Biology subjects

Zhou, X.

Publications and source records attributed to Zhou, X..

At least 289 records · Page 16Linked to original sources

bioRxiv2024

Structural basis of the bacterial flagellar motor rotational switching

The bacterial flagellar motor is a huge bidirectional rotary nanomachine that drives rotation of the flagellum for bacterial motility1-5. The cytoplasmic C ring of the flagellar motor functions as the switch complex for the rotational direction switching from counterclockwise to clockwise2,3,6-9. However, the structural basis of the rotational switching and how the C ring is assembled have long remained elusive. Here, we present two high-resolution cryo-electron microscopy structures of the C ring-containing flagellar motor-hook complex from Salmonella Typhimurium, which are in the default counterclockwise state and in a constitutively active CheY mutant-induced clockwise state, respectively. In both complexes, the C ring consists of four subrings, but is in two different conformations. The CheY proteins are bound into an open groove between two adjacent protomers on the surface of the middle subring of the C ring and interact with the FliG and FliM subunits. The binding of the CheY protein induces a significant upward shift of the C ring towards the MS ring and inward movements of its protomers towards the motor center, which eventually remodels the structures of the FliG subunits and reverses the orientations and surface electrostatic potential of the torque helices to trigger the counterclockwise-to-clockwise rotational switching. The conformational changes of the FliG subunits reveal that the stator units on the flagellar motors require a relocation process in the bacterial inner membrane during the rotational switching. This study provides unprecedented molecular insights into the rotational switching mechanism and a detailed overall structural view of the bacterial flagellar motors.

microbiology↗

bioRxiv2024

Evolution of a Eukaryotic Transcription Factor's co-TF Dependence Involves Multiple Intrinsically Disordered Regions Affecting Activation and Autoinhibition

Combinatorial control by transcription factors (TFs) is central to eukaryotic gene regulation, yet its mechanism, evolution, and regulatory impact are not well understood. Here we use natural variation in the yeast phosphate starvation (PHO) response to examine the genetic basis and species variation in TF interdependence. In Saccharomyces cerevisiae, main TF Pho4 relies on the co-TF Pho2 to regulate [~]28 genes, whereas in the related pathogen Candida glabrata, Pho4 has reduced Pho2 dependence and regulates [~]70 genes. We found C. glabrata Pho4 (CgPho4) binds the same motif with 3-4 fold higher affinity. Machine learning and yeast one-hybrid assay identify two intrinsically disordered regions (IDRs) in CgPho4 that boost its activation domains activity. In ScPho4, an IDR next to the DNA binding domain both allows for enhanced activity with Pho2 and inhibits activity without Pho2. This study reveals how IDR divergence drives TF interdependence evolution by influencing activation potential and autoinhibition.

evolutionary biology↗

bioRxiv2024

Structural basis for the inhibition of coronaviral main proteases by PF-00835231

The main protease (Mpro) of coronaviruses plays a key role in viral replication, thus serving as a hot target for drug design. It has been proven that PF-00835231 is promising inhibitor of SARS-CoV-2 Mpro. Here, we report the inhibition potency of PF-00835231 against SARS-CoV-2 Mpro and seven Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) from SARS-CoV-2 variants. The results confirm that PF-00835231 has broad-spectrum inhibition against various coronaviral Mpros. In addition, the crystal structures of SARS-CoV-2 Mpro, SARS-CoV Mpro, MERS-CoV Mpro, and seven SARS-CoV-2 Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) in complex with PF-00835231 are solved. A detailed analysis of these structures reveal key determinants essential for inhibition and elucidates the binding modes of different coronaviral Mpros. Given the importance of the main protease for the treatment of coronaviral infection, structural insights into the Mpro inhibition by PF-00835231 can accelerate the design of novel antivirals with broad-spectrum efficacy against different human coronaviruses.

molecular biology↗

bioRxiv2024

Nanocluster-antibody-drug conjugates (NADC) as an intravesical precision theranostic agent for interstitial cystitis

