Biology subjects

Jin, H.

Publications and source records attributed to Jin, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

TUCL-Seq for miRNA single molecule sequencing

MicroRNAs (miRNAs) are pivotal regulatory molecules in gene expression, and their precise quantification is essential for elucidating their roles in diverse biological processes. This research paper introduces a pioneering workflow for the single molecule sequencing (SMS) of miRNA, integrating advanced miRNA sample preparation with SMS sequencing. Central to our approach is TUCL-Seq, which is an automated, on-chip miRNA library preparation method designed to streamline the sequencing process. This innovative approach incorporates the addition of sequencing adaptor to RNA samples in solution, and converts it to cDNA on a sequencing flow cell for sequencing. Coupled with single molecule sequencing instrument and base-call software, our workflow significantly reduces sample-to-result time, and decreases the need for manual interventions compared to traditional SMS and next-generation sequencing methods. We demonstrate the efficacy of TUCL-Seq by accurately quantifying synthetic miRNA samples with a 24-hour workflow, showcasing its potential for high-throughput miRNA profiling. Our findings highlight the promise of this integrated workflow for advancing miRNA research and applications in diagnostics and therapeutics.

biochemistry↗

bioRxiv2025

Therapeutic Potential of 20E in Pulmonary Arterial Hypertension: Involvement of Mas Receptor and PI3K/Akt Pathway

BACKGROUND20-Hydroxyecdysone (20E), a natural polyhydroxylated steroid found in various edible plants, exhibits diverse pharmacological effects. This study aims to investigate the preventive and therapeutic effects of 20E on pulmonary arterial hypertension (PAH) and elucidate its underlying molecular mechanisms. METHODSA monocrotaline (MCT)-induced PAH rat model was utilized to evaluate the efficacy of 20E. The Mas receptor antagonist A779 and agonist AVE0991 were used to investigate the role of Mas in PAH progression and 20E-mediated prevention. Molecular docking and pull-down assays were conducted to confirm the interaction between 20E and the Mas receptor. In vitro, the effects of 20E on Ang II-induced proliferation and migration of human pulmonary arterial smooth muscle cells (HPASMCs) were assessed. The PI3K/Akt signaling pathway was analyzed by Western blot. RESULTS20E prevented PAH at 30 mg/kg and 90 mg/kg, while 90 mg/kg rescued pre-existing PAH. The protective effects of 20E were attenuated by A779. 20E upregulated Mas receptor expression and directly bound to it. In vitro, 20E inhibited Ang II-induced HPASMC proliferation and migration. It also downregulated p-PI3K, p-Akt, and p-mTOR while restoring P27 and P21 expression. Furthermore, knockdown of the Mas in HPASMCs abolished the effects of 20E on these processes. CONCLUSIONS20E inhibits PASMC proliferation and migration by activating the Mas receptor and modulating the downstream PI3K/Akt signaling pathway, thereby effectively preventing and rescuing PAH. It may be a promising pharmacological candidate for PAH treatment.

molecular biology↗

bioRxiv2025

KCa3.1 Contributes to Neuroinflammation and Nigral Dopaminergic Neurodegeneration in Experimental models of Parkinson's Disease

Chronic neuroinflammation and misfolded -synuclein (Syn) have been identified as key pathological correlates driving Parkinsons disease (PD) pathogenesis; however, the contribution of ion channels to microglia activation in the context of -synucleinopathy remains elusive. Herein, we show that KCa3.1, a calcium-activated potassium channel, is robustly upregulated within microglia in multiple preclinical models of PD and, most importantly, in human PD and dementia with Lewy bodies (DLB) brains. Pharmacological inhibition of KCa3.1 via senicapoc or TRAM-34 inhibits KCa3.1 channel activity and the associated reactive microglial phenotype in response to aggregated Syn, as well as ameliorates of PD like pathology in diverse PD mouse models. Additionally, proteomic and transcriptomic profiling of microglia revealed that senicapoc ameliorates aggregated Syn-induced, inflammation-associated pathways and dysregulated metabolism in primary microglial cells. Mechanistically, FYN kinase in a STAT1 dependent manner regulates KCa3.1 mediated the microglial reactive activation phenotype after -synucleinopathy. Moreover, reduced neuroinflammation and subsequent PD-like neuropathology were observed in SYN AAV inoculated KCa3.1 knockout mice. Together, these findings suggest that KCa3.1 inhibition represents a novel therapeutic strategy for treating patients with PD and related -synucleinopathies.

