Biology subjects

Cheng, Y.

Publications and source records attributed to Cheng, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Deconvolution to restore cryo-EM maps with anisotropic resolution

With technological advancements in recent years, single particle cryogenic electron microscopy (cryo-EM) has become a major methodology for structural biology. Structure determination by single particle cryo-EM is premised on randomly orientated particles embedded in a thin layer of vitreous ice to resolve high-resolution structure in all directions. In practice, preferentially distributed particle orientations and/or other imperfections in imaging and data processing deteriorate quality of obtained cryo-EM map. Here we present a deconvolution approach, named AR-Decon, that computationally improves the quality of cryo-EM maps. We tested and validated the procedure, compared its performance with that of machine learning based density modification method, and benchmarked its performance with a wide range of deposited maps. Our results show that AR-Decon is robust and is a generally applicable post-processing procedure for single particle cryo-EM.

biophysics↗

bioRxiv2025

Population-scale cellular GUIDE-seq-2 and biochemical CHANGE-seq-R profiles reveal human genetic variation frequently affects Cas9 off-target activity

Genome editing enzymes can introduce targeted changes to DNA in living cells1-4, transforming biological research and enabling the first approved gene editing therapy for sickle cell disease5. However, their activity can be altered by genetic variation at on- or off-target sites6-8, potentially impacting both their precision and therapeutic safety. Due to a lack of scalable methods to measure genome-wide editing activity in cells from large populations and diverse target libraries, the frequency and extent of these variant effects on editing remain unknown. Here, we present the first systematic, population-scale study of how genetic variation affects the cellular genome-wide activity of CRISPR-Cas9, enabled by a novel, sensitive, and unbiased cellular assay, GUIDE-seq-2, with improved scalability and accuracy compared to the original broadly adopted method9. Analyzing Cas9 genome-wide activity at 1,115 on- and off-target sites across six guide RNAs in cells from 95 individuals spanning four genetically diverse populations, we found that genetic variants frequently overlap off-target sites, with 14% significantly altering Cas9 editing activity. To understand the effect of mismatches in more diverse sequence contexts, we developed a novel method, combinatorial high-throughput analysis of nuclease cleavage effects (CHANCE-seq), the first massively parallel biochemical approach that can quantify Cas9 activity across millions of mismatched target sites. We leveraged this large-scale CHANCE-seq dataset to train a context-aware deep neural network model, CHANCE-net, to accurately predict and interpret the effects of single-nucleotide variants on off-targets with up to six mismatches. Our deep combinatorial profiling of Cas9 off-target activity revealed frequent, context-dependent synergistic effects of mismatches. Taken together, our findings illuminate an approach to accounting for genetic variation when designing genome-editing strategies for research and therapeutics.

genomics↗

bioRxiv2025

Chronically implantable μLED arrays for optogenetic cortical surface stimulation in mice

Cortical implants are a proven clinical neurotechnology with the potential to transform our understanding of cognitive processes. These processes rely on complex neuronal networks that are difficult to selectively probe or stimulate. Optogenetics offers cell-type specificity, but achieving the density and coverage required for chronic, high-resolution modulation remains a challenge. Here we present a 100-element {micro}LED array (200 {micro}m pixel pitch, 2 x 2 mm2 footprint) coupled into a miniaturised, flexible system suitable for chronic implantation and optogenetic stimulation of the surface of the mouse cortex. The {micro}LEDs can remain stable for over 300 hours continuous operation time in-vivo, allowing for months-long chronic experiments. Simultaneous electrophysiology recordings confirmed robust neuronal responses corresponding to low {micro}LED drive currents (<5 mA), minimising thermal effects and supporting future wireless operation. The spatial resolution of neuronal responses was consistent with a simulated model of light scattering in the cortical layers, enabling device optimisation. Behavioural experiments with chronically implanted mice demonstrated robust learning during discrimination tasks using spatially distinct optogenetic stimulation patterns.

neuroscience↗

bioRxiv2025

5' UTR-mediated retention of eIF3 on 80S ribosomes promotes co-translational folding of ER membrane proteins

