Biology subjects

Xi, Z.

Publications and source records attributed to Xi, Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Site Specific Hydration Contributes to the Thermal Stability of Proteins and Protein-Complexes

Water is integral to protein structure and stability, yet ensemble measurements rarely identify the hydration interactions associated with stabilization. Here, variable-temperature electrospray ionization ion mobility mass spectrometry, conformation-resolved thermodynamic analysis, and molecular dynamics simulations are combined to examine solvent-dependent thermal stability of chymotrypsin inhibitor 2 (CI-2), a model two-state folder, and the barnase-barstar (Bn-B*) complex, which contains a highly polar binding interface. Replacing H2O with D2O raises the thermal transition temperature of both systems by approximately 3 {degrees}C, favors compact conformations, and reduces the relative thermodynamic accessibility of extended states. The conformationally resolved enthalpy-entropy compensation reveals D2O increases enthalpy for CI-2 but reduces enthalpy for Bn-B* as a result of opposing effects on the individual subunits of the complex. Molecular dynamics simulations reveal that lower solvent-accessible surface areas and fewer protein-water hydrogen bonds in D2O coexist with longer water residence times at specific structural sites. In CI-2, the strongest changes localize to the second {beta}-strand/C-terminal region; in Bn-B*, persistent hydration is concentrated at the charged protein-protein interface. Together, these results support a model in which D2O dependent stabilization is associated not only with the extent of hydration, but the site and lifetime of water interactions. More broadly, this work provides an experimental framework for resolving solvent-dependent conformational thermodynamics of protein unfolding and interactions, enabling direct comparison of free-energy landscapes across solution environments.

biochemistry↗

bioRxiv2026

Lorentz Tangent Diffusion Adapter for Multi-label Prediction of Protein-Protein Interaction Types

Protein-protein interactions shape cellular organization and disease processes, but their prediction remains difficult because known interaction networks are incomplete, noisy and highly uneven across proteins. Hyperbolic graph learning is well suited to the hierarchical structure of such networks, yet most methods emphasize learning hyperbolic embeddings and pay less attention to how these embeddings are propagated and decoded for type prediction. Here we introduce the Lorentz Tangent Diffusion Adapter (LTDA), a post-embedding module for PPI type prediction, that is, for predicting which of several interaction types a candidate protein pair undergoes. LTDA learns relation-specific protein representations on the Lorentz manifold, projects them to tangent space, applies lightweight graph diffusion, and decodes the diffused representations directly in tangent space. This design uses the tangent space as a prediction-oriented interface between hierarchical representation learning and interaction classification. On SHS27K and SHS148K under BFS and DFS splits, LTDA consistently improves over representative PPI methods and a reproduced HI-PPI baseline, with average gains of 1.41 percentage points in Micro-F1, 3.21 in AUPR, 1.31 in AUC and 1.25 in accuracy. Ablation, degree-bin and biological-evidence analyses indicate that the gains are not explained by simple smoothing or hub correction. LTDA therefore shows that post-embedding adaptation can make Lorentz representations more effective for PPI type prediction.

bioinformatics↗

bioRxiv2026

Membrane-gated SNARE zippering focuses energy for fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive stagewise membrane fusion by zippering into membrane-bridging four-helix bundles. Yet the conformations underlying successive fusion stages and the coupling of folding energy to bilayer remodeling remain unclear. Using optical tweezers, we measured the intermediates, energetics, kinetics, and force dependence of individual synaptic SNARE complexes assembled in cis on single membranes and in trans between apposed membranes. Membrane-anchored cis-SNAREs assembled through N-terminal and cooperative C-terminal/linker-domain transitions, whereas their transmembrane domains showed little intrinsic dimerization. Syntaxin retained membrane-dependent helical continuity through its linker domain before zippering was complete. PIP2 strengthened but slowed late zippering. In trans, membrane repulsion arrested single trans-SNARE complexes in a half-zippered state. G{beta}{gamma} further clamped this intermediate and inhibited late zippering; G-GDP, but not G-GTP{gamma}S, relieved the clamp, revealing a nucleotide-dependent mechanism for GPCR-mediated inhibition of neurotransmitter release. Modeling suggests that cooperative late zippering, syntaxin linker helicity, and concerted action of multiple SNAREs focus folding energy released over a long distance onto short-range membrane apposition. Thus, mechanically gated SNARE zippering is regulated by membrane forces, lipids, and regulatory proteins.

