Biology subjects

Fu, T.

Publications and source records attributed to Fu, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Identifying brain-penetrant small molecule modulators of human microglia using a cellular model of synaptic pruning

Microglia dysregulation is implicated across a range of neurodevelopmental and neurodegenerative disorders, making their modulation a promising therapeutic target. Using PBMC-derived induced microglia-like cells (piMGLCs) in a scalable assay, we screened 489 CNS-penetrant compounds for modulation of microglial phagocytosis of human synaptosomes in a validated assay for microglia-mediated synaptic pruning. Compounds from the library that reduced phagocytosis by [≥]2 standard deviations across the library without cytotoxicity were validated in secondary screens, with 28 of them further confirmed to reduce phagocytosis by 50% or more. Image-based morphological measurements were calculated to measure the degree of ramified vs. amoeboid morphotype as an indicator of activation state. Additionally, transcriptomic profiling indicated divergent effects on cell signaling, metabolism, activation, and actin dynamics across confirmed compounds. In particular, multiple CNS-penetrant small molecules with prior FDA approval or demonstration of safety in vivo demonstrated modulatory effects on microglia. These potential disease-modifying agents represent high-priority candidates for repositioning studies in neurodevelopmental, neuroinflammatory, or neurodegenerative disorders.

neuroscience↗

bioRxiv2024

Inflammation-like environments limit the loss of quorum sensing in Pseudomonas aeruginosa

Within-host environments are complex and multidimensional, making it challenging to link evolutionary responses of colonizing pathogens to causal selective drivers. Loss of quorum sensing (QS) via mutation of the master regulator, lasR, is a common adaptation of Pseudomonas aeruginosa during chronic infections. Here, we use experimental evolution in host-mimicking media to show that loss of QS is constrained by environmental factors associated with host inflammation. Specifically, environments combining oxidative stress and abundant free amino acids limited loss of QS, whereas QS loss was rapid in the absence of oxidative stress regardless of amino acids. Under oxidative stress, lasR mutations were contingent upon first decoupling regulation of oxidative stress responses from QS via mutations in the promoter region of the primary catalase, katA, or in the oxidative stress regulator, oxyR, enabling maintenance of oxidative stress tolerance. Together, our findings suggest that host inflammatory responses likely delay the loss of QS whilst colonizers undergo stepwise evolution, first adapting to survive lethal stressors before responding to other nutritional selective drivers that favour loss of QS.

evolutionary biology↗

bioRxiv2024

Mettl5 coordinates protein production and degradation of PERIOD to regulate sleep in Drosophila

Sleep plays a critical role in animal physiology, primarily governed by the brain, and its disruption is prevalent in various brain disorders. Mettl5 is associated with intellectual disability (ID), which often includes sleep disturbances. However, the mechanism underlying these sleep disruptions in ID remains poorly understood. In this study, we investigated the sleep phenotypes resulting from Drosophila Mettl5 mutations. Rescue experiments revealed that Mettl5 functions predominantly within neurons and glia marked by Mettl5-Gal4 to regulate sleep. Previous work established that Mettl5 forms a complex with Trmt112 to influence rRNA methylation. Notably, a mutation in Trmt112 mutation recapitulated these sleep disturbances, implicating translational regulation by the Mettl5/Trmt112 complex. Subsequent RNA-seq and Ribo-seq analyses of Mettl51bp mutants uncovered downstream effects, including altered expression of proteasome components and clock genes. Rescue experiments confirmed that the net increase in PERIOD protein underlies the sleep phenotype. This study illuminates the interplay between ribosome function, clock genes, and the proteasome in sleep regulation, highlighting the integrated roles of protein synthesis and degradation. These findings could potentially provide an example for in vivo study of rRNA methylation function, expand our understanding of protein homeostasis in sleep and offer insights into the sleep phenotypes associated with ID.