Interstitial cystitis (IC) is a chronic inflammatory bladder disorder with recurring severe pain, lacking timely diagnostic and therapeutic options. Here, we propose a unitary theranostic nanocluster-antibody-drug conjugate (NADC) by covalently placing dihydroorotate dehydrogenase inhibitors (DHODHi) and ultrasmall gold quantum clusters (AuQCs) on a nerve growth factor (NGF) antagonistic antibody with simultaneous X-ray computed tomographic and near-infrared fluorescence imaging contrasts. Combining anti-inflammatory effects from all individual components, intravesical NADC specifically homed to mucosal lesions with NGF overexpression in voided bladder and capably alleviated inflammation in chronic, acute, and prophylactic IC models of rats, as revealed by behavioral and pathological evaluations. Transcriptomics unveiled cytokine modulation and concomitant inhibition of perturbed IL-17, NF-{kappa}B, TNF, and JAK-STAT signaling pathways. Notably, NADC indirectly remodeled the host bladder microbiota by differentially varying anti-inflammatory and pro-inflammatory bacteria diversities. Distinct from conventional nanoparticles conjugated with antibodies and drugs, NADC relies on the antibody framework, outperforms clinical standard-of-care agents, and represents an emerging category of precision theranostic agents with translational potential for IC theranostics in clinical practice.

microbiology↗

bioRxiv2024

InstructPLM: Aligning Protein Language Models to Follow Protein Structure Instructions

Large language models are renowned for their efficacy in capturing intricate patterns, including co-evolutionary relationships, and underlying protein languages. However, current methodologies often fall short in illustrating the emergence of genomic insertions, duplications, and insertion/deletions (indels), which account for approximately 14% of human pathogenic mutations. Given that structure dictates function, mutated proteins with similar structures are more likely to persist throughout biological evolution. Motivated by this, we leverage crossmodality alignment and instruct fine-tuning techniques inspired by large language models to align a generative protein language model with protein structure instructions. Specifically, we present a method for generating variable-length and diverse proteins to explore and simulate the complex evolution of life, thereby expanding the repertoire of options for protein engineering. Our proposed protein LM-based approach, InstructPLM, demonstrates significant performance enhancements both in silico and in vitro. On native protein backbones, it achieves a perplexity of 2.68 and a sequence recovery rate of 57.51, surpassing Protein-MPNN by 39.2% and 25.1%, respectively. Furthermore, we validate the efficacy of our model by redesigning PETase and L-MDH. For PETase, all fifteen designed variable-length PETase exhibit depolymerization activity, with eleven surpassing the activity levels of the wild type. Regarding L-MDH, an enzyme lacking an experimentally determined structure, InstructPLM is able to design functional enzymes with an AF2-predicted structure. Code and model weights of InstructPLM are publicly available*.

bioinformatics↗

bioRxiv2024

A New Large-Scale Extracellular Vesicle Production Strategy for Biomedical Drug Development

1.Extracellular Vesicles (EVs) are nanoscale to microscale membranous vesicles released by cells. They are vital for intercellular communications in physiological and pathological conditions, offering immense potential in diagnostics and therapeutical applications due to their ability to transfer diverse biological cargos between cells. While research of EVs is continuously gaining popularity in academia, large-scale manufacturing aiming for clinical applications remains challenging. Herein, we introduce a novel large-scale EV production strategy, including an upstream process with fed-batch suspension HEK293 cell culture, and a downstream process with chromatographic purification for ultra-pure EVs. Such promising EV production strategy enables the potential capability of large scale GMP manufacture of EVs for clinical applications.

bioengineering↗

bioRxiv2024

A Novel Simple ImmunoAssay for Quantification of Blood Anti-NMDAR1 Autoantibodies

High titers of anti-NMDAR1 autoantibodies in human brain cause anti-NMDAR1 encephalitis, a rare disease that displays a variety of psychiatric symptoms and neurological symptoms. Currently, immunohistochemical staining and cell-based assays are the standard methods for detection and semi-quantification of the anti-NMDAR1 autoantibodies. Low titers of blood circulating anti-NMDAR1 autoantibodies have been reported in a significant subset of the general human population. However, detection and quantification of these low titers of blood circulating anti-NMDAR1 autoantibodies are problematic because of high non-specific background from less diluted serum/plasma. Development of a new method to quantify these low titers of blood anti-NMDAR1 autoantibodies is necessary to understand their potential impacts on psychiatric symptoms and cognition. Based on our previous One-Step assay, we report the development of a novel simple immunoassay to quantify cross-species blood anti-NMDAR1 autoantibodies, and its validation with immunohistochemistry and cell-based assays in both humans and mice.

