Biology subjects

Gao, H.

Publications and source records attributed to Gao, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Ultrahigh Resolution Structure of the Heat-stable Form-IAq RuBisCO from the Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the initial carbon fixation reaction in the Calvin-Benson-Bassham cycle in plants and most microbial autotrophs. Among the many forms of RuBisCOs, form-I--a protein complex containing 8 large and 8 small subunits--is the most common, representing over 90% of all known RuBisCOs. Although many form-I RuBisCO structures have been determined, no structure has been reported for a form-IAq RuBisCO. Here, and at ultrahigh (1.55 [A]) resolution, we detail the structure of the heat-stable form-IAq RuBisCO from the thermophilic and anaerobic purple bacterium Thermochromatium (Tch.) tepidum. The overall structure of the Tch. tepidum form-IAq RuBisCO resembles both a form-IAc RuBisCO from a chemolithotrophic sulfur bacterium and a synthetic form-I RuBisCO reconstructed from ancestral sequences. However, despite significant structural and sequence similarities with other form-I RuBisCOs, the Tch. tepidum form-IAq RuBisCO shows significantly greater interactions between adjacent small subunits through their extended N-terminal domains that contain a characteristic six-residue insertion unique to form-IAq RuBisCOs. Structural differences of Tch. tepidum RuBisCO from its mesophilic relative Allochromatium vinosum suggests the mechanisms of its enhanced thermostability. In addition to small subunit interactions, key substitutions on the hydrophilic surface of the small subunits likely contribute to its thermostability as well. It has been proposed that form-I RuBisCOs first appeared in anaerobic thermophiles. Our structure of the Tch. tepidum RuBisCO represents the first such structure of a form-IAq enzyme, providing fresh clues for unraveling the evolutionary history of RuBisCO and new details for how this key enzyme remains active at elevated temperatures.

plant biology↗

bioRxiv2025

AI-driven discovery of host thioredoxin as a CRISPR enhancer of phage-encoded miniature Cas12 hacker nuclease

The evolutionary arms race between bacteriophages and their bacterial hosts has driven the evolution of sophisticated adaptive immune systems, such as CRISPR-Cas, as a crucial defense mechanism. While bacteriophages have developed various anti-CRISPR strategies to counteract these immune systems, the role of bacterial host factors in enhancing CRISPR-Cas functions has been relatively unexplored. In this study, we employ an artificial intelligence (AI)-driven approach to systematically analyze potential interactions between Escherichia coli (E. coli) proteins and fifteen previously uncharacterized Cas12 proteins, generating 65,715 predicted binary complex structures. Our findings reveal a previously unknown dimension of CRISPR immunity, demonstrating that the hosts ubiquitous redox enzyme, thioredoxin (TrxA), significantly enhances the DNA cleavage efficiency of a phage-encoded, miniature Cas12 nuclease (termed Cas12 hacker). This synergistic relationship represents a strategic inversion, where a bacteriophage hijacks a host protein to reinforce its own genome degradation machinery, possibly targeting rival nucleic acids. Through comprehensive biochemical characterizations, structural analyses of the Cas12 hacker-TrxA-sgRNA-dsDNA quaternary complex, and in vivo bacterial defense assays, we uncover an intricate association in which thioredoxin binds to and activates the Cas12 hacker nuclease, intensifying its DNA cleavage capacity and bolstering CRISPR immunity. Our findings expand the understanding of the molecular interactions underlying host-phage conflicts and highlight the potential for harnessing endogenous host factors to enhance the capabilities of CRISPR-based genetic engineering tools.

microbiology↗

bioRxiv2025

Cross-reactive sarbecovirus antibodies induced by mosaic RBD-nanoparticles

Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) spike proteins that recognize SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, we developed mosaic-8b RBD-nanoparticles presenting eight sarbecovirus RBDs arranged randomly on a 60-mer nanoparticle. Mosaic-8b immunizations protected animals from challenges from viruses whose RBDs were matched or mismatched to those on nanoparticles. Here, we describe neutralizing mAbs isolated from mosaic-8b-immunized rabbits, some on par with Pemgarda, the only currently FDA-approved therapeutic mAb. Deep mutational scanning, in vitro selection of spike resistance mutations, and single-particle cryo-electron microscopy structures of spike-antibody complexes demonstrated targeting of conserved RBD epitopes. Rabbit mAbs included critical D-gene segment RBD-recognizing features in common with human anti-RBD mAbs, despite rabbit genomes lacking an equivalent human D-gene segment, thus demonstrating that the immune systems of humans and other mammals can utilize different antibody gene segments to arrive at similar modes of antigen recognition. These results suggest that animal models can be used to elicit anti-RBD mAbs with similar properties to those raised in humans, which can then be humanized for therapeutic use, and that mosaic RBD-nanoparticle immunization coupled with multiplexed screening represents an efficient way to generate and select broadly cross-reactive therapeutic pan-sarbecovirus and pan-SARS-CoV-2 variant mAbs. Significance StatementSARS-CoV-2 variants and potential zoonotic sarbecovirus infections continue to threaten human health. Anti-SARS-CoV-2 mAbs that recognize conserved epitopes could be used prophylactically or therapeutically. We present approaches to elicit and identify cross-reactive mAbs using immunizations in animals with mosaic RBD-nanoparticles. We show that human and other mammalian immune systems can utilize different antibody gene segments to arrive at similar modes of antigen recognition, underscoring the flexibility of mammalian antibody repertoires and suggesting that experimental animals can be used to generate therapeutically-useful cross-reactive anti-RBD mAbs. The combination of mosaic-8b RBD-nanoparticles to focus the immune response and a multiplexed assay to select cross-reactive mAbs can be applied at larger scale, or against other pathogens, to identify mAbs of therapeutic and scientific potential.