genetics↗

bioRxiv2024

Topological rearrangements activate the HerA-DUF anti-phage defense system

Leveraging the rich structural information provided by AlphaFold, we used integrated experimental approaches to characterize the HerA-DUF4297 (DUF) anti-phage defense system, in which DUF is of unknown function. To infer the function of DUF, we performed structure-guided genomic analysis and found that DUF homologs are universally present in bacterial immune defense systems. One notable homolog of DUF is Cap4, a universal effector with nuclease activity in CBASS, the most prevalent anti-phage system in bacteria. To test the inferred nuclease function of DUF, we performed biochemical experiments and discovered that the DUF only exhibits activity against DNA substrates when it is bound by HerA. To understand how HerA activates DUF, we determined the structures of DUF and the HerA-DUF complex. DUF forms large oligomeric assemblies with or without HerA, suggesting that oligomerization per se is not sufficient for DUF activation. Instead, DUF activation requires dramatic topological rearrangements that propagate from HerA to the entire HerA-DUF complex, leading to reorganization of DUF for effective DNA cleavage. We further validated these structural insights by structure- guided mutagenesis. Together, these findings reveal dramatic topological rearrangements throughout the HerA-DUF complex, challenge the long-standing dogma that protein oligomerization alone activates immune signaling, and may inform the activation mechanism of CBASS.

biochemistry↗

bioRxiv2024

ANLN directly interacts with PCNA to regulate UV induced translesion synthesis

Anillin (ANLN) is a cytoskeletal binding protein involved in mitosis. ANLN is located in the nucleus during interphase and in the cytoplasmic contractile rings during mitosis. Our previous studies found that ANLN is abnormally overexpressed in esophageal squamous cell carcinoma (ESCC), promoting cell division by regulating contractile ring localization. However, the exact function of ANLN in the nucleus remains unclear. Here, we found that the expression of ANLN in the nucleus is associated with poor prognosis in ESCC patients, rather than in the cytoplasm. Protein mass spectrometry and bioinformatics analysis revealed that ANLN is related to DNA synthesis, and proliferating cell nuclear antigen (PCNA) is found to be a potential interacting protein of ANLN. PCNA directly interacts with the PIP box domain of ANLN and co-localizes in the nucleus. ANLN promotes DNA replication and S phase progression in a PCNA dependent manner and independent with the cytoskeletal function of ANLN. Importantly, ANLN is involved in transletion synthesis (TLS), a type of DNA synthesis under stress, by promoting PCNA monoubiquitination at K164 residue. Mechanistically, ANLN binds and recruits the E3 ligase RAD18 to promote PCNA monoubiquitination and DNA polymerase eta loading under UV radiation conditions. Consistently, depletion of ANLN leads to increased genomic instability and increased sensitivity to UV radiation. The findings of the study showed that ANLN in the nucleus as a protein scaffold is involved in UV induced DNA synthesis pathway, providing new insights into the function and mechanism of ANLN in cancer cells.

cancer biology↗

bioRxiv2024

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgo) and the DNA Defense Module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

biophysics↗

bioRxiv2024

TDC-2: Multimodal Foundation for Therapeutic Science

Drug discovery AI datasets and benchmarks have not traditionally included single-cell analysis biomarkers. While benchmarking efforts in single-cell analysis have recently released collections of single-cell tasks, they have yet to comprehensively release datasets, models, and benchmarks that integrate a broad range of therapeutic discovery tasks with cell-type-specific biomarkers. Therapeutics Commons (TDC-2) presents datasets, tools, models, and benchmarks integrating cell-type-specific contextual features with ML tasks across therapeutics. We present four tasks for contextual learning at single-cell resolution: drug-target nomination, genetic perturbation response prediction, chemical perturbation response prediction, and protein-peptide interaction prediction. We introduce datasets, models, and benchmarks for these four tasks. Finally, we detail the advancements and challenges in machine learning and biology that drove the implementation of TDC-2 and how they are reflected in its architecture, datasets and benchmarks, and foundation model tooling.

