Biology subjects

Gu, S.

Publications and source records attributed to Gu, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

The comprehensive detection of hemoglobinopathy variants via long-read sequencing

BACKGROUNDThe genetic complexity of hemoglobin genes, characterized by high GC content and homologous sequences, poses significant challenges for detecting hemoglobin variants in clinical settings. METHODSA long-read indexed PCR method utilizing the novel CycloneSEQ nanopore sequencing platform was developed to detect all variant types, including single nucleotide variants (SNVs), deletions, structural variants (SVs) in HBA, HBB, HBD, and HBG genes. The method was validated using 507 clinical samples to assess its performance. RESULTSThe long-read indexed PCR system employed 13 primers targeting the hemoglobin gene clusters. This design enabled the detection of 37 types of HBA deletions, 5 SV (3 multicopies (, anti3.7, anti4.2) and 2 fusion allele (HK and anti-HK)), 37 HBB deletions, and all SNVs in the targeted regions. Validation across 507 samples (84 with HBA variants, 60 with HBB variants, 256 with both HBA and HBB variants, and 107 with no known variants) demonstrated 100.0% sensitivity and specificity. Additionally, the long-read sequencing enabled phasing of variants within hemoglobin genes, providing insights critical for clinical interpretation. CONCLUSIONSThe long-read indexed PCR method, combined with the CycloneSEQ nanopore sequencing platform, proved to be a robust and efficient solution for detecting hemoglobinopathy variants. The integration of indexed primers and barcoding enhances scalability, making this method ideal for large-scale population screening programs in the future.

genomics↗

bioRxiv2024

Improving the value utilization of tuna peptide powder for the cosmetics field through ozone oxidation

To improve the utilization efficiency of small molecule peptides in marine proteins and promote their application in cosmetics, this study adopts ozone oxidation to conduct deodorization research on tuna small molecule peptides. To devise a method for the ozone oxidation and deodorization of tuna peptide powder suitable for the cosmetics field, orthogonal tests were carried out. The time, ozone concentration, and temperature were adjusted to determine the optimal conditions for ozonation and deodorization of tuna peptide powder. The results showed that at 50{degrees}C and an ozone concentration of 99.10 x 103 mg/m3, wet ozone oxidation for 40 min can also significantly reduce the fishy smell. Under these conditions, the color of tuna peptide powder treated with wet ozone was lighter. In addition, the main volatile compounds were detected by gas chromatography-mass spectrometry (GC-MS). The content of the main fishy volatile substance, hexanal, decreased by 66.4%, and two octyl aldehydes were observed. The results also showed that the protein activity of tuna peptide powder after ozonation remained unchanged. Thus, this study presents an approach to the ozone oxidation of tuna peptide powder that is suitable for cosmetics.

bioengineering↗

bioRxiv2024

Single-Molecule Barcoding Technology for Single-Cell Genomics

Recent advances in barcoding technologies have significantly enhanced the scalability of single-cell genomic experiments. However, large-scale experiments are still rare due to high costs, complex logistics, and laborintensive procedures. To facilitate the routine application of the largest scalability, it is critical to simplify the production and use of barcoding reagents. Here, we introduce AmpliDrop, a technology that initiates the barcoding process using a pool of inexpensive single-copy barcodes and integrates barcode multiplicity generation with tagging of cellular content into a single reaction driven by DNA polymerase during library preparation. The barcoding reactions are compartmentalized using an electronic pipette or a robotic or standalone liquid handling system. These innovations eliminate the need for barcoded beads and complex combinatorial indexing workflows and provide flexibility for a wide range of scales and tube formats, as well as compatibility with automation. We show that AmpliDrop is capable of capturing transcriptomes and chromatin accessibility, and it can also be adapted for user-customized applications, including antibody-based protein detection, bacterial or viral DNA detection, and CRISPR perturbations without dual guide RNA-expression vectors. We validated AmpliDrop by investigating the influence of short-term static culturing on cell composition in human forebrain organoids, revealing metabolic reprogramming in lineage progenitors.

