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Yu, Y.

Publications and source records attributed to Yu, Y..

At least 181 records · Page 10Linked to original sources
bioRxiv2025

Quantitative Analysis of Coupled Carbon and Energy Metabolism for Lignin Carbon Utilization in Pseudomonas putida

Soil Pseudomonas species, which can thrive on lignin-derived phenolic compounds, are widely explored for biotechnology applications. Yet, there is limited understanding of how the native metabolism coordinates phenolic carbon processing with cofactor generation. Here, we achieve quantitative understanding of this metabolic balance through a multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate as a non-aromatic reference, proteomics data reveal >140-fold increase in proteins for transport and initial catabolism of each phenolic substrate, but metabolomics profiling reveals that bottleneck nodes in initial phenolic compound catabolism maintain more favorable cellular energy state. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic 13C-metabolomics. Quantitative analysis by 13C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling via pyruvate carboxylase promotes fluxes in the tricarboxylic acid cycle to provide 50-60% NADPH yield and 60-80% NADH yield, resulting in 2-fold higher ATP yield than for succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. The quantitative blueprint elucidated here explains deficient versus sufficient cofactor rebalancing during manipulations of key metabolic nodes in lignin valorization.

microbiology↗
bioRxiv2025

Red Light-Activated Reversible Inhibition of Protein Functions by Assembled Trap

Red light offers strong tissue penetration and low phototoxicity, making it an attractive option for optogenetic applications. However, the available red light-inducible optogenetic tools are rather limited. Here, we present a novel red light-induced protein clustering system, namely R-LARIAT (Red Light-Activated Reversible Inhibition by Assembled Trap), that controls protein functions in a spatiotemporal manner. Our system capitalizes on the rapid and reversible binding of LDBs (nanobodies-based dimerization binders) to a bacterial phytochrome DrBphP, which uses a mammalian endogenous biliverdin chromophore to absorb red light. An anti-GFP nanobody fused with LDBs allows this method to quickly trap a wide variety of GFP-tagged proteins into light-induced protein clusters. Strikingly, our system exhibits an excellent performance in clustering efficiency with high light sensitivity and stability, it can function even when shielded by multiple glass plates. By utilizing the R-LARIAT system to trap and sequester tubulin, cell cycle progression can be blocked in HeLa cells. Therefore, the R-LARIAT system takes advantage of red light with greater tissue penetration and holds the potential to precisely control protein functions in living organisms.

cell biology↗
bioRxiv2025

AreTomoLive: Automated reconstruction of comprehensively-corrected and denoised cryo-electron tomograms in real-time and at high throughput

A high throughput processing pipeline that performs comprehensive corrections is needed to realize the full potential of cryo-electron tomography and subtomogram averaging. The fields fragmented software landscape remains a significant hurdle to this end. Here we present AreTomoLive, an automated real-time pipeline composed of two GPU-accelerated packages. The first, AreTomo3, streamlines tomographic alignment and reconstruction, with new features to fully account for sample geometry, locally correct the contrast transfer function, and curate data for downstream tasks. The second package, DenoisET, is a new implementation of the machine learning algorithm Noise2Noise and runs in parallel with AreTomo3 to perform contrast enhancement. To reduce barriers to routine use, AreTomoLive prioritizes automation: AreTomo3 autonomously pauses and reactivates processing depending on the status of data collection, while DenoisET algorithmically determines when to transition from training to inference. AreTomoLive endeavors to advance cryoET for in situ structural analysis with its comprehensive corrections and full automation.

cell biology↗
bioRxiv2025

Distinct forms of amyloid-β moderate sleep duration through NAD+-linked redox metabolism in Alzheimer's disease.