neuroscience↗

bioRxiv2024

A registration strategy to characterize DTI-observed changes in skeletal muscle architecture due to passive shortening

Skeletal muscle architecture is a key determinant of muscle function. Architectural properties such as fascicle length, pennation angle, and curvature can be characterized using Diffusion Tensor Imaging (DTI), but acquiring these data during a contraction is not currently feasible. However, an image registration-based strategy may be able to convert muscle architectural properties observed at rest to their contracted state. As an initial step toward this long-term objective, the aim of this study was to determine if an image registration strategy could be used to convert the whole-muscle average architectural properties observed in the extended joint position to those of a flexed position, following passive rotation. DTI and high-resolution fat/water scans were acquired in the lower leg of seven healthy participants on a 3T MR system in +20{degrees} (plantarflexion) and -10{degrees} (dorsiflexion) foot positions. The diffusion and anatomical images from the two positions were used to propagate DTI fiber-tracts from seed points along a mesh representation of the aponeurosis of fiber insertion. The -10{degrees} and +20{degrees} anatomical images were registered and the displacement fields were used to transform the mesh and fiber-tracts from the +20{degrees} to the -10{degrees} position. Students paired t-tests were used to compare the mean architectural parameters between the original and transformed fiber-tracts. The whole-muscle average fiber-tract length, pennation angle, curvature, and physiological cross-sectional areas estimates did not differ significantly. DTI fiber-tracts in plantarflexion can be transformed to dorsiflexion position without significantly affecting the average architectural characteristics of the fiber-tracts. In the future, a similar approach could be used to evaluate muscle architecture in a contracted state.

physiology↗

bioRxiv2024

Comprehensive evaluation and prediction of editing outcomes for near-PAMless adenine and cytosine base editors

Base editors enable the direct conversion of target bases without inducing double-strand breaks, showing great potential for disease modeling and gene therapy. Yet, their applicability has been constrained by the necessity for specific protospacer adjacent motif (PAM). We generated four versions of near-PAMless base editors and systematically evaluated their editing patterns and efficiencies using an sgRNA-target library of 45,747 sequences. Near-PAMless base editors significantly expanded the targeting scope, with both PAM and target flanking sequences as determinants for editing outcomes. We developed BEguider, a deep learning model to accurately predict editing results for near-PAMless base editors. We also provided experimentally measured editing outcomes of 20,541 ClinVar sites, demonstrating that variants previously inaccessible by NGG PAM base editors can now be precisely generated or corrected. We have made our predictive tool and data available online to facilitate development and application of near-PAMless base editors in both research and clinical settings.

bioinformatics↗

bioRxiv2024

The distribution of Apis laboriosa revisited: range extensions and biogeographic affinities

Apis laboriosa, the Himalayan giant honeybee, inhabits the foothills of the Himalaya and neighboring mountainous regions. Here we revise its distribution in light of recent reports and discoveries. The range now extends from longitude 105.9{degrees}E in Cao Bang, Vietnam, in the east to 74.4{degrees}E in the Pir Panjal Range of western Himalaya, a linear distance of 3300 km, with the most notable new localities in northeastern Vietnam, central Myanmar, northern Thailand, and AJK-Pakistan. The species generally occurs at lower elevations in the eastern part of its range than in Nepal, northern India, and the border region between India and Pakistan. Most but not all of the new localities are within the range predicted by species distribution modelling. We discuss the new localities that fall outside of the predicted range, the biotic characteristics of the terrestrial ecoregions in which the species occurs, and the remaining regions that may harbor this spectacular honey bee species.