molecular biology↗

bioRxiv2024

Transfer RNA acetylation regulates in vivo mammalian stress signaling

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2 phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

developmental biology↗

bioRxiv2024

Molecular differences between neonatal and adult stria vascularis from organotypic explants and transcriptomics

SummaryThe stria vascularis (SV), part of the blood-labyrinth barrier, is an essential component of the inner ear that regulates the ionic environment required for hearing. SV degeneration disrupts cochlear homeostasis, leading to irreversible hearing loss, yet a comprehensive understanding of the SV, and consequently therapeutic availability for SV degeneration, is lacking. We developed a whole-tissue explant model from neonatal and adult mice to create a robust platform for SV research. We validated our model by demonstrating that the proliferative behaviour of the SV in vitro mimics SV in vivo, providing a representative model and advancing high-throughput SV research. We also provided evidence for pharmacological intervention in our system by investigating the role of Wnt/{beta}-catenin signaling in SV proliferation. Finally, we performed single-cell RNA sequencing from in vivo neonatal and adult mouse SV and revealed key genes and pathways that may play a role in SV proliferation and maintenance. Together, our results contribute new insights into investigating biological solutions for SV-associated hearing loss. SignificanceHearing loss impairs our ability to communicate with people and interact with our environment. This can lead to social isolation, depression, cognitive deficits, and dementia. Inner ear degeneration is a primary cause of hearing loss, and our study provides an in depth look at one of the major sites of inner ear degeneration: the stria vascularis. The stria vascularis and associated blood-labyrinth barrier maintain the functional integrity of the auditory system, yet it is relatively understudied. By developing a new in vitro model for the young and adult stria vascularis and using single cell RNA sequencing, our study provides a novel approach to studying this tissue, contributing new insights and widespread implications for auditory neuroscience and regenerative medicine. Highlights- We established an organotypic explant system of the neonatal and adult stria vascularis with an intact blood-labyrinth barrier. - Proliferation of the stria vascularis decreases with age in vitro, modelling its proliferative behaviour in vivo. - Pharmacological studies using our in vitro SV model open possibilities for testing injury paradigms and therapeutic interventions. - Inhibition of Wnt signalling decreases proliferation in neonatal stria vascularis. - We identified key genes and transcription factors unique to developing and mature SV cell types using single cell RNA sequencing.

molecular biology↗

bioRxiv2024

A POLR3B-variant reveals a Pol III transcriptome response dependent on La protein/SSB

RNA polymerase III (Pol III, POLR3) synthesizes tRNAs and other small non-coding RNAs. Human POLR3 pathogenic variants cause a range of developmental disorders, recapitulated in part by mouse models, yet some aspects of POLR3 deficiency have not been explored. We characterized a human POLR3B:c.1625A>G;p.(Asn542Ser) disease variant that was found to cause mis-splicing of POLR3B. Genome-edited POLR3B1625A>G HEK293 cells acquired the mis-splicing with decreases in multiple POLR3 subunits and TFIIIB, although display auto-upregulation of the Pol III termination-reinitiation subunit POLR3E. La protein was increased relative to its abundant pre-tRNA ligands which bind via their U(n)U-3-termini. Assays for cellular transcription revealed greater deficiencies for tRNA genes bearing terminators comprised of 4Ts than of [≥]5Ts. La-knockdown decreased Pol III ncRNA expression unlinked to RNA stability. Consistent with these effects, small-RNAseq showed that POLR3B1625A>Gand patient fibroblasts express more tRNA fragments (tRFs) derived from pre-tRNA 3-trailers (tRF-1) than from mature-tRFs, and higher levels of multiple miRNAs, relative to control cells. The data indicate that decreased levels of Pol III transcripts can lead to functional excess of La protein which reshapes small ncRNA profiles revealing new depth in the Pol III system. Finally, patient cell RNA analysis uncovered a strategy for tRF-1/tRF-3 as POLR3-deficiency biomarkers.

molecular biology↗

bioRxiv2024

Characterization of Cancer Stem Cells in Laryngeal Squamous Cell Carcinoma by Single-Cell RNA Sequencing