bioinformatics↗

bioRxiv2024

Global genomic diversity of Pseudomonas aeruginosa in bronchiectasis

BackgroundPseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis (CF) and bronchiectasis, associated with poorer outcomes. Unlike CF, bronchiectasis has been a neglected disease. More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P. aeruginosa infections to guide novel and improved treatments. MethodsWe have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections. ResultsWe observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms. ConclusionsOur findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes. SummaryThe largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection.

genomics↗

bioRxiv2024

Blocker-SELEX: A Structure-guided Strategy for Developing Inhibitory Aptamers Disrupting Undruggable Transcription Factor Interactions

Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges associated with modulating their protein-protein and protein-DNA interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs do not favor the use of small-molecule inhibitors, or neutral antibodies, in blocking TF interactions. Aptamers are short oligonucleotides exhibiting high affinity and specificity for a diverse range of targets. Large molecular weights, expansive blocking surfaces and efficient cellular internalization make aptamers a compelling molecular tool for use as traditional TF interaction modulators. Here, we report a structure-guided design strategy called Blocker-SELEX to develop inhibitory aptamers (iAptamer) that selectively block TF interactions. Our approach led to the discovery of iAptamers that cooperatively disrupts SCAF4/SCAF8-RNA Polymerase II (RNAP2) interactions, thereby dysregulating RNAP2-dependent gene expression and splicing and, in turn, leading to the impairment of cell proliferation. This approach was further applied to develop iAptamers to efficiently block WDR5-MYC interaction with a nexus in cancer. Taken together, our study highlights the potential of Blocker-SELEX in developing iAptamers that effectively disrupt potentially pathogenic TF interactions with attendant implications for iAptamers as chemical tools for use in the study of biological functions of TF interactions, but also for potential use in nucleic acids drug discovery.

biochemistry↗

bioRxiv2023

Molecular basis of Gabija anti-phage supramolecular assemblies

As one of the most prevalent anti-phage defense systems in prokaryotes, Gabija consists of a Gabija protein A (GajA) and a Gabija protein B (GajB). The assembly and function of the Gabija system remain unclear. Here we present cryo-EM structures of GajA and the GajAB complex, revealing tetrameric and octameric assemblies, respectively. In the center of the complex, GajA assembles into a symmetric tetramer, which recruits two sets of GajB dimer at opposite sides of the complex, resulting in a 4:4 GajAB supramolecular complex for anti-phage defense. Further biochemical analysis showed that GajA alone is sufficient to cut double-stranded DNA and plasmid DNA, which can be inhibited by ATP. Unexpectedly, the GajAB displays enhanced activity for plasmid DNA, suggesting a role of substrate selection by GajB. Together, our study defines a framework for understanding anti-phage immune defense by the GajAB complex.

immunology↗

bioRxiv2022

Loss of function in the autism and schizophrenia-associated gene CYFIP1 in human microglia supports a role in synaptic pruning

BackgroundThe CYFIP1 gene, located in the neurodevelopmental risk locus 15q11.2, is highly expressed in microglia, but its role in human microglial function as it relates to neurodevelopment is not well understood. MethodsWe generated multiple CRISPR knockouts of CYFIP1 in patient-derived models of microglia to characterize function and phenotype. Using microglia-like cells reprogrammed from peripheral blood mononuclear cells, we quantified phagocytosis of synaptosomes (isolated and purified synaptic vesicles) from human iPSC-derived neuronal cultures as an in vitro model of synaptic pruning. We repeated these analyses in human iPSC-derived microglia, and characterized microglial development and function through morphology and motility. ResultsCYFIP1 knockout using orthogonal CRISPR constructs in multiple patient-derived cell lines was associated with statistically significant decrease in synaptic vesicle phagocytosis in microglia models derived from both PBMCs and iPSCs (p<0.0001). Morphology was also shifted toward a more ramified profile (p<0.0001), and motility was significantly reduced (p<0.0001). However, iPSC-CYFIP1 knockout lines retained the ability to differentiate to functional microglia. Conclusion: The changes in microglial phenotype and function from loss of CYFIP1 may contribute to pruning abnormalities observed in CYFIP1-associated neurodevelopmental disorders. Investigating risk genes in a range of CNS cell types may be required to fully understand the way in which common and rare variants intersect to yield neuropsychiatric disorders.