neuroscience↗

bioRxiv2025

Canonical Hedgehog Signaling Controls Astral Microtubules and Mitotic Spindle Orientation in Neural Progenitors and iPSCs

Mitotic spindle orientation is crucial for cell fate determination and tissue organization. Although the intracellular machinery governing spindle orientation is well characterized, whether and how secreted factors, such as morphogens, regulate this process remains poorly understood. This study investigated the role of Hedgehog (HH) signaling in modulating mitotic spindle orientation in neural progenitor cells and in induced pluripotent stem cells (iPSCs). Time-lapse microscopy of cerebral organoids and iPSCs revealed that HH signaling increases the angle of the mitotic spindle relative to the apical surface, prolongs mitosis, and enhances spindle rotation. Mechanistically, HH signaling reduces both the number and the length of astral microtubules, key regulators of spindle orientation. This reduction correlates with increased spindle angle in iPSCs. Furthermore, we show that canonical HH signaling, involving GLI-dependent transcriptional regulation, contributes to these effects. RNA sequencing and gene set enrichment analysis (GSEA) revealed that HH signaling upregulates genes associated with microtubule depolymerization, suggesting a transcriptional mechanism by which HH signaling influences astral microtubule dynamics and, consequently, mitotic spindle orientation. These findings highlight a novel link between a morphogen, transcriptional regulation, and the control of mitotic spindle orientation, with implications for development and tissue homeostasis.

developmental biology↗

bioRxiv2025

Rationally Designed PKD1 Activator Protects Against Neurodegeneration in Pre-clinical Models of Parkinson's Disease

Oxidative stress leads to degeneration in Parkinsons disease (PD). The key signal transduction and regulatory networks that are involved during this degenerative process in PD are currently being investigated for novel neuro-protective strategies. We recently discovered that the activation of Protein Kinase D1 (PKD1) acts as a novel compensatory mechanism in PD models and positive modulation of PKD1 can be a therapeutic strategy. Therefore, the purpose of the present study was to take a translational approach by developing a PKD1 activator and characterizing the protective function in pre-clinical models of PD. Positive genetic modulation of PKD1 by overexpression of constitutively active PKD1 protected against MPP+ induced dopaminergic neurotoxicity. Pharmacological activation by Rosiglitazone protected, whereas inhibition by kb NB 142-70 exacerbated against MPP+ and 6-OHDA toxicity in cell culture PD models. Importantly, peptides were rationally designed and screened for their ability to activate PKD1 using our screening methods. Peptide AK-P4 was identified to activate PKD1 specifically and protect against MPP+ and 6-OHDA in both N27 cells and primary mesencephalic neurons. Further AK-P4 tagged with TAT sequence (AK-P4T) delivered using intra-venous injections activated PKD1 in mice. The neuro-protective effects of AK-P4T were tested using the sub-chronic MPTP mice model. Co-treatment with AK-P4T significantly restored the neurotransmitter levels and the behavioral and locomotory activities of the MPTP mouse model of PD. Collectively, our results demonstrate that rationally designed PKD1 activator peptide AK-P4T positively modulated PKD1 and protected against neurodegeneration in the pre-clinical models of PD. Our results suggest that positive modulation of the PKD1 using AK-P4T shows promise as a potential therapeutic agent against PD.