Cancer stem cells (CSCs) constitute a pivotal element within the tumor microenvironment (TME), driving the initiation and progression of cancer. However, the identification of CSCs and their underlying molecular mechanisms in laryngeal squamous cell carcinoma (LSCC) remains a formidable challenge. We employed single-cell RNA sequencing of matched primary tumor tissues, paracancerous tissues, and local lymph nodes from three LSCC patients. Two distinct clusters of stem cells originating from epithelial populations were delineated and verified as CSCs and normal stem cells (NSCs) respectively. CSCs were abundant in the paracancerous tissues compared to the tumor tissues. CSCs showed high expression of stem cell marker genes such as PROM1, ALDH1A1, and SOX4, and increased activity of tumor-related hypoxia, Wnt/{beta}-catenin, and notch signaling pathways. We then explored the intricate crosstalk between CSCs and the TME cells and identified targets within the TME that related with CSCs. We also find eight marker genes of CSCs that correlated significantly with the prognosis of LSCC patients. Furthermore, bioinformatics analyses showed that drugs such as erlotinib, OSI-027, and ibrutinib selectively targeted the CSC-specifically expressed genes. In conclusion, our results represent the first comprehensive characterization of CSCs properties in LSCC at the single-cell level.

bioinformatics↗

bioRxiv2024

Direct Measurement of 8OG syn-anti Flips in Mutagenic 8OG·A and Long-Range Damage-Dependent Hoogsteen Breathing Dynamics Using 1H CEST NMR

Elucidating how damage impacts DNA dynamics is essential for understanding the mechanisms of damage recognition and repair. Many DNA lesions alter the propensities to form lowly-populated and short-lived conformational states. However, NMR methods to measure these dynamics require isotopic enrichment, which is difficult for damaged nucleotides. Here, we demonstrate the utility of the 1H chemical exchange saturation transfer (CEST) NMR experiment in measuring the dynamics of oxidatively damaged 8-oxoguanine (8OG) in the mutagenic 8OGsyn*Aanti mismatch. Using 8OG-H7 as an NMR probe of the damaged base, we directly measured 8OG syn-anti flips to form a lowly-populated (pop. [~] 5%) and short-lived (lifetime [~] 50 ms) non-mutagenic 8OGanti*Aanti. These exchange parameters were in quantitative agreement with values from 13C off-resonance R1{rho} and CEST on a labeled partner adenine. The Watson-Crick-like 8OGsyn*Aanti mismatch also rescued the kinetics of Hoogsteen motions at distance A-T base pairs, which the G*A mismatch had slowed down. The results lend further support for 8OGanti*Aanti as a minor conformational state of 8OG*A, reveal that 8OG damage can impact Hoogsteen dynamics at a distance, and demonstrate the utility of 1H CEST for measuring damage-dependent dynamics in unlabeled DNA.

biochemistry↗

bioRxiv2023

IGHMBP2 deletion suppresses translation and activates the integrated stress response

IGHMBP2 is a non-essential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 upregulation. With recent studies showing the ISR can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

molecular biology↗

bioRxiv2023

Host signature as major driver of root and rhizosphere core microbiomes that differently affect plant functional traits

BackgroundPlant can evolve with a core root microbiome that maintains essential functions for host performance. However, the relative importance of plant traits and soil factors on the structure, assembly, co-occurrence networks of the core root microbiomes and their relevance for plant characteristics remain elusive. Here, we investigated how plant species identity and soil environment affect the core bacterial communities in the bulk soil, rhizosphere and root endosphere of four plants with a gradient of Cd/Zn accumulation capacity under controlled and field environments. We further tested on the role of the core bacterial isolates in plant growth and accumulation of metal and nutrients. ResultsWe identified root compartment and plant species rather than environmental parameters as the primary driver of Cd-accumulator root microbiome. Stochastic processes were more important for the assembly of endosphere generalists (58.5%) than rhizosphere counterparts (45.2%), indicating that generalists were more robust to environmental changes. Increasing host selection from epiphytes to endophytes resulted in the existence of the endosphere and rhizosphere generalist core microbiota common to different plants under varying growth environments, highlighting that shared environmental and physiological features of host plants are decisive for core microbiome establishment. Further, endophytic core microbiota conferred greater biotic connectivity within networks and was more important predictors of plant metal accumulation, whereas the rhizosphere cores were more closely linked to plant biomass and nutrient status. The divergent functions of rhizosphere and endosphere core microbes on plant characteristics were also validated by inoculating the synthetic communities comprising bacterial isolates belonging to the core microbiota. ConclusionThis study indicated the pivotal role of plant trait in the assembly of conserved and functionally important core microbiome common to different Cd-accumulators, which brings us closer to manipulating the persistent root microbial associations to accelerate the rejuvenation of metal-disturbed soils through host genetics.