Sleep disruptions precede a clinical diagnosis of Alzheimers disease (AD) by several years. However, how AD pathologies affect sleep remains unclear. Here, we integrate epidemiological data with insights from Drosophila models of AD to investigate how AD progression could be linked to sleep disruption. We found that individuals with a high risk of AD report a shorter sleep duration than do those with a clinical diagnosis of AD. We showed that the expression of different forms of amyloid-{beta} in flies can replicate these variations in sleep duration. Analysis of the metabolome and proteome of these flies revealed distinct changes in NAD+-linked redox metabolism and levels of hyperkinetic (Hk), a redox-sensing sleep homeostat. We showed that the genetic upregulation of Hk is neuroprotective and increased KCNAB2 expression in the brain decreases AD risk in humans. Overall, our data provides a new mechanism linking the disruption of NAD+-linked redox sensing and sleep disruption in AD.

neuroscience↗
bioRxiv2025

Targeting tGLI1, a novel mediator of tumor therapeutic resistance, using Ketoconazole sensitizes glioblastoma to CDK4/6 therapy and Chemoradiation

Glioblastoma (GBM) remains the most aggressive primary brain tumor in adults, with no effective treatments. While cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) show clinical promise in some cancers, they have not significantly improved survival in GBM patients. This lack of response is attributed to the treatment-resistant glioma stem cell (GSC) population. We previously identified truncated glioma-associated oncogene homolog 1 (tGLI1) as a novel transcription factor promoting GSCs; however, its role in CDK4/6i resistance has never been investigated in any cancer type. Here, we found positive correlations between tGLI1 and CDK4/6 therapeutic resistance in patient datasets and in vitro studies. Pharmacological inhibition of tGLI1 using FDA-approved ketoconazole (KCZ), a tGLI1-specific inhibitor, sensitized GBM and GSCs to CDK4/6is. KCZ+CDK4/6i combination therapy demonstrated synergistic anti-proliferative effects, significantly inhibiting GBM stemness and cell cycle progression while increasing apoptosis. The combination was more efficacious than monotherapies in two orthotopic GBM mouse models. tGLI1 promoted GBM resistance to radiation therapy and temozolomide, while KCZ potentiated effects of these treatments. Collectively, we report for the first time that tGLI1 is a novel mediator of GBM resistance to CDK4/6is, and KCZ sensitizes GBM to CDK4/6is, thereby supporting future clinical utility of novel KCZ+CDK4/6i combinatorial therapy for GBM patients.

cancer biology↗
bioRxiv2025

In vitro and in vivo Antiviral Activity of the Acyclic Nucleoside Phosphonate Prodrug LAVR-289 against Poxvirus and African Swine Fever Virus Replication

Poxviruses are double-stranded DNA viruses including relevant zoonotic pathogens with high morbidity. Although African swine fever virus (ASFV) belongs to the Asfarviridae family and is not strictly classified as a member of the Poxviridae, both fall within the same class of Pokkesviricetes that replicate in the cytoplasm, and some poxviruses pose potential biological warfare threats. Among compounds targeting these viruses, acyclic nucleoside phosphonate prodrugs are nucleoside analogues inhibitors of viral DNA polymerases that have been identified as promising agents. However, some limitations related to their toxicity and the rapid emergence of resistance highlight the need for new antiviral molecules. In this study, the new nucleoside analogue LAVR-289 was shown to effectively inhibit the viral replication by intervening early in the viral replication step, targeting a specific domain of the poxvirus DNA polymerase. Using monkeypox virus models, the subcutaneous or oral administration of LAVR-289 demonstrates protective efficacy in infected animal models without toxicity or behavioral modification. The stability in vivo, long shelf-life and efficacy make LAVR-289 a promising candidate for further development and stockpiling as a medical countermeasure against dsDNA virus outbreaks. Its broad-spectrum efficacy is a real asset in a context of recurrent viral epidemics, risk of bioterrorism and emergence of resistance strains in the population. HighlightsO_LILAVR-289 is a unique acyclic nucleoside phosphonate prodrug targeting viral DNA polymerases. C_LIO_LILAVR-289 displays antiviral activity against dsDNA viruses, ASFV and poxviruses. C_LIO_LIFirst report of in vivo evaluation of LAVR-289 against MPXV by subcutaneous and oral administration. C_LIO_LILAVR-289 reduces clinical signs and increase survival in animal models. C_LI

microbiology↗
bioRxiv2025

Biological function and mechanism of flpL gene in regulating pathogenicity of Aeromonas hydrophila