neuroscience↗

bioRxiv2025

A dWDR45 knock-out Drosophila model to decipher the role of autophagy in BPAN

Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurological disorder characterized by severe cognitive and motor impairments. BPAN is caused by de novo pathogenic variants in the WDR45 gene on the X chromosome. WDR45 gene encodes the protein WDR45/WIPI4, a known regulator of autophagy. A defective autophagy has been observed in cellular models of BPAN disease and is associated with neurological dysfunctions in wdr45 knockout (KO) mice. However, it remains unclear whether the autophagic defect directly contributes to all WDR45 loss-induced phenotypes or whether other WDR45-dependent cellular functions are involved. To investigate this, we generated a CRISPR/Cas9-mediated KO of CG11975 (dwdr45 KO), the Drosophila homolog of WDR45. Our analysis revealed that dwdr45 KO flies display BPAN-like phenotypes, including impaired locomotor function, seizure-like behavior, autophagy dysregulation and iron dyshomeostasis. Additionally, dwdr45 KO flies exhibit shortened lifespan compared to control flies. These findings demonstrate that dwdr45 KO fly is a relevant in-vivo model for investigating the key cellular and molecular mechanisms underlying BPAN-associated phenotypes. Here we showed that induction of autophagy in dwdr45 KO flies improved both the shortened lifespan and the seizure-like behavior, but did not restore locomotor function. This suggests that defective autophagy contributes to some, but not all, aspects of the phenotypes resulting from loss of dWdr45 function.

cell biology↗

bioRxiv2025

Molecular and structural basis of a subfamily of PrfH rescuing both the damaged and intact ribosomes stalled in translation

In bacteria, spontaneous mRNAs degradation and ribotoxin-induced RNA damage are two main biological events that lead to the stall of protein translation. The ubiquitous trans-translation system as well as several alternative rescue factors (Arfs) are responsible for rescuing the stalled ribosomes caused by truncated mRNAs that lack the stop codons. To date, protein release factor homolog (PrfH) is the only factor known to rescue the stalled ribosome damaged by ribotoxins. Here we show that a subfamily of PrfH, exemplified by PrfH from Capnocytophaga gingivalis (CgPrfH), rescues both types of stalled ribosomes described above. Our in vitro biochemical assays demonstrate that CgPrfH hydrolyzes the peptides attached to P-site tRNAs when in complex with both the damaged and intact ribosomes. Two cryo-EM structures of CgPrfH in complex with the damaged and intact 70S ribosomes revealed that CgPrfH employs two different regions of the protein to recognize two different stalled ribosomes to orient the GGQ motif for peptide hydrolysis. Thus, using a combination of bioinformatic, biochemical, and structural characterization described here, we have uncovered a family of ribosome rescue factors that possesses dual activities to resolve two distinct stalled protein translation in bacteria.

biochemistry↗

bioRxiv2024

Geometry-based BERT: an experimentally validated deep learning model for molecular property prediction in drug discovery

Various deep learning based methods have significantly impacted the realm of drug discovery. The development of deep learning methods for identifying novel structural types of active compounds has become an urgent challenge. In this paper, we introduce a self-supervised representation learning framework, i.e., GEO-BERT. GEO-BERT considers the information of atoms and chemical bonds in chemical structures as the input, and integrates the positional information of the three-dimensional conformation of the molecule for training. Specifically, GEO-BERT enhances its ability to characterize molecular structures by introducing three different positional relationships: atom-atom, bond-bond, and atom-bond. By benchmarking study, GEO-BERT has demonstrated optimal performance on multiple benchmarks. We also performed prospective study to validate the GEO-BERT model, with screening for DYRK1A inhibitors as a case. Two potent and novel DYRK1A inhibitors (IC50: <1 M) were ultimately discovered at a hit rate of 10%. Taken together, we have developed the Geometry-based BERT model for molecular property prediction and proved its practical utility in early-stage drug discovery. Graphical Abstract GEO-BERT is a model pretrained on large-scale drug molecule data, while improving the accuracy of property prediction by using three-dimensional structural information within the molecule. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/630211v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1e9bc54org.highwire.dtl.DTLVardef@4b6832org.highwire.dtl.DTLVardef@a5d8eeorg.highwire.dtl.DTLVardef@44f7be_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Domain Structure and Interface Control of Mechanical Stiffness in Sustainable Cellulose Bio-nanocomposites

Renewable and biodegradable plastics derived from soy protein isolate (SPI) offer a promising alternative to conventional petroleum-based plastics, particularly for film-grade bioplastics applications such as plastic bags. However, even with reinforcement from cellulose nanocrystals (CNCs), their mechanical properties including stiffness lag behind those of petroleum-based plastics. To identify pathways for improving CNC-reinforced SPI composites, we studied stiffening mechanisms by interpreting experimental data using homogenization models that accounted for CNC agglomeration and the formation of CNC/SPI interphases. To model effects of surface modification of CNCs with polydopamine (polyDOPA), we incorporated two key mechanisms: enhanced CNC dispersion and modified CNC-SPI interfacial interactions. Models accounted for interphases surrounding CNCs, arising from physicochemical interactions with the polyDOPA-modified CNC surfaces. Consistent wih experimental observations of polyDOPA modification enhancing mechanical properties through both increased spatial distribution of CNCs and matrix-filler interactions, results demonstrated that improved dispersion and interfacial bonding contribute to increased composite stiffness. Results highlight the potential of biodegradable CNC/SPI bio-nanocomposites as sustainable plastic alternatives, and suggest pathways for further enhancing their mechanical properties.