ecology↗

bioRxiv2024

ROS-Induced Oxidative Stress Causes Enrichment and Emergence of Antibacterial Resistance in Conventional Activated Sludge Process

Since the Great Oxidation Event 2.4 billion years ago, microorganisms have evolved sophisticated responses to oxidative stress. These ancient adaptations remain relevant in modern engineered systems, particularly in conventional activated sludge (CAS) processes, which serve as significant reservoirs of antibiotic resistance genes (ARGs). While ROS-induced stress responses are known to promote ARG enrichment/emergence in pure cultures, their impact on ARG dynamics in wastewater treatment processes remains unexplored. Shotgun-metagenomics analysis of two hospital wastewater treatment plants showed that only 35-53% of hospital effluent resistome was retained in final effluent. Despite this reduction, approximately 30% of ARGs in CAS showed higher abundance than upstream stages, of which 22% emerged de novo. Beta-lactamases and efflux pumps constituted nearly 50% of these enriched ARGs. These ARGs exhibited significant correlations (p < 0.05) with ROS stress response genes (oxyR, soxR, sodAB, katG and ahpCF). The CAS resistome determined 58-75% of the effluent ARG profiles, indicating treatment processes outweigh influent composition in shaping final resistome. Proof-of-concept batch reactor experiments confirmed increased ROS and ARG levels under high dissolved oxygen (8 mg/L) compared to low oxygen (2 mg/L). Untargeted metaproteomics revealed higher expression of resistant proteins (e.g., OXA-184, OXA-576, PME-1, RpoB2, Tet(W/32/O)) under elevated ROS levels. Our findings demonstrate that CAS processes actively shape effluent resistome through ROS-mediated selection, indicating that treatment processes, rather than initial wastewater composition, determine final ARG profiles. This study indicates that the emergence of ARGs needs to be considered as an integral aspect of wastewater treatment design and operation to prevent antibiotic resistance dissemination. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/585878v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@379e42org.highwire.dtl.DTLVardef@1772832org.highwire.dtl.DTLVardef@1f025d7org.highwire.dtl.DTLVardef@144507a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIApproximately 30% of ARGs enriched in CAS versus upstream stages, of which 22% emerged de novo C_LIO_LIBeta-lactamases and efflux pumps comprise [~]50% of enriched ARGs in CAS C_LIO_LICAS resistome shapes 58-75% of effluent ARGs, outweighing influent composition C_LIO_LIHigh DO levels increase ROS production and ARG abundance in CAS systems C_LIO_LIMetaproteomics confirms higher expression of resistant proteins under elevated ROS levels C_LI

genomics↗

bioRxiv2024

Exploration of the link between COVID-19 and gastric cancer from the perspective of bioinformatics and systems biology

BackgroundCoronavirus disease 2019 (COVID-19), an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has caused a global pandemic. Gastric cancer (GC) poses a great threat to peoples health, which is a high- risk factor for COVID-19. Previous studies have found some associations between GC and COVID-19, whereas the underlying molecular mechanisms are not well understood. MethodsWe used a bioinformatics and systems biology approach to investigate the relationship between GC and COVID-19. The gene expression profiles of COVID-19 (GSE196822) and GC (GSE179252) were downloaded from the Gene Expression Omnibus (GEO) database. After identifying the shared differentially expressed genes (DEGs) for GC and COVID-19, functional annotation, protein-protein interaction (PPI) network, hub genes, transcriptional regulatory networks and candidate drugs were analyzed. ResultsA total of 209 shared DEGs were identified to explore the linkages between COVID-19 and GC. Functional analyses showed that Immune-related pathway collectively participated in the development and progression of COVID-19 and GC. In addition, there are selected 10 hub genes including CDK1, KIF20A, TPX2, UBE2C, HJURP, CENPA, PLK1, MKI67, IFI6, and IFIT2. The transcription factor/gene and miRNA/gene interaction networks identified 38 transcription factors (TFs) and 234 miRNAs. More importantly, we identified ten potential therapeutic agents, including ciclopirox, resveratrol, etoposide, methotrexate, trifluridine, enterolactone, troglitazone, calcitriol, dasatinib and deferoxamine, some of which have been reported to improve and treat GC and COVID-19. This study also provides insight into the diseases most associated with mutual DEGs, which may provide new ideas for research on the treatment of COVID-19. ConclusionsThis research has the possibility to be contributed to effective therapeutic in COVID-19 and GC.