immunology↗

bioRxiv2024

Benchmarking AI Models for In Silico Gene Perturbation of Cells

Understanding perturbations at the single-cell level is essential for unraveling cellular mechanisms and their implications in health and disease. The growing availability of biological data has driven the development of a variety of in silico perturbation methods designed for single-cell analysis, which offer a means to address many inherent limitations of experimental approaches. However, these computational methods are often tailored to specific scenarios and validated on limited datasets and metrics, making their evaluation and comparison challenging. In this work, we introduce a comprehensive benchmarking framework to systematically evaluate in silico perturbation methods across four key scenarios: predicting effects of unseen perturbations in known cell types, predicting effects of observed perturbations in unseen cell types, zero-shot transfer to bulk RNA-seq of cell lines, and application to real-world biological cases. For each scenario, we curated diverse and abundant datasets, standardizing them into flexible formats to enable efficient analysis. Additionally, we developed multiple metrics tailored to each scenario, facilitating a thorough and comparative evaluation of these methods. Our benchmarking study assessed 10 methods, ranging from linear baselines to advanced machine learning approaches, across these scenarios. While some methods demonstrated surprising efficacy in specific contexts, significant challenges remain, particularly in zero-shot predictions and the modeling of complex biological processes. This work provides a valuable resource for evaluating and improving in silico perturbation methods, serving as a foundation for bridging computational predictions with experimental validation and real-world biological applications.

bioinformatics↗

bioRxiv2024

TGFβ1 and RGD Cooperatively Regulate SMAD 2/3 Mediated Oncogenic Effects in Prostate Cancer Cells in Bioorthogonally Constructed Hydrogels

To recapitulate prostate cancer metastasis, DU145 cells were cultured in a hyaluronic acid-based, bioorthogonally constructed, protease-degradable hydrogel. In the presence of covalently conjugated integrin-binding peptide (GRGDSP), DU145 cells formed tumoroids and exhibited small protrusions. Upon addition of soluble transforming growth factor beta 1 (TGF{beta}1), cells underwent morphological changes to form extended interconnected cellular networks. Contrarily, in RGD-free hydrogels, cells maintained spherical structures even in the presence of TGF{beta}1. In RGD-conjugated hydrogels, TGF{beta}1 induced nuclear localization of SMAD2/3, upregulating a wide range of TGF{beta}1 target genes and proteins. Prolonged exposure to TGF{beta}1 led to matrix remodeling and induced epithelial-to-mesenchymal transition in DU145 cells, with loss of epithelial markers and gain of mesenchymal markers. TGF{beta}RI/ALK5 inhibitor SB-431542 attenuated TGF{beta}1-induced morphological changes, abrogated nuclear localization of SMAD2/3, and restored the expression of key epithelial markers. Our findings highlight the cooperative role of TGF{beta}1 signaling and integrin binding peptide in the acquisition of aggressive phenotype and promoting tumor progression. TEASERPhysiologically relevant 3D cell culture platforms enabled mechanistic investigation of growth factor signaling related to prostate cancer metastasis.

bioengineering↗

bioRxiv2024

Immunofocusing on the conserved fusion peptide of HIV envelope glycoprotein in rhesus macaques

SummaryDuring infection, the fusion peptide (FP) of HIV envelope glycoprotein (Env) serves a central role in viral fusion with the host cell. As such, the FP is highly conserved and therefore an attractive epitope for vaccine design. Here, we describe a vaccination study in non-human primates (NHPs) where glycan deletions were made on soluble HIV Env to increase FP epitope exposure. When delivered via implantable osmotic pumps, this immunogen primed immune responses against the FP, which were then boosted with heterologous trimers resulting in a focused immune response targeting the conserved FP epitope. Although autologous immunizations did not elicit high affinity FP-targeting antibodies, the conserved FP epitope on a heterologous trimer further matured the lower affinity, FP-targeting B cells. This study suggests using epitope conservation strategies on distinct Env trimer immunogens can focus humoral responses on desired neutralizing epitopes and suppress immune-distracting antibody responses against non-neutralizing epitopes.

immunology↗

bioRxiv2024

Engineered mitochondrial A-to-G editors with enhanced efficiency and targeting scope