Co-translational folding of nascent polypeptides is essential for protein function and cellular homeostasis. Ribosome-associated chaperones assist in this process, but coordination between their recruitment and translation initiation remains poorly understood. We report here that specific binding sites for eukaryotic translation initiation factor eIF3 within the 5 untranslated regions (5UTRs) of mRNAs promote its retention on 80S ribosomes during the synthesis of select endoplasmic reticulum (ER) membrane proteins. Disruption of these eIF3 binding sites leads to misfolding and sequestration of newly synthesized membrane proteins into ER whorls. Sequestration into ER whorls is exacerbated by HSP70 inhibition but can be rescued by overexpressing HSPA8 and HSPA1. Cross-linking assays reveal that 5UTR binding sites stabilize eIF3-80S interactions during early elongation, facilitating recruitment of HSPA8 to ribosomes. These findings indicate that genetic instructions within 5UTRs direct eIF3-mediated chaperone recruitment, ensuring proper co-translational folding of ER membrane proteins.

cell biology↗

bioRxiv2025

Formation and Gelation of Elastin-like Polypeptide Complex Coacervates

Protein liquid-liquid phase separation underlies the formation of membraneless organelles in cells and performs a key role in the assembly process of natural materials such as the assembly of tropoelastin into elastic fibers. Here, we engineered a series of charged elastin-like polypeptides (ELPs) that form complex coacervates, providing a rapid method to concentrate proteins into a fluid state. Compared to coacervates formed from simple coacervation, complex coacervates exhibited greater fluidity, likely due to differences between electrostatic interactions and hydrophobic forces. We designed these ELPs to further contain crosslinking domains compatible with tyrosinase or transglutaminase and found that crosslinking was enhanced when proteins were in a complex coacervate compared to free in solution. Crosslinking the ELP complex coacervates led to the formation of gels with distinct properties dependent on the nature of the crosslinking. This work expands the design space of ELP hydrogels, offering a novel strategy for forming crosslinked networks from complex coacervates and providing opportunity for future use in tissue engineering and biocompatible biomaterials applications.

synthetic biology↗

bioRxiv2025

TRIM21-NUP98 interface accommodates structurally diverse molecular glue degraders

Molecular glue degraders enable targeted protein degradation by bridging interactions between target proteins and E3 ubiquitin ligases. Whereas some protein-protein interfaces exhibit the capacity to accommodate structurally diverse degraders, the extent of this adaptability across molecular glue targets remains unclear. We recently identified (S)-ACE-OH as a molecular glue degrader that recruits the E3 ubiquitin ligase TRIM21 to the nuclear pore complex by recognizing NUP98, thereby inducing the degradation of nuclear pore proteins. Here, we analyzed public compound toxicity data across a large collection of cell lines and identified two additional molecular glue degraders, PRLX 93936 and BMS-214662, that engage the TRIM21-NUP98 interface to induce the degradation of nucleopore proteins. Additionally, we confirmed that HGC652, another TRIM21-dependent molecular glue degrader, also binds at this interface. Together with our previously characterized degrader (S)-ACE-OH, these findings demonstrate that the TRIM21-NUP98 interface can accommodate structurally diverse molecular glue degraders, expanding the potential for its therapeutic exploitation.

biochemistry↗

bioRxiv2024

Modeling and predicting single-cell multi-gene perturbation responses with scLAMBDA

Understanding cellular responses to genetic perturbations is essential for understanding gene regulation and phenotype formation. While high-throughput single-cell RNA-sequencing has facilitated detailed profiling of heterogeneous transcriptional responses to perturbations at the single-cell level, there remains a pressing need for computational models that can decode the mechanisms driving these responses and accurately predict outcomes to prioritize target genes for experimental design. Here, we present scLAMBDA, a deep generative learning framework designed to model and predict single-cell transcriptional responses to genetic perturbations, including single-gene and combinatorial multi-gene perturbations. By leveraging gene embeddings derived from large language models, scLAMBDA effectively integrates prior biological knowledge and disentangles basal cell states from perturbation-specific salient representations. Through comprehensive evaluations on multiple single-cell CRISPR Perturb-seq datasets, scLAMBDA consistently outperformed state-of-the-art methods in predicting perturbation outcomes, achieving higher prediction accuracy. Notably, scLAMBDA demonstrated robust generalization to unseen target genes and perturbations, and its predictions captured both average expression changes and the heterogeneity of single-cell responses. Furthermore, its predictions enable diverse downstream analyses, including the identification of differentially expressed genes and the exploration of genetic interactions, demonstrating its utility and versatility.