Aeromonas hydrophila is human-animal commensal bacterium that seriously threatens the development of aquaculture and human health. Type IV pili (T4P) are essential for bacterial physiological function. The relationship between the molecular mechanism of flpL gene affiliated to T4P and the pathogenicity is still unclear. In the research, A. hydrophila with a genetically stable deletion of flpL gene ({Delta}flpL) was constructed. The median lethal dose of {Delta}flpL for Crucian carp was 4.87 times higher compared to wild-type strain, suggesting that {Delta}flpL significantly reduced pathogenicity. The attenuation may be attributed to a reduced capacity for biofilm formation and downregulation of the expression levels of virulence-related genes in {Delta}flpL. Meanwhile, the significant increase in swimming capacity and adhesion can be related to the upregulation of flpC, tapM, tapB and tapP genes. In conclusion, flpL gene plays essential role in the pathogenicity, adhesion, motility, and capacity for biofilm formation in A. hydrophila. The research aims to clarify the pathogenesis of A. hydrophila, and lay the foundation for developing live attenuated vaccines through the targeted modification flpL gene.

microbiology↗
bioRxiv2025

Tumor suppressor collateral damage screens reveal mRNA homeostasis protein HBS1L as a novel vulnerability in ch9p21 driven FOCAD deleted cancer

Chromosomal deletion of tumor suppressor genes often occurs in an imprecise manner, leading to co-deletion of neighboring genes. This collateral damage can create novel dependencies specific to the co-deleted context. One notable example is the dependency on PRMT5 activity in tumors with MTAP deletion, which co-occurs with CDKN2A/B loss, leading to the development of MTA-cooperative PRMT5 inhibitors. To identify additional collateral damage context/target pairs for chromosome 9p and other common loci of chromosomal deletions, we conducted a combinatorial CRISPR screen knocking out frequently co-deleted genes in combination with a focused target library. We identified the gene encoding the ribosome rescue factor PELO as synthetic lethal with loss of gene encoding the SKI complex interacting protein FOCAD, which is frequently co-deleted alongside MTAP and CDKN2A/B on chromosome 9p. A genome-wide screen in FOCAD isogenic cells further identified the ribosome rescue GTPase and PELO binding partner HBS1L as the top synthetic lethal target for FOCAD loss. Analysis of publicly available data and genetic manipulation of HBS1L using orthogonal modalities validated this interaction. HBS1L dependency in FOCAD-deleted cells was rescued by FOCAD re-expression, and FOCAD intact cells could be rendered HBS1L-dependent by FOCAD knockout, demonstrating the context specificity of this interaction. Mechanistically, HBS1L loss led to translational arrest and activated the unfolded protein response in FOCAD-deleted cells. In vivo, HBS1L deletion eliminated growth of FOCAD-deleted tumors. Here we propose a model where the FOCAD/SKI complex and HBS1L/PELO work together to resolve aberrant mRNA-induced ribosomal stalling, making the HBS1L/PELO complex an intriguing novel target for treating FOCAD-deleted tumors.

cancer biology↗
bioRxiv2025

From Mechanistic Interpretability to Mechanistic Biology: Training, Evaluating, and Interpreting Sparse Autoencoders on Protein Language Models

Protein language models (pLMs) are powerful predictors of protein structure and function, learning through unsupervised training on millions of protein sequences. pLMs are thought to capture common motifs in protein sequences, but the specifics of pLM features are not well understood. Identifying these features would not only shed light on how pLMs work, but potentially uncover novel protein biology--studying the model to study the biology. Motivated by this, we train sparse autoencoders (SAEs) on the residual stream of a pLM, ESM-2. By characterizing SAE features, we determine that pLMs use a combination of generic features and family-specific features to represent a protein. In addition, we demonstrate how known sequence determinants of properties such as thermostability and subcellular localization can be identified by linear probing of SAE features. For predictive features without known functional associations, we hypothesize their role in unknown mechanisms and provide visualization tools to aid their interpretation. Our study gives a better understanding of the limitations of pLMs, and demonstrates how SAE features can be used to help generate hypotheses for biological mechanisms. We release our code, model weights and feature visualizer.