microbiology↗

bioRxiv2023

Siderophore Piracy Promotes Dynamical Coexistence in Microbial Community

Niche construction drives ecological dynamics, yet the tragedy of the commons predicts that non-contributing cheaters will undermine cooperation. Here, we studied microbial iron competition by combining dynamic modeling with benefit flow graphs, demonstrating that moderate cheating is not merely tolerated but essential for diversity. In small communities, mutual exploitation forms closed loops enabling steady or dynamic coexistence. In larger communities, we uncovered a paradox: increasing cheating breadth promotes community-level extinction, yet fosters higher biodiversity in surviving communities. We resolve this paradox by mapping ecological dynamics onto the topology of the "Maximal Benefit Transfer Graph", which predicts community fate through its core structure. Broad cheating eliminates the self-loop core that drives competitive exclusion, but increases "terminator" sinks that cause collapse. However, when communities avoid these sinks, cheating aggregates the network and generates cyclic loops to enable coexistence. Thus, structured exploitation acts not as destabilizing vulnerability but as necessary architecture for biodiversity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/568182v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@143f144org.highwire.dtl.DTLVardef@194c2adorg.highwire.dtl.DTLVardef@1792d4aorg.highwire.dtl.DTLVardef@8c96a7_HPS_FORMAT_FIGEXP M_FIG C_FIG How does cheating affect microbial biodiversity? By mapping the strongest benefit flows between species, we discovered a topological rule for survival. While too much cheating creates dead-ends that crash the system, moderate cheating connects species into self-sustaining loops. These "exploitation cycles" act as a scaffold, supporting high diversity and complex population.

systems biology↗

bioRxiv2023

Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

pharmacology and toxicology↗

bioRxiv2023

Cross-Dataset Identification of Human Disease-Specific Cell Subtypes Enabled by the Gene Print-based Algorithm--gPRINT

Despite extensive efforts in developing cell annotation algorithms for single cell RNA sequencing results, most algorithms fail to achieve cross-dataset mapping of cell subtypes due to factors such as batch effects between datasets. This limitation is particularly evident when rapidly annotating disease-specific cell subtypes across multiple datasets. In this study, we present gPRINT, a machine learning tool that utilizes the unique one-dimensional "gene print" expression patterns of individual cells. gPRINT is capable of automatically predicting cell types and annotating disease-specific cell subtypes. The development of gPRINT involved curation and harmonization of public datasets, algorithm validation within and across datasets, and the annotation of disease-specific fibroblast subtypes across various disease subgroups and datasets. Additionally, we created a preliminary single-cell atlas of human tendinopathy fibroblasts and successfully achieved automatic prediction of disease-specific cell subtypes in tendon disease. Furthermore, we conducted an exploration of key targets and related drugs specific to this subtype in tendon disease. The proposed approach offers an automated and unified method for identifying disease-specific cell subtypes across datasets, serving as a valuable reference for annotating fibroblast-specific subtypes in different disease states and facilitating the exploration of therapeutic targets in tendon disease.

bioinformatics↗

bioRxiv2023

From sequence to ecology: siderophore-receptor coevolution algorithm predicts bacterial interactions in complex communities