cell biology↗

bioRxiv2026

An AI System for Autonomous Algorithm Evolution in Drug Development

Artificial intelligence (AI) is increasingly permeating the drug development pipeline. Numerous algorithms for accelerating this multi-stage and multi-task process have been constructed, which depends heavily on expert design and labor-intensive task-specific optimization. Given that AI-driven acceleration of drug development is recognized as a cumulative, often synergistic, effect across multiple stages, the autonomous evolution of existing algorithms across the entire pipeline is demanded to achieve a holistic advancement. Here, we present DrugEvolve, a multi-role large language model system for systematic and autonomous algorithm evolution in drug development. DrugEvolve realizes a closed-loop evolution process by incorporating Researcher, Engineer, and Analyst domains, and enables an iterative design, implementation, evaluation, and refinement of algorithm by leveraging scientific knowledge and accumulated evolutionary experience. Across eleven representative tasks spanning target identification, drug discovery, preclinical study, and clinical trial, DrugEvolve autonomously evolved the corresponding task-specific algorithms and achieved substantial performance enhancement on 120 benchmark test sets. Moreover, it showed robust generalizabilities across heterogeneous data modalities (ranging from biological sequence and graph to molecular topology and textual language), and realized gains in both predictive and generative tasks. Collectively, this AI system can serve not only as an algorithmic infrastructure for drug development, but also as a transferable paradigm for broader scientific domains.

pharmacology and toxicology↗

bioRxiv2026

Focal Transcranial Magnetic Stimulation of the Rat Anterior Cingulate Cortex Inhibits Incubation of Opioid Craving after Voluntary Abstinence

Relapse remains a major challenge in opioid addiction treatment, underscoring the need for innovative therapies. Progress in neuromodulation therapies has been limited by insufficient mechanistic understanding of stimulation engagement and disease-related changes in the brain. We used a novel, focal transcranial magnetic stimulation (TMS) system to deliver high-density theta burst stimulation (hdTBS) combined with resting-state fMRI to test whether anterior cingulate cortex (ACC) stimulation reduces relapse-like behavior and alters functional circuitry in a rodent model of opiate dependence. The coil focality and stimulation parameters approximate human TMS protocols, and the targeted region represents a functional homolog of the human ACC. We trained rats to self-administer oxycodone intravenously for 14 days. We then introduced an electric barrier for 13 days, which caused cessation of drug self-administration. We assessed relapse to oxycodone seeking immediately after training (early abstinence) and after electric-barrier exposure (late abstinence). We administered daily hdTBS or sham stimulation for 7 days before the late-abstinence test. Sham-treated rats showed a time-dependent increase in oxycodone seeking during abstinence (incubation of oxycodone craving) and reduced ACC functional connectivity. In contrast, hdTBS prevented the incubation of oxycodone craving and restored ACC connectivity with the dorsal and ventral striatum. Tracer-based axonal-projection data further showed that stimulation-induced effects aligned with regions receiving dense projections from the stimulation site, suggesting that the projection architecture is critical to the propagation of focal stimulation across distributed networks. These findings identify ACC-centered circuits as mechanistically informed targets for TMS-based interventions that aim to reduce opioid relapse during abstinence. One sentence SummaryPrefrontal TMS stimulation reduced relapse-like behavior and restored corticostriatal circuits, highlighting translational targets for addiction treatment.