bioinformatics↗
bioRxiv2025

Construction of a low-temperature degrading synthetic bacterial community of corn straw

The stability of the cellulose structure in straw creates a challenge for short-term degradation in the natural environment, especially in the cold regions of north China. Therefore, to secure strains that can degrade straw at low temperatures, we selected soil samples from straw-returning land and used cellulose as the only carbon source. After conducting preliminary screening and further re-screening, we ultimately selected three strains that exhibited efficient degradation of corn straw at a temperature of 4 . Subsequently, the synthetic bacterial community C was constructed using these strains: Stenotrophomonas maltophilia, Flavobacterium johnsoniae and Pantoea rodasii. We conducted a 45-day solid-state and liquid-state fermentation experiment at low-temperature (12 ). The activity of lignocellulase was determined, and the enzyme production conditions were optimized. The results show that the liquid fermentation degradation rate of straw increased by 40 % compared with the control group in the natural state (without adding C). Similarly, the solid-state fermentation degradation rate of straw increased by 30 %, which was significantly faster than that of the control group. Finally, the enzyme production conditions of strain C were optimized as follows: culture time 96 h, inoculation amount 4 %, temperature 12 , pH 6.0, and Alkaline Carboxymethyl Cellulase (CMCase) activity 24.34 U/mL. The microbial system C efficiently degrades straw at low temperature, which is valuable for improving the comprehensive utilization rate of straw and has a broad application prospect in improving soil fertility.

microbiology↗
bioRxiv2025

DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

The SARS-CoV-2 pandemic and the emergence of novel variants underscore the need to understand host-virus interactions and identify host factors that restrict viral infection. Here, we perform a genome-wide CRISPR knockout screen to identify host restriction factors for SARS-CoV-2, revealing DAZAP2 as a potent antiviral gene. DAZAP2, previously implicated in SARS-CoV-2 restriction, is found to inhibit viral entry by blocking virion fusion with both endolysosomal and plasma membranes. Additionally, DAZAP2 suppresses genomic RNA replication without affecting the primary translation of viral replicases. We demonstrate that DAZAP2 functions as a pan-coronavirus restriction factor across four genera of coronaviruses. Importantly, knockout of DAZAP2 enhances SARS-CoV-2 infection in mouse models and in human primary airway epithelial cells, confirming its physiological relevance. Mechanistically, antiviral activity of DAZAP2 appears to be indirect, potentially through the regulation of host gene expression, as it primarily localizes to the nucleus. Our findings provide new insights into the host defense system against coronaviruses and highlight DAZAP2 as a potential target for host-directed antiviral therapies. IMPORTANCEDuring viral infection, the host defense response is mediated by a variety of host factors through distinct mechanisms that have yet to be fully elucidated. Although DAZAP2 was previously implicated in SARS-CoV-2 restriction, its mechanisms of action and in vivo relevance remain unclear. In this study, we identify the DAZAP2 as a potent pan-coronavirus restriction factor that inhibits viral infection through dual mechanisms: blocking virion fusion with both endolysosomal and plasma membranes, and suppressing genomic RNA replication. We confirm its physiological relevance in host defense using mouse models and primary cell cultures. This study advances our understanding of host-pathogen interactions. Targeting DAZAP2 or its regulatory pathways could provide a new approach to enhance host defense against current and future coronavirus threats.

microbiology↗
bioRxiv2025

Isostere 18F-protein post-translational editing enables dynamic tracking of neurodegeneration biomarkers