biophysics↗

bioRxiv2024

cancerSimCraft: A Multi-resolution Cancer Genome Simulator with Comprehensive Ground Truth Tracking

Cancer evolution follows complex trajectories involving diverse genomic alterations and clonal dynamics, making it challenging to validate computational methods for single-cell DNA sequencing analysis. Here we present cancerSimCraft, a comprehensive framework for simulating cancer genome data at both clonal and single-cell resolution. cancerSimCraft combines deterministic rules with stochastic processes to model various genomic events including CNVs, SNVs, and WGDs. The framework enables integration of real cancer genome patterns with user-defined parameters, supporting customizable simulation designs that reflect both empirical data and theoretical models. Through systematic benchmarking, we demonstrate cancerSimCrafts utility in evaluating computational methods under various conditions, particularly focusing on the impact of dataset size and parameter sensitivity. We further demonstrated its application in exploring clonal evolution and mutation patterns through controlled in silico experiments. These comprehensive simulation capabilities make cancerSimCraft a valuable resource for both computational method development and theoretical studies in cancer genomics.

bioinformatics↗

bioRxiv2024

Nitrogen response and growth trajectory of sorghum CRISPR-Cas9 mutants using high-throughput phenotyping

Inorganic nitrogen (N) fertilizer has emerged as one of the key factors driving increased crop yields in the past several decades; however, the overuse of chemical N fertilizer has led to severe ecological and environmental burdens. Understanding how crops respond to N fertilizer has become a central topic in plant science and plant genetics, with the ultimate goal of enhancing N use efficiency (NUE) in crop production. As one of the most essential macronutrients, N significantly influences crop performance across different developmental stages of plant, phenotypic traits result from the accumulative effects of genetic factors, prevailing environmental conditions (specifically N availability), and their complex interactions. To characterize the targeting N-responsiveness and growth trajectory, we employed CRISPR-Cas9 technique to generate sorghum mutants using CRISPR technology. Using a LemnaTec plant imaging system, we obtained time series imagery data from 29 to 130 days after sowing (DAS) for these CRISPR-edited mutants under high N and low N greenhouse conditions. After imagery data analysis, we extracted a number of morphological and greenness index traits as a proxy of plant growth and N responses. Subsequently, we employed two different methods to model the temporal N-responsive traits, allowing us to estimate seven key parameters from the growth curve. Our findings revealed that the wildtype and the edited sorghum lines exhibited differences in N responses for several of the key growth-related parameters. The high-throughput N phenotyping pipeline paves the way for a better understanding of the N responses of edited lines in a dynamic manner and sheds light on further improvements in crop NUE.

plant biology↗

bioRxiv2024

p300/CBP-catalyzed acetylation safeguards minor zygotic genome activation via activating DUX

Zygotic genome activation (ZGA), the first transcriptional event at the onset of life, is crucial for early embryonic development. However, the mechanisms that initiate and regulate ZGA in mammals remain poorly understood. In this study, we demonstrate that p300/CBP-catalyzed acetylation plays a vital role in regulating ZGA. Inhibition of p300/CBP acetyltransferase activity results in impaired minor ZGA and a subsequent 2-cell arrest. By profiling the dynamic landscape of p300 during the ZGA stage, we elucidate the distinct patterns of p300 establishment in promoter and enhancer regions. Moreover, p300 recruits RNA polymerase (Pol II) to pre-configure in the promoter regions of ZGA genes, and orchestrates the gradual establishment of enhancer activity during the early 2-cell stage to activate ZGA. Furthermore, p300 enhances minor ZGA by promoting the expression of DUX. Restoring DUX levels, which are diminished due to p300/CBP inhibition, rescues ZGA and embryonic development by re-establishing Pol II enrichment on minor ZGA genes. Overall, our findings elucidate the mechanism by which p300/CBP regulates ZGA through acetylation and activation of the key transcription factor DUX. HighlightsO_LIp300/CBP plays a stage-specific role during preimplantation development phase. C_LIO_LIp300 displays distinctive dynamic changes in promoter and enhancer regions during ZGA stage. C_LIO_LIp300/CBP-catalyzed acetylation facilitates the pre-configuration and elongation of Pol II at ZGA gene regions. C_LIO_LIDUX recruits p300 and Pol II to activate minor ZGA genes independently of p300s catalytic activity. C_LI