neuroscience↗

bioRxiv2022

scAllele: a versatile tool for the detection and analysis of variants in scRNA-seq

Single-cell RNA sequencing (scRNA-seq) data contain rich information at the gene, transcript, and nucleotide levels. Most analyses of scRNA-seq have focused on gene expression profiles, and it remains challenging to extract nucleotide variants and isoform-specific information. Here, we present scAllele, an integrative approach that detects single nucleotide variants, insertions, deletions, and their allelic linkage with splicing patterns in scRNA-seq. We demonstrate that scAllele achieves better performance in identifying nucleotide variants than other commonly used tools. In addition, the read-specific variant calls by scAllele enables allele-specific splicing analysis, a unique feature not afforded by other methods. Applied to a lung cancer scRNA-seq data set, scAllele identified variants with strong allelic linkage to alternative splicing, some of which being cancer-specific. scAllele represents a versatile tool to uncover multi-layer information and novel biological insights from scRNA-seq data.

bioinformatics↗

bioRxiv2022

L-GIREMI uncovers RNA editing sites in long-read RNA-seq

Using third-generation sequencers, long-read RNA-seq is increasingly applied in transcriptomic studies given its major advantage in characterizing full-length transcripts. A number of methods have been developed to analyze this new type of data for transcript isoforms and their abundance. Another application, which is significantly under-explored, is to identify and analyze single nucleotide variants (SNVs) in the RNA. Identification of SNVs, such as genetic mutations or RNA editing sites, is fundamental to many biomedical questions. In long-read RNA-seq, SNV analysis presents significant challenges, due to the well-known relatively high error rates of the third-generation sequencers. Here, we present the first study to detect and analyze RNA editing sites in long-read RNA-seq. Our new method, L-GIREMI, effectively handles sequencing errors and biases in the reads, and uses a model-based approach to score RNA editing sites. Applied to PacBio long-read RNA-seq data, L-GIREMI affords a high accuracy in RNA editing identification. In addition, the unique advantage of long reads allowed us to uncover novel insights about RNA editing occurrences in single molecules and double-stranded RNA (dsRNA) structures. L-GIREMI provides a valuable means to study RNA nucleotide variants in long-read RNA-seq.

bioinformatics↗

bioRxiv2021

Generating Electricity from Water Evaporation Through Microbial Biofilms

Sustainable strategies for energy production are required to reduce reliance on fossil fuels and to power electronics without generating toxic waste.1-7 Generating electricity from water evaporation through engineered materials is a promising approach,8,9 but power outputs have been low and the materials employed were not sustainably produced. Microorganisms can be mass produced with renewable feedstocks. Here, we demonstrate that it is possible to engineer microbial biofilms as a cohesive, flexible material for long-term continuous electricity production from evaporating water. The biofilm sheets were the functional component in devices that continuously produced power densities ([~]1 W/cm2) higher than that achieved with non-biological materials. Current production scaled directly with biofilm-sheet size and skin-patch devices harvested sufficient electricity from the moisture on skin to continuously power wearable devices. The results demonstrate that appropriately engineered biofilms can perform as robust functional materials without the need for further processing or maintaining cell viability. Biofilm-based hydroelectric current production was comparable to that achieved with similar sized biofilms catalyzing current production in microbial fuel cells,10,11 without the need for an organic feedstock or maintaining cell viability. The ubiquity of biofilms in nature suggests the possibility of additional sources of biomaterial for evaporation-based electricity generation and the possibility of harvesting electricity from diverse aqueous environments.

microbiology↗

bioRxiv2021

Reference genomes of 545 silkworms enable high-throughput exploring genotype-phenotype relationships