The neurofilament light chain protein (NfL) is a suggested general marker for neuronal loss. Its release from brain parenchyma into cerebral spinal fluid, and presumed detection in blood has seen it established as a first blood-based marker of disease activity and drug efficacy in multiple sclerosis (MS) and in the presymptomatic diagnosis and assessment of disease course for other neurodegenerative disorders.1 However, the lack of characterisation of its behaviour in circulation, largely due to its antibody-dependent measurement, have hampered the biological interpretation of these measurements, especially after acute injury such as in MS relapse or head trauma.2 Here, we describe a strategy for exploiting positron emission tomography (PET) imaging using isosteric protein mimics following the installation of a fluorine-18 label that is benign enough to provide sensitive, real-time information on the dynamics and trafficking of NfL protein. This circumvents the limits of current methods that integrate 18F into proteins through the bio-conjugation of bulky, unnatural groups, which we show perturb NfLs assembly and functional properties from those in the natural state. We use a visible-light-driven reaction to access radioactive isostere proteins that are unperturbed and so closely resemble their native form. In this way, generation of [18F]fluoroalkyl radicals that can be rapidly reacted at pre-defined sites on proteins creates mimics of proteinogenic side chains bearing near-zero-size labels to probe proteins in functionally true form. These prosthetic-free, protein radiotracers can be generated in excellent radiochemical yield (up to 67%) via a semi-automated protocol in just 15 mins. High associated molar activities (precursor up to 102 GBq mol-1) allowed high sensitivity dynamic observations in blood, brain and cerebrospinal fluid, enabling even the first unambiguous observations of spinal flow kinetics using proteins. These dynamics, including the high rate of spinal flow (on the order of mm per min) and drainage of NfL from CSF into sacral lymph nodes, now provides evidence that the slow fall rate of antibody-detected markers that is observed after acute neural insults is not due to a long half-life, but rather reflects sustained neuronal loss. This discovery will now help to better correlate clinical and radiological features of disease with NfL blood levels. Our methodology now demonstrates the broad potential of a near-zero-size labelling method for the functional study of proteins in whole organisms without interfering with their biological activity and native assembly.

synthetic biology↗
bioRxiv2025

Transferring functional chromosomes between intergeneric yeast generates monochromosomal hybrids with improved phenotypes and transcriptional responses

Interspecific genetic exchanges caused by natural hybridization or horizontal gene transfer can lead to enhanced phenotypes, which are often of interest for industrial applications and evolutionary research. However, transferring genetic materials between distantly related species, such as intergeneric yeasts, presents technical challenges. In this study, we established a method to transfer individual chromosomes from Saccharomyces cerevisiae (Sc) into Kluyveromyces marxianus (Km), an emerging model for bioproduction. The Sc chromosome of interest was circularized, genetically modified to carry Km centromeres and replication origins, and transformed into Km via protoplast transformation. Using this method, we generated two synthetic strains, each containing a full set of Km chromosomes and either Sc chromosome I or III. The Sc chromosomes exhibited normal replication, segregation, and active transcription after the transfer. The synthetic strains displayed enhanced phenotypes in flocculation and salt tolerance, which were found to be caused by transgressive expression of FLO9 and SPS22 on the transferred Sc chromosomes, respectively. Transcriptomic analysis revealed that transgressive expression was prevalent among the transferred Sc genes, suggesting evolution of lineage-specific cis- and trans-regulatory interactions across a long evolutionary timescale. Our strategy has potential applications in optimizing cell factories, constructing synthetic genomes, and advancing evolutionary research.

synthetic biology↗
bioRxiv2025

ATP-sensitive isoforms of P2X5: A reevaluation of its function across species

The P2X receptor family comprises ATP-gated non-selective cation channels central to physiological processes across the nervous, immune, cardiovascular, respiratory, and reproductive systems. While P2X1, P2X2, P2X3, P2X4, and P2X7 are well-established as ATP-sensitive, P2X5 isoforms and transcript variants (TVs) have traditionally been considered ATP-insensitive, limiting their functional exploration. This study identifies previously overlooked ATP-sensitive P2X5 isoforms across diverse species. Through gene profiling and next-generation sequencing (NGS), we found that the zfP2X5TV2 isoform dominates ATP-sensitive forms in zebrafish. In mice, ATP-sensitive mP2X5G317 (mP2X5.1) comprises [~]90% of transcripts, while in rats, exon 3-containing rP2X5F191 accounts for over 70%. In human cell lines, ATP-sensitive isoforms retaining exons 3, 7, and 10 represent [~]15-30% of P2X5 transcripts. RNA-seq from human tissues confirms frequent retention of exons 3 and 7 and polymorphic exon 10 expression. ATP-sensitive P2X5 variants were also identified in chickens, bullfrogs, dogs, cows, and naked mole-rats. These findings challenge the prevailing assumption of ATP-insensitivity, highlighting the need to reassess P2X5s roles in physiological and pathological contexts.