developmental biology↗

bioRxiv2024

Aurora kinase A promotes trained immunity via regulation of endogenous S-adenosylmethionine metabolism

Innate immune cells can acquire a memory phenotype, termed trained immunity, but the mechanism underlying the regulation of trained immunity remains largely elusive. Here, we demonstrate that inhibition of Aurora kinase A (AurA) dampens trained immunity induced by {beta}-glucan. ATAC-seq and RNA-seq analysis reveals that AurA inhibition restricts chromatin accessibility of genes associated with inflammatory pathways such as JAK-STAT, TNF and NF-{kappa}B pathways. Specifically, AurA inhibition promotes nuclear localization of FOXO3 and the expression of glycine N-methyltransferase (GNMT), a key enzyme responsible for S-adenosylmethionine (SAM) consumption. Metabolomic analysis confirms a reduction in SAM level upon AurA inhibition. As a result of SAM deficiency, trained mouse macrophages exhibit decreased H3K4me3 and H3K36me3 enrichment on gene regions of Il6 and Tnf. Additionally, the tumor inhibition effect of {beta}-glucan is notably abolished by AurA inhibition. Together, our findings identify an essential role of AurA in regulating trained immunity via a methylation-dependent manner by maintaining endogenous SAM level through mTOR-FOXO3-GNMT axis.

immunology↗

bioRxiv2024

Mitochondrial dysfunction Impairs the Nuclear Pore Complex in Parkinson's Disease Pathogenesis

The mislocalization of transcription factors and irregularities in the nuclear envelope of dopaminergic neurons affected by Parkinsons disease (PD) implicates the nuclear pore in disease pathogenesis. While mitochondrial dysfunction is an integral component of PD pathophysiology, the involvement of channel-forming nucleoporins (Nups) in mitochondrial dysfunction-related neurodegeneration has not been investigated. Here we have identified pathological changes in the levels and distribution of a set of Nups, which are key structural and functional components of the nuclear pore complex, in dopaminergic neuronal models of PD. We observed that mitochondrial dysfunction reduces the expression of these Nups and disrupts the localization of Ran GTPase in both in vitro and in vivo dopaminergic neuron models. Furthermore, the nuclear pore central channel component, Nup62, mislocalizes and accumulates in the cytoplasm of these neurons under mitochondrial stress conditions. Mitochondrial stress also interferes with classical nuclear export of proteins in dopaminergic neural cells. Notably, we observed Nup pathology and Ran gradient loss in nigral dopaminergic neurons of PD patient brains, which highlights the clinical relevance of nuclear pore dysfunction as a disease mechanism. These findings provide direct evidence of the critical role of Nup-related abnormalities in mitochondrial dysfunction-induced degeneration of dopaminergic neurons, ultimately connecting nuclear pore complex and nucleocytoplasmic transport dysregulation to cell death in the development of PD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/622143v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@193d8acorg.highwire.dtl.DTLVardef@176135forg.highwire.dtl.DTLVardef@f1edceorg.highwire.dtl.DTLVardef@1338d9d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Mitochondrial Stress Disassembles Nuclear Architecture through Proteolytic Activation of PKCδ and Lamin B1 Phosphorylation in Neuronal Cells: Implications for Pathogenesis of Age-related Neurodegenerative Diseases