bioinformatics↗

bioRxiv2024

Causal role of frontocentral beta oscillation in comprehending linguistic communicative functions

Linguistic communication is often considered as an action serving the function of conveying the speakers goal to the addressee. Although neuroimaging studies have suggested a role of the motor system in comprehending communicative functions, the underlying mechanism is yet to be specified. Here, by 2 EEG experiments and a tACS experiment, we demonstrate that the frontocentral beta oscillation, which represents action states, plays a crucial part in linguistic communication understanding. Participants read scripts involving 2 interlocutors and rated the interlocutors attitudes. Each script included a critical sentence said by the speaker expressing a context-dependent function of either promise, request, or reply to the addressees query. These functions were behaviorally discriminated, with higher addressees will rating for the promise than for the reply and higher speakers will rating for the request than for the reply. EEG multivariate analyses showed that different communicative functions were represented by different patterns of frontocentral beta activity but not by patterns of alpha activity. Further tACS results showed that, relative to alpha tACS and sham stimulation, beta tACS improved the predictability of communicative functions of request or reply, as measured by the speakers will rating. These results convergently suggest a causal role of the frontocentral beta activities in comprehending linguistic communications. HighlightsO_LIThe frontocentral beta activity patterns discriminate communicative functions. C_LIO_LIThe frontocentral beta activity plays a causal role in comprehending the functions. C_LIO_LIThe comprehension could be subserved by the mental simulation of the communication. C_LIO_LIOur findings connect the idea of embedded semantics with the speech act theory. C_LI

neuroscience↗

bioRxiv2024

HHIP protein interactions in lung cells provide insight into COPD pathogenesis

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. The primary causes of COPD are environmental, including cigarette smoking; however, genetic susceptibility also contributes to COPD risk. Genome-Wide Association Studies (GWASes) have revealed more than 80 genetic loci associated with COPD, leading to the identification of multiple COPD GWAS genes. However, the biological relationships between the identified COPD susceptibility genes are largely unknown. Genes associated with a complex disease are often in close network proximity, i.e. their protein products often interact directly with each other and/or similar proteins. In this study, we use affinity purification mass spectrometry (AP-MS) to identify protein interactions with HHIP, a well-established COPD GWAS gene which is part of the sonic hedgehog pathway, in two disease-relevant lung cell lines (IMR90 and 16HBE). To better understand the network neighborhood of HHIP, its proximity to the protein products of other COPD GWAS genes, and its functional role in COPD pathogenesis, we create HUBRIS, a protein-protein interaction network compiled from 8 publicly available databases. We identified both common and cell type-specific protein-protein interactors of HHIP. We find that our newly identified interactions shorten the network distance between HHIP and the protein products of several COPD GWAS genes, including DSP, MFAP2, TET2, and FBLN5. These new shorter paths include proteins that are encoded by genes involved in extracellular matrix and tissue organization. We found and validated interactions to proteins that provide new insights into COPD pathobiology, including CAVIN1 (IMR90) and TP53 (16HBE). The newly discovered HHIP interactions with CAVIN1 and TP53 implicate HHIP in response to oxidative stress.

genetics↗

bioRxiv2024

Single-cell genomics and regulatory networks for 388 human brains

Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized [~]250 disease-risk genes and drug targets with associated cell types. Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/585576v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@70b24org.highwire.dtl.DTLVardef@6c9eddorg.highwire.dtl.DTLVardef@131fae2org.highwire.dtl.DTLVardef@4e0f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells - The Impact of Ligands and Membrane Disruptors