The silkworm Bombyx mori is a domestic insect for silk production and a lepidopteran model. The currently available genomes limit a full understanding of its genetic and phenotypic diversity. Here we assembled long-read genomes of 545 domestic and wild silkworms and constructed a high-resolution pan-genome dataset. We found that the silkworm population harbors extremely variable genomes containing 7,308 new gene families, 4,260 (22%) core gene families, and 3,432,266 non-redundant SVs. We deciphered a series of causal genes and variants associated with domestication, breeding, and ecological adaptation traits, and experimentally validated two of those genes using CRISPR-Cas9 or RNA interference. This unprecedented large-scale genomic resource allows for high-throughput screening of interesting traits for functional genomic research and breeding improvement of silkworms and may serve as a guideline for traits decoding in other species.

genomics↗

bioRxiv2021

Network pharmacology and chemoproteomics unveil the mechanism of Total Flavonoid Aglycones Extract inhibiting EMT in A549 cell line

Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC.

pharmacology and toxicology↗

bioRxiv2021

BnaA03.WRKY28, interacting with BnaA09.VQ12, acts as a brake factor of activated BnWRKY33-mediated resistance outburst against Sclerotinia sclerotiorum in Brassica napus

Sclerotinia sclerotiorum causes substantial damage to the growth of Brassica napus (rapeseed) and makes a significant loss of crop yield. The plant innate immune system may be the primary solution to defense against S. sclerotiorum for rapeseed. Here, we identify that BnWRKY33, a transcription factor in the innate immune pathway, can be rapidly phosphorylated and activated by the MAPK cascade after rapeseed is infected with S. sclerotiorum. In the MAPK cascade, activated BnaA03.MKK4 phosphorylates and activates BnaA06.MPK3 and BnaC03.MPK3. The activated BnMPK3 acts on the substrate BnWRKY33 to enhance its transcriptional activity and trigger a transcriptional burst of BnWRKY33, which helps plants effectively resist the pathogenic fungi by enhancing the expression of phytoalexin synthesis-related genes. With constant infection, BnaA03.WRKY28 and BnaA09.VQ12 are induced, and BnaA03.WRKY28 physically interacts with BnaA09.VQ12 to form a protein complex. BnaA03.WRKY28 preferentially binds to the promoter of BnWRKY33 with the help of BnaA09.VQ12. Compared with activated BnWRKY33, BnaA03.WRKY28 has a lower transcriptional activity on downstream BnWRKY33, which leads to weaker resistance against S. sclerotiorum for rapeseed in the later stage of infection. Furthermore, the induced BnaA03.WRKY28 may promote axillary bud activity and axillary meristem initiation by regulating the expression of branching-related genes (such as BnBRC1), thus promoting the formation of branches in the leaf axils. One-sentence summaryUnder constant infection by Sclerotinia sclerotiorum, BnaA03.WRKY28 interacts with BnaA09.VQ12 and takes precedence over phosphorylated BnWRKY33 to bind to the BnWRKY33 promoter, thereby weakening resistance but promoting branching.

plant biology↗

bioRxiv2020

Shotgun lipidomics and mass spectrometry imaging unveil diversity and dynamics in lipid composition in Gammarus fossarum

Sentinel species are playing an indispensable role in monitoring environmental pollution in aquatic ecosystems. Many pollutants found in water prove to be endocrine disrupting chemicals that could cause disruptions in lipid homeostasis in aquatic species. A comprehensive profiling of the lipidome of these species is thus an essential step towards understanding the mechanism of toxicity induced by pollutants. We here extensively examined both the composition and spatial distribution of lipids in freshwater crustacean Gammarus fossarum. The baseline lipidome of gammarids of different gender and reproductive stage was established by high throughput shotgun lipidomics. Spatial lipid mapping by high resolution mass spectrometry imaging led to the discovery of sulfate-based lipids in hepatopancreas and their accumulation in mature oocytes. We uncovered in G. fossarum a diverse and dynamic lipid composition that deepens our understanding of the biochemical changes during development and which could serve as a reference for future ecotoxicological studies.

biochemistry↗