Mitochondrial dysfunction and oxidative stress are hallmarks of pathophysiological processes in age-related neurodegenerative diseases including Parkinsons, Alzheimers and Huntingtons diseases. Neuronal cells are highly vulnerable to mitochondrial stress, however, the cellular and molecular mechanisms underlying the enhanced vulnerability are not well understood. Previously, we demonstrated that the novel PKC isoform PKC{delta} is highly expressed in dopamin(DA)ergic neurons and plays a key role in inducing apoptotic cell death during neurotoxic stress via caspase-3-mediated proteolytic activation. Herein, we further uncovered a key downstream molecular event of PKC{delta} signaling following mitochondrial dysfunction that governs neuronal cell death by dissembling nuclear architecture. Exposing N27 DAergic cell line to the mitochondrial complex-1 inhibitor tebufenpyrad induced PKC{delta} phosphorylation at the T505 activation loop accompanied by caspase-3-dependent proteolytic activation of the kinase. Subcellular analysis using high-resolution 3D confocal microscopy revealed that proteolytically activated cleaved PKC{delta} translocates to the nuclear compartment, colocalizing with Lamin B1. Electron microscopy also enabled the visualization of nuclear membrane damage triggered by subjecting the DAergic neuronal cells by Tebufenpyrad (Tebu) toxicity. In silico analyses identified that the threonine site on Lamin B1 (T575) is likely phosphorylated by PKC{delta}, suggesting that Lamin B1 serves as a key downstream target of the kinase. Interestingly, N27 DAergic cells stably expressing the PKC{delta} proteolytic cleavage site-resistant mutant failed to induce nuclear damage, PKC{delta} activation, and Lamin B1 phosphorylation. Furthermore, CRISPR/Cas9-based stable knockdown of PKC{delta} greatly attenuated Tebu-induced Lamin B1 phosphorylation. Also, studies using Lamin B1T575G mutated at phosphorylation and PKC{delta}-{Delta}NLS-overexpressing N27 cells showed that PKC{delta} activation and translocation to the nuclear membrane are critically required for phosphorylating Lamin B1 at T575 to induce nuclear membrane damage during Tebu insult. Additionally, Tebu failed to induce Lamin B1 damage and Lamin B1 phosphorylation in organotypic midbrain slices cultured from PKC{delta}-/- mouse pups. More importantly, we observed higher PKC{delta} activation, Lamin B1 phosphorylation and Lamin B1 loss in nigral DAergic neurons from the postmortem brains of PD patients as compared to age-matched healthy control brains, thus providing translational relevance of our finding. Collectively, our data reveal that PKC{delta} functions as a Lamin B1 kinase to disassemble the nuclear membrane during the neuronal cell death process triggered by mitochondrial stress. This mechanistic insight may have important implications for the etiology of age-related neurodegenerative diseases resulting from mitochondrial dysfunction as well as for the development of novel treatment strategies.

neuroscience↗

bioRxiv2024

The Arabidopsis amino acid transporter UmamiT20 confers susceptibility to B. cinerea

O_LIInduction of SWEET sugar transporters by bacterial pathogens via transcription activator-like (TAL) effectors is necessary for successful blight infection of rice, cassava and cotton, - likely providing sugars for bacterial propagation. C_LIO_LIHere, we show that infection of Arabidopsis by the necrotrophic fungus Botrytis cinerea causes increased accumulation of amino acid transporter UmamiT20 mRNA in leaves. UmamiT20 protein accumulates in leaf veins surrounding the lesions after infection. Consistent with a role during infection, umamiT20 knock-out mutants were less susceptible to B. cinerea. C_LIO_LIFunctional assays demonstrate that UmamiT20 mediates amino acid transport of a wide range of amino acid substrates. C_LIO_LIPathogen-induced UmamiT20 mRNA and protein accumulation support the hypothesis that transporter-mediated pathogen susceptibility is not unique to SWEETs in bacterial blight of rice but also for a necrotrophic fungus and implicate nutrients other than sucrose, i.e., amino acids, in nutrition or nutrient signaling related to immunity. We hypothesize that stacking of mutations in different types of susceptibility-related nutrient carriers to interfere with access to several nutrients may enable engineering robust pathogen resistance in a wide range of plant-pathogen systems. C_LI Lay AbstractPathogens infect plants to gain access to their nutrient resources, enabling the pathogens to cause disease and reproduce efficiently. Here we find that an amino acid transporter constitutes a susceptibility factor for the fungal pathogen B. cinerea.

plant biology↗

bioRxiv2024

Unveil the Molecular Interplay between Aminoglycosides and Pseudouridine in IRES Translation