physiology↗
bioRxiv2025

Biased sampling confounds machine learning prediction of antimicrobial resistance

Antimicrobial resistance (AMR) poses a growing threat to human health. Increasingly, genome sequencing is being applied for the surveillance of bacterial pathogens, producing a wealth of data to train machine learning (ML) applications to predict AMR and identify resistance determinants. However, bacterial populations are highly structured and sampling is biased towards human disease isolates, meaning samples and derived features are not independent. This is rarely considered in applications of ML to AMR. Here, we demonstrate the confounding effects of sample structure by analyzing over 24,000 whole genome sequences and AMR phenotypes from five diverse pathogens, using pathological training data where resistance is confounded with phylogeny. We show resulting ML models perform poorly, and increasing the training sample size fails to rescue performance. A comprehensive analysis of 6,740 models identifies species- and drug-specific effects on model accuracy. We provide concrete recommendations for evaluating future ML approaches to AMR.

bioinformatics↗
bioRxiv2025

Discovery of Expression-Governing Residues in Proteins

Understanding how amino acids influence protein expression is crucial for advancements in biotechnology and synthetic biology. In this study, we introduce Venus-TIGER, a deep learning model designed to accurately identify amino acids critical for expression. By constructing a two-dimensional matrix that links model representations to experimental fitness, Venus-TIGER achieves improved predictive accuracy and enhanced extrapolation capability. We validated our approach on both public deep mutational scanning datasets and low-throughput experimental datasets, demonstrating notable performance compared to traditional methods. Venus-TIGER exhibits robust trans-ferability in zero-shot predicting scenarios and enhanced predictive performance in few-shot learning, even with limited experimental data. This capability is particularly valuable for protein design aimed at enhancing expression, where generating large datasets can be costly and time-consuming. Additionally, we conducted a statistical analysis to identify expression-associated features, such as sequence and structural preferences, distinguishing between those linked to high and low expression. Our investigation also revealed a correlation among stability, activity and expression, providing insight into their interconnected roles and underlying mechanisms.

bioengineering↗
bioRxiv2024

Virological and antigenic characteristics of SARS-CoV-2 variants LF.7.2.1, NP.1, and LP.8.1

XEC and KP.3.1.1 have surpassed KP.3 to become the globally dominant lineages due to their unique NTD mutations. However, several emerging JN.1 sublineages, such as LF.7.2.1, MC.10.1, NP.1, and, especially, LP.8.1, have demonstrated superior growth advantages compared to XEC. It is critical to access the virological and antigenic characteristics of these emerging SARS-CoV-2 variants. Here, we found that LF.7.2.1 is significantly more immune invasive than XEC, primarily due to the A475V mutation, which enabled the evasion of Class 1 neutralizing antibodies. However, LF.7.2.1s weak ACE2 binding affinity substantially impaired its fitness. Likewise, MC.10.1 and NP.1 exhibited strong antibody immune evasion due to the A435S mutation, but their limited ACE2 engagement efficiency restricted their growth advantage, suggesting that A435S may regulate the Spike conformation, similar to the NTD glycosylation mutations found in KP.3.1.1 and XEC. Most importantly, we found that LP.8.1 showed comparable humoral immune evasion to XEC but demonstrated much increased ACE2 engagement efficiency, supporting its rapid growth. These findings highlight the trade-off between immune evasion and ACE2 engagement efficiency in SARS-CoV-2 evolution, and underscore the importance of monitoring LP.8.1 and its descend lineages.

immunology↗