neuroscience↗

bioRxiv2025

Unveiling the Hidden: Dissecting Liraglutide Oligomerization Dual Pathways via Direct Mass Technology, Electron-Capture Dissociation, and Molecular Dynamics

Peptide therapeutics have revolutionized drug design strategies, yet the inherent structural flexibility and conjugated moieties of drug molecules present challenges in discovery, rational design, and manufacturing. Liraglutide, a GLP-1 receptor agonist conjugated with palmitic acid at its lysine residue, exemplifies these challenges by forming oligomers, which may compromise efficacy through progressive formation of aggregates. Here, we incorporate native mass spectrometry platforms including electron-capture dissociation (ECD), direct mass technology (DMT), and molecular dynamics (MD) to capture the early oligomerization process of liraglutide. Our findings reveal a restricted C-terminal region upon oligomer formation, as indicated by the reduced release of z-ions in ECD analysis. Additionally, we identified the formation of higher-order oligomers (n=25-62) by DMT, primarily stabilized by hydrophilic interactions involving preformed stable oligomers (n=14). Together, these integrative mass spectrometry results delineate a dual-pathway oligomerization process for liraglutide, demonstrating the power of mass spectrometry in uncovering hidden pathways of self-association. This approach underscores the potential of mass spectrometry as a key tool in the rational design and optimization of peptide-based therapeutics.

biophysics↗

bioRxiv2024

Fitness compatibility and dengue virus inhibition in a Bangladeshi strain of Aedes aegypti infected with the Wolbachia strain wAlbB

Dengue cases in Bangladesh have surged in recent years. The existing insecticide-based control program, implemented in parts of the country, is challenged by issues of insufficient household coverage and high levels of insecticide resistance in the primary dengue virus (DENV) vector, Aedes aegypti. A more sustainable, effective alternative could be the implementation of a Wolbachia-mediated disease management strategy. Infecting mosquitoes with Wolbachia can change their reproductive compatibility and their ability to transmit DENV. These new phenotypes can be exploited to suppress or replace wild-type Ae. aegypti populations. Such strategies require the development of well-characterised Wolbachia-infected strains with biological characteristics that are comparable with local mosquitoes. We created and characterised a Wolbachia-infected Ae. aegypti strain with a Dhaka wild-type genetic background, and compared its reproductive compatibility, maternal inheritance, fitness, and virus-blocking ability to the parental strains (Dhaka wild-type and wAlbB2-F4). The new Ae. aegypti strain wAlbB2-Dhaka demonstrated complete cytoplasmic incompatibility with the wild-type strain and complete maternal transmission, retaining levels of pyrethroid resistance of the Dhaka wild-type (70% survival to 10 times the dose of permethrin expected to kill susceptible mosquitoes). No significant fitness costs were detected during laboratory comparisons of fecundity, fertility, survival, mating competitiveness, or desiccation tolerance. Compared to the wild-type strain, wAlbB2-Dhaka mosquitoes had a significantly reduced number of DENV genome copies in the bodies (44.4%, p = 0.0034); two-fold reduction in dissemination to legs and wings (47.6%, p < 0.0001); and >13-fold reduction of DENV in saliva expectorates (proxy of transmission potential) (92.7%, p < 0.0001) 14 days after ingesting dengue-infected blood. Our work indicates that the wAlbB2-Dhaka strain could be used for Ae. aegypti suppression or replacement strategies for dengue management in Bangladesh. Author summaryBangladesh is currently grappling with a series of severe dengue outbreaks, resulting in more than 300 thousand infections, 1700 fatalities, and a significant influx of patients requiring hospitalisation in 2023. These outbreaks coincide with the emergence of high levels of insecticide resistance in the Ae. aegypti population, which is contributing to the failure of conventional, insecticidal control strategies. In search of more sustainable, effective dengue control strategies, approaches using the endosymbiotic bacteria Wolbachia that render mosquitoes resistant to arbovirus infection are being trialled in a number of countries. We transinfected Dhaka wild-type Ae. aegypti with the Wolbachia strain wAlbB2 by backcrossing it with a Wolbachia wAlbB2-infected Ae. aegypti strain and assessed the new strains suitability for field release. We compared the fitness and virus-blocking ability of the backcrossed strain with its parental strains. We demonstrated that the new strain, wAlbB2-Dhaka, is as fit as the Dhaka wild-type strain and blocked [~]92% of dengue virus transmission. This strain has great potential for Wolbachia-mediated dengue management strategies in Bangladesh.