Eukaryotic ribosomes are enriched with pseudouridine, particularly at the functional centers targeted by antibiotics. Here we investigated the roles of pseudouridine in aminoglycoside-mediated translation inhibition by comparing the structural and functional properties of the wild-type ribosomes and those lacking pseudouridine (cbf5-D95A). We showed that the cbf5-D95A ribosomes have decreased thermostability and high sensitivity to aminoglycosides. When presented with an internal ribosome entry site (IRES) RNA, elongation factor eEF2, GTP, sordarin, hygromycin B preferentially binds to the cbf5-D95A ribosomes during initiation by blocking eEF2 binding and stalls the ribosomes in a non-rotated conformation, further hindering translocation. Hygromycin B binds to the inter-subunit bridge B2a that is known to be sensitive to pseudouridine, revealing a functional link between pseudouridine and aminoglycoside inhibition. Our results suggest that pseudouridine enhances both thermostability and conformational fitness of the ribosomes, thereby influencing their susceptibility to aminoglycosides. HighlightsO_LILoss of pseudouridine increases cell sensitivity to aminoglycosides C_LIO_LIPseudouridine enhances ribosome thermostability C_LIO_LIHygromycin B competes with eEF2 for the non-rotated ribosome C_LIO_LIHygromycin B deforms the codon-anticodon duplex C_LI

biochemistry↗

bioRxiv2024

Discovery of novel 1,4-dicarbonylthiosemicarbazides as DNA gyrase inhibitors for the treatment of MRSA infection

Antibiotic resistance has become a serious threat to public health, thus novel antibiotics are urgently needed to combat drug-resistant bacteria including MRSA (methicillin-resistant S. aureus). The 1,4-dicarbonylthiosemicarbazide is an interesting chemotype that could exhibit antibacterial activity. However, the currently available compounds are not as potent as clinical antibiotics. Herein, we adopted the computer-aided drug design strategy, substructure search, to retrieve antibacterial 1,4-dicarbonylthiosemicarbazide derivatives, and identified compound B5 (Specs ID: AG-690/15432331) from the Specs chemical library that exhibited moderate activity (minimum inhibitory concentration (MIC): 6.25 {micro}g/mL) against S. aureus (ATCC 29213). Based on that compound, we further designed and synthesized 45 derivatives, and evaluated their antibacterial activity. Eight derivatives were more potent than or equivalent to vancomycin (MIC: 1.56 {micro}g/mL). We compared the three most potent ones for their cytotoxicity to HepG2 and HUVEC cells and selected compound 1b as our lead compound for comprehensive biological evaluation. As a result, compound 1b exhibited a bacteriostatic mode, and was active against a panel of gram-positive bacteria strains, metabolically stable, and effective to protect the mice from MRSA infection. More importantly, we applied 2D similarity calculation and reverse docking to predict potential targets of compound 1b. Through experimental validation and molecular dynamics simulation, we were able to confirm that compound 1b inhibited S. aureus DNA gyrase (IC50: 1.81 {micro}M) and DNA supercoiling, potentially by binding to the ATPase domain, where ASP81, GLU58 and GLN91 formed key hydrogen bonds. Taken together, we have discovered a new class of DNA gyrase inhibitors represented by compound 1b for the treatment of MRSA infection, through the design, synthesis, and biological evaluation of novel 1,4-dicarbonylthiosemicarbazides. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/611349v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@549dadorg.highwire.dtl.DTLVardef@13bff64org.highwire.dtl.DTLVardef@1fc2c00org.highwire.dtl.DTLVardef@acee76_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAn interesting 1,4-dicarbonylthiosemicarbazide derivative B5 with moderate antibacterial activity for S. aureus was discovered by substructure search. C_LIO_LIBased on B5, 45 new 1,4-dicarbonylthiosemicarbazides were designed, synthesized and biologically evaluated, which led to the discovery of a promising lead compound 1b. C_LIO_LICompound 1b exhibited good antibacterial activity for a panel of gram-positive bacteria strains, and was effective to protect mice from MRSA infection. C_LIO_LIThe 1,4-dicarbonylthiosemicarbazides represented by compound 1b were discovered as a new class of DNA gyrase inhibitors, by target prediction and experimental validation. C_LI

pharmacology and toxicology↗