bioinformatics↗

bioRxiv2024

Integration of spatial multiplexed protein imaging and transcriptomics in the human kidney tracks the regenerative potential timeline of proximal tubules

The organizational principles of nephronal segments are based on longstanding anatomical and physiological attributes that are closely linked to the homeostatic functions of the kidney. Novel molecular approaches have recently uncovered layers of deeper signatures and states in tubular cells that arise at various timepoints on the spectrum between health and disease. For example, a dedifferentiated state of proximal tubular cells with mesenchymal stemness markers is frequently seen after injury. The persistence of such a state is associated with failed repair. Here, we introduce a novel analytical pipeline applied to highly multiplexed spatial protein imaging to characterize proximal tubular subpopulations and neighborhoods in reference and disease human kidney tissue. The results were validated and extended through integration with spatial and single cell transcriptomics. We demonstrate that, in reference tissue, a large proportion of S1 and S2 proximal tubular epithelial cells express THY1, a mesenchymal stromal and stem cell marker that regulates differentiation. Kidney disease is associated with loss of THY1 and transition towards expression of PROM1, another stem cell marker shown recently to be linked to failed repair. We demonstrate that the trajectory of proximal tubular cells to THY1 expression is clearly distinct from that of PROM1, and that a state with PROM1 expression is associated with niches of inflammation. Our data support a model in which the interplay between THY1 and PROM1 expression in proximal tubules associates with their regenerative potential and marks the timeline of disease progression.

bioinformatics↗

bioRxiv2024

CNV-Profile Regression: A New Approach for Copy Number Variant Association Analysis in Whole Genome Sequencing Data

Copy number variants (CNVs) are DNA gains or losses involving >50 base pairs. Assessing CNV effects on disease risk requires consideration of several factors. First, there are no natural definitions for CNV loci. Second, CNV effects can depend on dosage and length. Third, CNV effects can be more accurately estimated when all CNV events in a genomic region are analyzed together to assess their joint effects. We propose a new framework for association analysis that directly models an individuals entire CNV profile within a genomic region. This framework represents an individuals CNVs using a CNV profile curve to capture variations in CNV length and dosage and to bypass the need to predefine CNV loci. CNV effects are estimated at each genome position, making the results comparable across different studies. To jointly estimate the effects of all CNVs, we use a Lasso penalty to select CNVs associated with the trait and integrate a weighted L2-fusion penalty to encourage similar effects of adjacent CNVs when supported by the data. Simulations show that the proposed model can more effectively identify causal CNVs while maintaining false positive rates comparable to baseline methods and yield more precise effect-size estimates across different settings. When applied to CNV derived from whole genome sequencing data of the Alzheimers Disease Sequencing Project, the proposed methods identify additional CNVs associated with Alzheimers Disease (AD). These identified CNVs overlap with several known AD-risk genes and are significantly enriched by biological processes related to neuron structures and functions crucial in AD development.

bioinformatics↗

bioRxiv2024

Exploring the Hypothetical Role of Bacteroides Species in Depression Progression: Insights from Metagenomic Analysis

Depression, a psychiatric disorder with significant morbidity and mortality, has a complex etiology. Recent advances in microbiome research have highlighted the potential role of gut microbiota in depression pathogenesis. This study utilized shotgun metagenomic sequencing to compare the fecal microbiota of 28 depression patients and 26 healthy individuals. Significant differences in gut microbiota composition were observed between the two groups. We generated 350 non-redundant high-quality metagenome-assembled Genomes (MAGs) by binning and conducted comparisons between the depression and control groups. Notably, we found that the MAGs enriched in people with depression mostly belonged to Bactendicating a close link between Bacteroides abundance and the development of depression, suggesting that Bacteroides might be a potential culprit for deroides, ipression. In the depression group, we found that the module of nitric oxide synthesis was remarkably enriched, and all Bacteroides MAGs were annotated to nitric oxide synthase, suggesting that increased levels of Bacteroides may contribute to elevated nitric oxide synthesis. Specifically, the mean relative abundance about the genomes of Bacteroides xylanisolvens, Bacteroides caccae, Bacteroides fragilis, Bacteroides stercoris and Bacteroides ovatus showed strong discriminatory power in distinguishing depressed patients from healthy individuals (AUC=0.834). This research shed light on the potential role of gut microbiota in depression and highlights specific metabolic pathways and microbial markers for further investigation. ImprotanceThis research highlighted significant differences in the composition and function of gut microbiota between individuals with depression and healthy individuals, particularly the enrichment of Bacteroides MAGs in depression patients. The upregulation of the nitric oxide synthesis pathway associated with these MAGs belong to Bacteroides in the gut of depression patients had also been observed. The mean relative abundance of a specific set of Bacteroides MAGs had been identified, which could accurately classify individuals with depression from healthy individuals (AUC=0.834). Our results suggest the importance of exploring microbial markers as potential diagnostic and therapeutic targets in managing depression.