zoology↗

bioRxiv2024

TUG protein acts through a disordered region to organize the early secretory pathway

The endoplasmic reticulum (ER)-Golgi Intermediate Compartment (ERGIC) is a distinct compartment in mammalian cells, which forms in part by homotypic fusion of ER-derived vesicles and gives rise to the cis cisterna of the Golgi ribbon. How the ERGIC is regulated is not well understood. Here we show that the TUG protein is essential to maintain this compartment as a distinct organelle. TUG (UBXN9, Aspscr1) is known to regulate the cell type -specific trafficking of GLUT4 glucose transporters, but its role in more ubiquitous trafficking pathways has not been well characterized. TUG localized to the ERGIC and in fibroblasts its deletion enhanced anterograde flux and increased resorption of ERGIC markers into the cis-Golgi, perturbing membrane homeostasis in the early secretory pathway. TUG knockout cells had a compacted Golgi morphology, and ultrastructural studies revealed dilated cisterna with surrounding small vesicles. A central disordered region in TUG mediated its recruitment to ERGIC membranes, and an amino terminal domain was sufficient to induce oligomerization in cells. In TUG knockout cells, ERGIC-dependent processes such as autophagy are disrupted and model cargoes such as CFTR are missorted. Together, these results reveal a novel, TUG-dependent regulatory mechanism in the early secretory pathway, which modulates ERGIC organization and anterograde trafficking. This function is co-opted by GLUT4 and other proteins that employ an unconventional secretion pathway to the plasma membrane.

cell biology↗

bioRxiv2023

Aberration correction for deformable mirror based remote focusing enables high-accuracy whole-cell super-resolution imaging

Single-molecule localization microscopy (SMLM) enables three-dimensional (3D) investigation of nanoscale structures in biological samples, offering unique insights into their organization. However, traditional 3D super-resolution microscopy using high numerical aperture (NA) objectives is limited by imaging depth of field (DOF), restricting their practical application to relatively thin biological samples. Here, we developed a unified solution for thick sample super-resolution imaging using a deformable mirror (DM) which was served for fast remote focusing, optimized point spread function (PSF) engineering and accurate aberration correction. By effectively correcting the system aberrations introduced during remote focusing and sample aberrations at different imaging depths, we achieved high-accuracy, large DOF imaging of the whole-cell organelles [i.e. nuclear pore complex (NPC), microtubules, and mitochondria] with a nearly uniform resolution of approximately 30 nm across the entire cellular volume.

bioengineering↗

bioRxiv2023

Identification and Characterization of CtUGT3 as the Key Player of Astragalin Biosynthesis in Safflower

Safflower (Carthamus tinctorius L.) flowers are used as a traditional Chinese medicine for a long history. Flavonoids are the main bioactive components in safflower flowers, and most of them exist in the form of flavonoid glycosides. Only few glycosyltransferases have been identified in safflower. To reveal the uridine diphosphate glycosyltransferase (UGT) involved in flavonoid glycosides biosynthesis in safflower, a metabolomics and transcriptome analysis was performed by using the flowers under different light qualities treatments. Three differentially expressed UGT genes were screened, and their expressions were significantly related with concentrations of 9 flavonoid O-glycosides. Safflower corolla protoplasts were further used to confirm flavonoid O-glycosylation ability of UGT candidates. The astragalin (kaempferol 3-O-glucoside) content was only significantly increased when CtUGT3 was overexpressed in protoplasts. The biochemical properties and kinetic parameters of CtUGT3 were determined. CtUGT3 also showed flavonoid 3-OH and 7-OH glycosylation activities in vitro. Molecular modeling and site-directed mutagenesis revealed that E384 and S276 were critical catalytic residues for the 3-OH glycosylation of CtUGT3. These results demonstrate that CtUGT3 has a flavonoid 3-OH glycosylation function and is involved in the biosynthesis of astragalin in safflower. This study provides insights into the catalytic mechanisms of flavonoid O-glycosyltransferases, and makes a reference for flavonoid biosynthesis genes research in medicinal plants.