Predicting bacterial social interactions from genome sequences is notoriously difficult. Here, we developed bioinformatic tools to predict whether secreted iron-scavenging siderophores stimulate or inhibit the growth of community members. Siderophores are chemically diverse and can be stimulatory or inhibitory depending on whether bacteria possess or lack corresponding uptake receptors. We focused on 1928 representative Pseudomonas genomes and developed a co-evolution algorithm to match all encoded siderophore synthetases to corresponding receptor gene groups with >90% accuracy based on experimental validation. We derived community-level iron interaction networks to show that selection for siderophore-mediated interactions differs across habitats and lifestyles. Specifically, dense networks of siderophore sharing and competition were observed among environmental (soil/water/plant) strains and non-pathogenic species, while only fragmented networks occurred among human-derived strains and pathogenic species. Altogether, our sequence-to-ecology approach empowers the analyses of social interactions among thousands of bacterial strains and uncovers ways for targeted intervention to microbial communities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/565711v3_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@60d907org.highwire.dtl.DTLVardef@48756eorg.highwire.dtl.DTLVardef@115aac1org.highwire.dtl.DTLVardef@17d76e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics

CC chemokine receptor 5 (CCR5) promotes inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A CCR5 inhibitor has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of other diseases has had limited success, likely because of the complexity of CCR5 pharmacology and biology. CCR5 is activated by natural and engineered chemokines that elicit distinct signaling and trafficking responses, including receptor sequestration inside the cell. Intracellular sequestration may be therapeutically exploitable as a strategy for receptor inhibition, but the mechanisms by which different ligands promote receptor retention in the cell versus presence on the cell membrane are poorly understood. We employed live cell ascorbic acid peroxidase (APEX2) proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveal distinct ligand-dependent CCR5 trafficking patterns with temporal and spatial resolution. All three chemokines internalize CCR5 via {beta}-arrestin-dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the cellular mechanisms behind CCR5 intracellular sequestration and suggest how trafficking can be exploited for the development of functional antagonists of CCR5. Significance StatementCCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and transmission of HIV. It responds to different ligands with distinct signaling and trafficking behaviors; notably some ligands induce retention of the receptor inside the cell. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the cellular basis for receptor sequestration that can be exploited as a therapeutic strategy for inhibiting CCR5 function.

cell biology↗

bioRxiv2023

From sequence to molecules: Feature sequence-based genome mining uncovers the hidden diversity of bacterial siderophore pathways

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/564663v5_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1dedad4org.highwire.dtl.DTLVardef@1f0a39eorg.highwire.dtl.DTLVardef@185e345org.highwire.dtl.DTLVardef@10beec6_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Determinants of DDX3X sensitivity uncovered using a helicase activity in translation reporter

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5' UTRs to measure DDX3X mediated translational activity in cells. To dissect the structural underpinnings of DDX3X dependent translation, we first used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then employed HART to investigate how their perturbation impacts DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3Xs interaction with the translational machinery. HART revealed that both DDX3Xs association with the ribosome complex as well as its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally encoded measurement of DDX3X-dependent translation in cells.

molecular biology↗

bioRxiv2023

SIDERITE: Unveiling Hidden Siderophore Diversity in the Chemical Space Through Digital Exploration

Siderophores, a highly diverse family of secondary metabolites, play a crucial role in facilitating the acquisition of the essential iron. However, the current discovery of siderophore relies largely on manual approaches. In this work, we introduced SIDERTE, a digitized siderophore information database containing 872 siderophore records with 649 unique structures. Leveraging this digitalized dataset, we gained a systematic overview of siderophores by their clustering patterns in the chemical space. Building upon this, we developed a functional group-based method for predicting new iron-binding molecules. Applying this method to 4,314 natural product molecules from TargetMols Natural Product Library for high throughput screening, we experimentally confirmed that 40 out of the 48 molecules predicted as siderophore candidates possessed iron-binding abilities. Expanding our approach to the COCONUT natural product database, we predicted a staggering 3,199 siderophore candidates, showcasing remarkable structure diversity that are largely unexplored. Our study provides a valuable resource for accelerating the discovery of novel iron-binding molecules and advancing our understanding towards siderophores.

biochemistry↗