genomics↗

bioRxiv2024

Arabidopsis SUFB and CLP protease regulate SUFBC2D to manipulate iron-sulfur cluster biosynthesis in chloroplast

Iron-sulfur (Fe-S) clusters are essential cofactors for Fe-S proteins. SUFBC2D complex is the scaffold responsible for Fe-S cluster assembly in chloroplasts. However, the regulatory mechanism on SUFBC2D remains elusive. In this study, we report that the transcription of SUFB responds rapidly to leaf senescence, whereas the transcription of SUFC and SUFD does not. Intriguingly, their protein contents remain stable during leaf senescence. We further found that leaf death was occurred only when SUFB RNAi was induced, and SUFB and SUFC contents decreased much faster in the SUFB-RNAi lines than in the SUFC-RNAi lines, indicating that SUFB had a faster turnover rate than SUFC. Moreover, overexpressing SUFB increased the contents of SUFC and SUFD, and SUFBC2D, whereas overexpressing SUFC did not increase SUFB and SUFD. Our findings reveal that SUFB stabilizes SUFC and SUFD via forming SUFBC2D, whereas SUFC lacks this function. Furthermore, SUFB expression was sharply downregulated when the plants were subjected to iron deficiency, whereas SUFC and SUFD expression was not. Interestingly, the contents of all three SUF members decreased, indicating that plants degrade SUFBC2D in response to iron deficiency by downregulating SUFB transcription. We subsequently studied the degradation mechanism of SUFBC2D. Our results indicated that all SUFs are substrates of the caseinolytic protease (CLP) because they all accumulated in the CLP impaired mutant, and they physically interact with CLPS1, the substrate recognition adaptor of CLP. Collectively, our findings provide novel insights into how plants regulate SUFBC2D complex via SUFB to adapt to leaf senescence and iron deficiency.

plant biology↗

bioRxiv2024

In vivo genome editing of human hematopoietic stem cells for treatment of blood disorders by mRNA delivery

Ex vivo autologous hematopoietic stem cells (HSCs) gene therapy provides promising new treatments for hematological disorders. However, current methods involve complex processes and chemotherapeutic conditioning, leading to limited accessibility for treatment and significant side effects. Here, we developed an antibody-free targeted lipid nanoparticles (LNPs) for mRNA delivery to HSCs in vivo, enabling efficient base editing of the HBG target in human HSCs to reactivate fetal hemoglobin in derived erythroid cells. Delivery of ABE8e/sgRNA mRNA with optimized structure LNPs achieves efficient in vivo base editing of HBG in transfusion-dependent {beta}-thalassemia (TDT) patients derived HSCs engrafted in immunodeficient NCG-X mice, showing restored globin chain balance in erythroid cells. Our research indicated that utilizing LNPs for delivery of genome editor achieves efficient editing of endogenous genes of human HSCs. Notably, this non-viral delivery system eliminates the need for harvesting or mobilizing HSCs, providing a potent and one-time treatment potential for blood disorders like sickle cell disease (SCD) and TDT. HighlightsO_LIAntibody-free targeted LNPs system achieves efficient mRNA delivery and base editing in human bone marrow cells including HSCs via one-time intravenous injection. C_LIO_LIDelivery of ABE8e/sgRNA mRNA with optimized LNPs achieves efficient in vivo base editing of HBG in TDT patients derived HSCs engrafted in immunodeficient NCG-X mice, showing restored globin chain balance in erythroid cells. C_LIO_LILNPs efficiently deliver gene editing system to the BM for in vivo editing of human HSCs, providing preclinical evidence for the next generation non-viral gene therapy for blood disorders. C_LI

bioengineering↗

bioRxiv2024

deadtrees.earth - An Open-Access and Interactive Database for Centimeter-Scale Aerial Imagery to Uncover Global Tree Mortality Dynamics