plant biology↗

bioRxiv2023

VHL Mutation Drives Human Clear Cell Renal Cell Carcinoma Progression Through PI3K/AKT-Dependent Cholesteryl Ester Accumulation

BackgroundCholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. MethodsEnabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. FindingsSurprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIF and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. InterpretationCollectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. FundingThis work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No.2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

cancer biology↗

bioRxiv2022

Wolbachia wAlbB inhibit dengue and Zika infection in the mosquito Aedes aegypti with an Australian background

Biological control of mosquito vectors using the insect-specific bacteria Wolbachia is an emerging strategy for the management of human arboviral diseases. We recently described the development of a strain of Ae. aegypti infected with the Wolbachia strain wAlbB (referred to as the wAlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a wAlbB infected Ae. aegypti strain from the USA. Field releases of male wAlbB2-F4 mosquitoes resulted in the successful suppression of a wild population of mosquitoes in the trial sites by exploiting the strains Wolbachia-induced cytoplasmic incompatibility. We now demonstrate that the strain is resistant to infection by dengue and Zika viruses and is genetically similar to endemic Queensland populations. There was a fourfold reduction in the proportion of wAlbB2-F4 mosquitoes that became infected following a blood meal containing dengue 2 virus (16.7%) compared to wild type mosquitoes (69.2%) and a 6-7 fold reduction in the proportion of wAlbB2-F4 mosquitoes producing virus in saliva following a blood meal containing an epidemic strain of Zika virus (8.7% in comparison to 58.3% in wild type mosquitoes). Restriction-site Associated DNA (RAD) sequencing revealed that wAlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the wAlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the wAlbB2-F4 strain retains the insecticide susceptibility phenotype and genotype of the Australian mosquitoes. We demonstrate that the Wolbachia wAlbB2-F4, in addition to being suitable for suppression programs, can be effective in population replacement programs given its high inhibition of virus infection in mosquitoes. The ease at which a target mosquito population can be transfected with wAlbB2, while retaining genotypes and phenotypes of the target population, shows the robustness of this strain as a biocontrol agent against the Ae. aegypti mosquito itself as well as the pathogens it transmits. IMPORTANCEEpidemics of arthopod-borne virus (arbovirus) diseases affect millions of people and are becoming more frequent and widespread. A successful strategy to control these diseases is by infecting mosquito populations with benign, insect-specific Wolbachia bacteria that render mosquitoes refractory to infection with pathogenic arboviruses. Here we show that a strain of the major mosquito vector Ae. aegypti that was infected with Wolbachia following a simple back-cross mating procedure is refractory to infection with dengue and Zika viruses. Importantly, the genetic background of the strain is equivalent to the target population, which is important for persistence of the strain and regulatory approval.

microbiology↗

bioRxiv2022

Regulation of EGF-stimulated activation of the PI-3K/AKT pathway by exocyst-mediated exocytosis

