophysics↗

bioRxiv2024

Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance

SARS-CoV-2 main protease, Mpro, is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. Mpro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g. E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g. P252L, T21I, L50F), which restore the fitness of viral replication. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a Mpro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length Mpro protein as substrates, we demonstrate that the binding of Mpro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the Mpro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of Mpro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying Mpro and substrate interactions, and offers important insights into Mpro function, resistance development, and inhibitor design.

molecular biology↗

bioRxiv2024

APOE regulates the transport of GM1

Apolipoprotein E (APOE) is responsible for lipid transport, including cholesterol transport and clearance. While the {varepsilon}4 allele of APOE (APOE4) is associated with a significant genetic risk factor for late-onset Alzheimers disease (AD), no mechanistic understanding of its contribution to AD etiology has been established yet. In addition to cholesterol, monosialotetrahexosylganglioside (GM1) is a crucial lipid component in cell membranes and has been implicated in promoting the aggregation of amyloid beta protein (A{beta}), a key protein associated with AD. Here, we ask whether there are direct interactions between APOE and GM1 that further impact AD pathology. We find that both APOE3 and APOE4 exhibit superior binding affinity to GM1 compared to cholesterol and have an enhanced cellular uptake to GM1 lipid structures than cholesterol lipid structures. APOE regulates the transport process of GM1 depending on the cell type, which is influenced by the expression of APOE receptors in different cell lines and alters GM1 contents in cell membranes. We also find that the presence of GM1 alters the secondary structure of APOE3 and APOE4 and enhances the binding affinity between APOE and its receptor low-density lipoprotein receptor (LDLR), consequently promoting the cellular uptake of lipid structures in the presence of APOE. To understand the enhanced cellular uptake observed in lipid structures containing 20% GM1, we determined the distribution of GM1 on the membrane and found that GM1 clustering in lipid rafts, thereby supporting the physiological interaction between APOE and GM1. Overall, we find that APOE plays a regulatory role in GM1 transport, and the presence of GM1 on the lipid structures influences this transport process. Our studies introduce a plausible direct link between APOE and AD etiology, wherein APOE regulates GM1, which, in turn, promotes A{beta} oligomerization and aggregation.

neuroscience↗

bioRxiv2024

Development of a Two-Leaf Photosynthetic Model Sensitive to Chlorophyll and Its Coupling with a Wheat Growth Model

Crop growth models (CGMs) commonly simulates the response of photosynthetic rate to nitrogen (N) dynamic by calculating critical N concentration. However, critical N concentration makes it hard to describe the physiological effect of N dynamic on photosynthesis. Meanwhile, the effect of diffuse light on photosynthesis was limited in previous studies. In this study, we introduced a Two-leaf Photosynthetic Model Sensitive to Chlorophyll Content (TPMSCC) and coupled it with the crop growth model (WheatGrow) to enhance our understanding of how N dynamic and diffuse light affects photosynthesis. By coupling the Farquhar-von Caemmerer-Berry (FvCB) C3 photosynthesis model with a canopy radiative transfer model (PROSAIL), TPMSCC simulated the light interception of direct and diffuse light, and employed leaf chlorophyll content (LCC) to simulate the light absorption and electron transfer rate of leaves. Result showed that TPMSCC well simulated the light absorption of the wheat canopy and found the canopy photosynthetic rate benefited from the increase of diffuse radiation fraction (DRF) except for when the condition of a dense canopy at a high solar zenith angle was present. Research found the logarithmic and linear relationships of LCC to the initial light use efficiency ([a]) and the maximum photosynthetic rate (Amax), respectively, which followed the field measurements. Additionally, the optimized WheatGrow model outperforms its predecessor in describing the response of N application rate on photosynthesis.

biophysics↗

bioRxiv2024

CRISPR/Cas9 gene editing in Drosophila via visual selection in a summer classroom

CRISPR/Cas9 methods are a powerful in vivo approach to edit the genome of Drosophila melanogaster. To convert existing Drosophila GAL4 lines to LexA driver lines in a secondary school classroom setting, we applied the CRISPR-based genetic approach to a collection of Gal4 driver lines. The integration of the yellow+ coat color marker into homology-assisted CRISPR knock-in (HACK) enabled visual selection of Gal4-to-LexA conversions using brightfield stereo-microscopy available in a broader set of standard classrooms. Here, we report the successful conversion of eleven Gal4 lines with expression in neuropeptide-expressing cells into corresponding, novel LexA drivers. The conversion was confirmed by LexA- and Gal4-specific GFP reporter gene expression. This curriculum was successfully implemented in a summer course running 16 hours/week for seven weeks. The modularity, flexibility, and compactness of this course should enable development of similar classes in secondary schools and undergraduate curricula, to provide opportunities for experience-based science instruction, and university-secondary school collaborations that simultaneously fulfill research needs in the community of science.

genetics↗