Excessive tree mortality is a global concern and remains poorly understood as it is a complex phenomenon. We lack global and temporally continuous coverage on tree mortality data. Ground-based observations on tree mortality, e.g., derived from national inventories, are very sparse, not standardized and not spatially explicit. Earth observation data, combined with supervised machine learning, offer a promising approach to map tree mortality over time. However, global-scale machine learning requires broad training data covering a wide range of environmental settings and forest types. Drones provide a cost-effective source of training data by capturing high-resolution orthophotos of tree mortality events at sub-centimeter resolution. Here, we introduce deadtrees.earth, an open-access platform hosting more than a thousand centimeter-resolution orthophotos, covering already more than 300,000 ha, of which more than 58,000 ha are fully annotated. This community-sourced and rigorously curated dataset shall serve as a foundation for a global initiative to gather comprehensive reference data. In concert with Earth observation data and machine learning it will serve to uncover tree mortality patterns from local to global scales. This will provide the foundation to attribute tree mortality patterns to environmental changes or project tree mortality dynamics to the future. Thus, the open and interactive nature of deadtrees.earth together with the collective effort of the community is meant to continuously increase our capacity to uncover and understand tree mortality patterns.

ecology↗

bioRxiv2024

Role of dopamine in reward expectation and predictability during execution of action sequences

BackgroundMesolimbic dopamine (DA) neurons are central to cue guided reward seeking and action sequence learning. Yet, the mechanisms by which cue-induced DA neural activity drives goal-directed or habitual sequence execution remain unknown. MethodsWe designed two novel tasks to isolate the effect of sequence-delineating cues on DA-driven behavioral strategies and learning. In the lever insertion fixed-ratio 5 task (LI5), the lever insertion marked sequence initiation. In the lever retraction fixed-ratio 5 task (LR5), the lever retraction served as both sequence termination and reward-predictive cue. ResultsWe found that sequence initiation and termination cues differentially affect reward expectation during action sequences, with only the termination cue contributing to greater outcome devaluation insensitivity, automaticity and behavioral chunking. Mesolimbic fiber photometry recording revealed that this habit-like behavior was associated with a rapid backpropagation in DA signals from the reward to the immediately preceding cue and with attenuated DA reward prediction error signals, which reflected greater behavioral inflexibility. Finally, in absence of external cues, brief optogenetic stimulation of VTA DA neurons at sequence termination was sufficient to drive automaticity and, to some extent, behavioral chunking. ConclusionOur results highlight the critical role of cue-evoked DA signals at sequence termination in mediating credit assignment and driving the development of habitual action sequence execution.

neuroscience↗

bioRxiv2024

Fentanyl reinforcement history has sex-specific effects on multi-step decision-making

It is commonly thought that drug addiction involves a transition to habitual control of action, where the choice to consume drugs becomes automatized and reflects a failure to deliberate over possible negative outcomes. Determining whether the pursuit of addictive drugs is habitual is hampered by a lack of behavior assessments suitable for use during a bout of actual drug seeking. Therefore, to understand how variable histories of drug reinforcement might affect goal-directed and habitual pursuit of drug, we trained rats to perform a multi-step decision-making task to earn oral fentanyl and sucrose rewards following extensive pretraining with either fentanyl or sucrose. Importantly, this task allowed for independent measurements of goal-directed and habitual choice characteristics during online pursuit of rewards, and habitual choice could be further categorized into perseverative and reward-guided components. Chronic fentanyl led to a bias for reward-guided habitual choice specifically in females, and a high degree of perseveration in both sexes. These behavioral changes after chronic fentanyl pretraining generalized across fentanyl and sucrose seeking. In contrast, acute fentanyl selectively increased perseveration in females, and blunted the gradual within-session improvement in goal-directed choice in both sexes. These results show that chronic fentanyl reinforcement promotes habits that generalize across drug and non-drug reward seeking, and that female rats are especially susceptible to habitual control induced by both chronic and acute fentanyl reinforcement.