The phosphoinositide-3 kinase (PI-3K)/AKT cell survival pathway is an important pathway activated by EGFR signaling. Here we show, that in addition to previously described critical components of this pathway, i.e., the docking protein Gab1, the PI-3K/AKT pathway in epithelial cells is regulated by the exocyst complex, which is a vesicle tether that is essential for exocytosis. Using live-cell imaging, we demonstrate that PI(3,4,5)P3 levels fluctuate at the membrane on a minutes time scale and that these fluctuations are associated with local PI(3,4,5)P3 increases at sites where recycling vesicles undergo exocytic fusion. Supporting a role for exocytosis in PI(3,4,5)P3 generation, acute promotion of exocytosis by optogenetically driving exocyst-mediated vesicle tethering upregulates PI(3,4,5)P3 production and AKT activation. Conversely, acute inhibition of exocytosis using Endosidin2, a small-molecule inhibitor of the exocyst subunit Exo70, impairs PI(3,4,5)P3 production and AKT activation induced by EGF stimulation of epithelial cells. Moreover, prolonged inhibition of EGF signaling by EGFR tyrosine kinase inhibitors results in spontaneous reactivation of AKT without a concomitant relief of EGFR inhibition. However, this reactivation can be negated by acutely inhibiting the exocyst. These experiments demonstrate that exocyst-mediated exocytosis - by regulating PI(3,4,5)P3 levels at the plasma membrane - subserves activation of the PI-3K/AKT pathway by EGFR in epithelial cells.

cell biology↗

bioRxiv2022

Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes

Multiple Wolbachia strains can block pathogen infection, replication, and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase 2 (Man2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed Man2 knockouts and examined effects on both Wolbachia and virus levels, using both dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titers were significantly elevated in Man2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the Man2 KO mutation was associated with decreased DENV titers, but in a Wolbachia-infected background, the Man2 KO mutation significantly modulated virus blocking. In contrast, the Man2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that Man2 modulates arbovirus infection in Ae. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.

microbiology↗

bioRxiv2021

Quantitative single-cell transcriptome-based ranking of engineered AAVs in human retinal explants

Gene therapy is a rapidly developing field, and adeno-associated virus (AAV) is a leading viral vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models, due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue, and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wildtype and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA-Seq AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers, except for a cone population with altered expression of cone-specific genes. The efficiency and tropism of AAVs in human explants were quantified, with single-cell resolution. The top performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants, and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/461256v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e110ddorg.highwire.dtl.DTLVardef@11a2c7eorg.highwire.dtl.DTLVardef@15f1633org.highwire.dtl.DTLVardef@1aa741a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Using a Quadruplet Codon to Expand the Genetic Code of an Animal

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.

synthetic biology↗

bioRxiv2021

The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila homologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and spermatocytes, the only two Drosophila cell types bearing motile cilia/flagella. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly. SUMMARY STATEMENTDNAAF3 function as a dynein assembly factor for motile cilia is conserved in Drosophila.

cell biology↗

bioRxiv2021

WGDI: A user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes

Evidence of whole-genome duplications (WGDs) and subsequent karyotype changes has been detected in most major lineages of life on Earth. To clarify the complex resulting multiple-layered patterns of gene collinearity in genome analyses there is a need for convenient and accurate toolkits. To meet this need, we introduce here WGDI (Whole-Genome Duplication Integrated analysis), a Python-based command-line tool that facilitates comprehensive analysis of recursive polyploidizations and cross-species genome alignments. WGDI supports three main workflows (polyploid inference, hierarchical inference of genomic homology, and ancestral chromosomal karyotyping) that can improve detection of WGD and characterization of related events. It incorporates a more sensitive and accurate collinearity detection algorithm than previous softwares, and can accelerate WGD-related karyotype research. As a freely available toolkit at GitHub (https://github.com/SunPengChuan/wgdi), WGDI outperforms similar tools in terms of efficiency, flexibility and scalability. In an illustrative example of its application, WGDI convincingly clarified karyotype evolution in Aquilegia coerulea and Vitis vinifera following WGDs and rejected the hypothesis that Aquilegia contributed as a parental lineage to the allopolyploid origin of core dicots.

bioinformatics↗