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different liganded states together with spectroscopic studies and molecular dynamics simulations have revealed a rich conformational landscape of GPCRs. However, their supramolecular spatio-temporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile ([~]0.01 m2/s), slow ([~]0.04 m2/s), and fast ([~]0.14 m2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6-ceramide led to significant changes for CD86 while leaving the diffusion of M1R unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression, and the binding of orthosteric ligands; in particular the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

biophysics↗

bioRxiv2024

Insight from Wild Yeast Isolates into the Molecular Mechanisms of Lifespan Variation Mediated by Caloric Restriction

Caloric restriction (CR) is known to extend lifespan across different species and holds great promise for preventing human age-onset pathologies. However, two major challenges exist. First, despite extensive research, the mechanisms of lifespan extension in response to CR remain elusive. Second, genetic differences causing variations in response to CR and genetic factors contributing to variability of CR response on lifespan are largely unknown. Here, we took advantage of natural genetic variation across 46 diploid wild yeast isolates of Saccharomyces species and the lifespan variation under CR conditions to uncover the molecular factors associated with CR response types. We identified genes and metabolic pathways differentially regulated in CR-responsive versus non-responsive strains. Our analysis revealed that altered mitochondrial function and activation of GCN4-mediated environmental stress response are inevitably linked to lifespan variation in response to CR and a unique mitochondrial metabolite might be utilized as a predictive marker for CR response rate. In sum, our data suggests that the effects of CR on longevity may not be universal, even among the closely related species or strains of a single species. Since mitochondrial-mediated signaling pathways are evolutionarily conserved, the dissection of related genetic pathways will be relevant to understanding the mechanism by which CR elicits its longevity effect. Author summaryCaloric restriction (CR) is an energy-balanced nutrient intake without malnutrition to reduce food intake by 20-40%. CR leads to distinct metabolic reprogramming and adaptive changes in gene expression and, as a result, increases health and lifespan in various model organisms, from yeast to most likely primates. Besides extending lifespan, CR also holds great promise for treating many human age-onset pathologies, and the molecules underlying its effects are sought as targets of pharmaceutical aging therapeutics. However, despite extensive research, the mechanisms of lifespan extension in response to CR remain elusive. In addition, several studies in different aging models have now demonstrated that the longevity effect of CR can vary dramatically across different genotypes within a population. As such, CR might be beneficial for some yet detrimental for others, and the mechanisms underlying such genotype-dependent variation are not clear. In this study, we meet these challenges by dissecting molecular response to CR in diverse wild isolates of yeast strains, aiming to characterize pathways and molecules mediating CRs effects on replicative lifespan (RLS) diversity. We found that the RLS significantly differs across genetically diverse wild yeast isolates under CR conditions. Examining the relationships among the RLS phenotypes under CR and non-CR conditions, transcript, and metabolite provided insights into the role of mitochondrial functions in CR-mediated lifespan extension.

molecular biology↗

bioRxiv2024

High-fidelity enhanced AsCas12a knock-in mice for efficient multiplexed gene editing, disease modeling and orthogonal immunogenetics

The advancement of CRISPR gene editing technology, especially the development of Cas9 knock-in mice, has significantly boosted the functional discovery of various genetics factors in diverse fields including genetics, genomics, immunology, and the biology of cancer. However, the pleiotropic effects on human disease and the complex nature of gene interaction networks require a knock-in mouse model capable of simultaneous multiplexed gene perturbation. Here, we present the generation and applications of Cre-dependent conditional and constitutive high-fidelity, enhanced AsCas12a (enAsCas12a-HF1) Rosa26-knock-in mice in the C57BL/6 background. With these mouse strains, we demonstrate highly efficient and multiplexed in vivo and ex vivo genome engineering as applied to lipid nanoparticle (LNP)-RNA-based liver protein targeting, AAV-based tumor modeling, and retrovirus-based immune cell engineering. By integrating with a dCas9-SPH CRISPR activation transgenic strain, we establish a simultaneous dual gene activation and knockout (DAKO) system that showcases the modular potential of these enAsCas12a-HF1 mice. Importantly, constitutive expression of enAsCas12a-HF1 does not lead to any discernable pathological differences as compared to the C57BL/6 background strain. These knock-in mice and the accompanying delivery methods would empower the deconvolution of complex gene interaction networks in broad areas of research.

bioengineering↗