animal behavior and cognition↗

bioRxiv2024

Neuregulin-1 Attenuates Myocardial Ischemia/Reperfusion Injury by Activating the UCP2/PINK1/LC3B-mediated Mitophagy

BACKGROUNDIschemia/reperfusion (I/R) injury may significantly affect the treatment outcomes and prognosis of patients with acute myocardial infarction following coronary artery recanalization. Available evidence suggests that neuregulin-1 (NRG-1) can provide a protection against myocardial I/R injury and is involved in various cardioprotective interventions by potential regulation of mitophagy. However, the molecular mechanisms linking NRG-1 and mitophagy remain to be clarified. This experiment aimed to determine whether NRG-1 postconditioning attenuated myocardial I/R injury through the regulation of mitophagy and to explore the underlying mechanisms. METHODBoth an in vivo myocardial I/R injury model of rats and an in vitro hypoxia/reoxygenation (H/R) model of H9C2 cardiomyocytes were applied. NRG-1 treatment was conducted immediately after I/R or H/R intervention. In the in vivo experiment, cardioprotective effects of NRG-1 were determined by infarct size, cardiac enzyme and histopathologic examinations. The potential downstream pathways and molecular targets of NRG-1 were screened by the RNA sequencing and the Protein-Protein Interaction Networks (PPI). The expression levels of mitochondrial uncoupling protein 2 (UCP2) and mitophagy-related protein in both the I/R myocardium and H/R cardiomyocytes were measured by immunofluorescence staining and Western blots. The activation of mitophagy was observed with the transmission electron microscopy (TEM) and JC-1 staining. RESULTSThe KEGG and GSEA analyses showed that the mitophagy-related pathways were enriched in the I/R myocardium treated with NRG-1, and UCP2 exhibited a significant correlation between mitophagy and interaction with PINK1. Meanwhile, the treatment with mitophagy inhibitor Mdivi-1 significant eliminated the cardioprotective effects of NRG-1 postconditioning in vivo, and the challenge with UCP2 inhibitor genipin could also attenuate the activating effect of NRG-1 postconditioning on mitophagy. Consistently, the in vitro experiment using H9C2 cardiomyocytes showd that NRG-1 treatment significantly up-regulated the expression levels of UCP2 and mitophagy-related proteins, and activated the mitophagy, whereas the challenge with small interfering RNA (siRNA)-mediated UCP2 knockdown abolished the effects of NRG-1. CONCLUSIONSNRG-1 postconditioning can produce a protection against the myocardial I/R injury by activating mitophagy through the UCP2/PINK1/LC3B signaling pathway.

molecular biology↗

bioRxiv2024

Fatty acid desaturation guides cellular decisions between ferroptosis and cellular senescence

When subject to damage or stress, cells develop responses in order to maintain tissue homeostasis. Two such decisions are ferroptosis and cellular senescence, but how cells decide between these outcomes remains unclear. Here we show that senescent cells increase levels of multiple membrane-bound polyunsaturated fatty acids (PUFAs), but a specific PUFA, dihomo-gamma-linolenic acid (DGLA, 20:3{omega}-3) is reduced. Exogenous repletion of DGLA or inhibition of delta-5-desaturase, the enzyme that metabolizes DGLA, instead results in cell death by ferroptosis. Senescent cells had elevated levels of other fezzroptosis sensitizers, including labile iron and expression of lipoxygenases - but also increased Gpx4 levels to prevent ferroptosis. Oral DGLA lowered senescent cell burden in aged mice and improved age-related functional outcomes. Finally, obese humans with lowered DGLA desaturation rates showed lower markers of adipose tissue senescence. Together, our data implicate DGLA and its desaturation as a major driver of decisions between senescence and ferroptosis.

cell biology↗

bioRxiv2024

Latent-TGF-β has a domain swapped architecture

The multifunctional cytokine TGF-{beta} is produced in a latent form (L-TGF-{beta}) where a RGD containing homodimeric prodomain forms a "ring" encircling mature TGF-{beta} shielding it from its receptors. Thus L-TGF-{beta} must be activated to function, a process driven by dynamic allostery resulting from integrin binding the L-TGF-{beta} RGD motif. Here we provide critical evidence that defines a domain-swapped architecture of L-TGF-{beta}, an essential component in the dynamic allostery mechanism of L-TGF-{beta} activation.

biophysics↗