Mitochondrial base editing is a powerful technology, but the current A-to-G mitochondrial DNA (mtDNA) base editors are reluctant to achieve high efficiency, which is critical for mtDNA engineering. Through laboratory evolution, TadA-8e variants were discovered with substantially increased activity and expanded targeting compatibility, especially for previously unfavorite sequence contexts, when applied to both nuclear and mitochondrial ABEs. Further engineering of the mtDNA editors (eTd-mtABEs) dramatically reduced both DNA and RNA off-targeting effects and enhanced strand-selective A-to-G editing with substitution of DddA to DNA nickases. Moreover, the eTd-mtABEs induced up to 145-fold editing frequencies compared to previous mtDNA adenine base editors in rat cells, and installed targeted mutations in all injected rat embryos with up to 74% efficiency in founders, resulting generation of SNHL and Leigh syndrome models with severe defects. It suggests that eTd-mtABEs are promising mtDNA engineering technology for basic research and for translational studies. HighlightsO_LIEngineered TadA variants enhance nuclear and mitochondrial ABE activity in all sequence contexts. C_LIO_LIAdditional mutations in eTd-mtABEs further minimize both DNA and RNA off-targeting effects. C_LIO_LIEngineered TadA variants are compatible to DNA nickase assisted strand-selective mtDNA editing. C_LIO_LIeTd-mtABEs enable highly efficient generation of rat mtDNA disease models with severe phenotypes. C_LI

bioengineering↗

bioRxiv2024

Universal function of WRI1 involved in integrated regulation of multiple latex metabolism pathways in Natural Rubber

Hevea brasiliensis and Taraxacum kok-saghyz are sources of nature rubber (NR) and recent research has focused on finding a method to increase NR quality and production. NR consists mainly of cis-1,4-polyisoprene, which is synthesized from sucrose by a series of reactions during carbon metabolism. WRINKLED1 (WRI1) is a transcription factor (TF) that coordinates many genes involved in carbon metabolism and lipid biosynthesis. Here, we isolated and characterized two orthologues of WRI in rubber-producing plants H. brasiliensisa and T. kok-saghyz, which are highly expressed in their latex. Subcellular localization and ectopic expression in Arabidopsis thaliana indicated that the TFs, HbWRI1 and TkWRI1, are involved in lipid accumulation. Overexpression of HbWRI1 and TkWRI1 in T. kok-saghyz substantially enhanced NR quality and production, including dry rubber content, molecular weight, and the diameter of rubber particles in the latex. Conversely, they were decreased in TkWRI1 repression. Furthermore, based on the activated expression in transgenic T. kok-saghyz latex and the existence of AW-box element in the promoter regions, 16 direct downstream genes of HbWRI1 and TkWRI1 were identified by a dual-luciferase reporter assay and yeast one-hybrid assay, and their products were responsible for both NR biosynthesis and lipid metabolism. These results reveal a regulatory module that HbWRI1 and TkWRI1 TFs can positively regulate NR quality and production in T. kok-saghyz latex by synergistically regulating the entire NR biosynthesis. This module is expected to be utilized to develop superior varieties to enhance NR quality and production.

biochemistry↗

bioRxiv2024

The E3 ligase NEDD4L prevents colorectal cancer liver metastasis via degradation of PRMT5 to inhibit the AKT/mTOR signaling pathway

Colorectal cancer is the second most common cause of cancer mortality worldwide, and liver metastasis is the major cause of death in patients with colorectal cancer. Dysfunctional E3 ligase activity has recently been shown to be associated with colorectal cancer. However, the key E3 ligase affecting colorectal cancer liver metastasis remains unknown. Therefore, we used an shRNA library targeting 156 E3 ubiquitin ligases to perform an in vivo loss-of-function screen of a human colorectal cancer cell line in a mouse model of colorectal cancer liver metastasis. The screen revealed that NEDD4L knockdown promoted colorectal cancer liver metastasis. Moreover, overexpression of NEDD4L prevented colorectal cancer liver metastasis. Mechanistic studies revealed that NEDD4L bound to the PPNAY motif in PRMT5 and ubiquitinated PRMT5 to promote its degradation. PRMT5 degradation attenuated the methylation of an arginine residue in AKT1 to inhibit the AKT/mTOR signaling pathway, consequently decreasing colorectal cancer cell proliferation. We are the first to show that PRMT5 is a substrate protein of the E3 ligase NEDD4L and reveals not only the metastasis-inhibiting function of NEDD4L but also a novel mechanism by which NEDD4L suppresses colorectal cancer liver metastasis. These findings may provide a new preventive strategy for liver metastasis.

cancer biology↗

bioRxiv2024

Weekly inhaled salmeterol xinafoate, a selective β2 adrenergic receptor agonist drug, therapeutically inhibits cancer growth and prolongs survival in vivo

Humans have spent approximately 99.5% of the 2.5 million years of evolutionary history as hunter-gatherers, which helped to evolutionarily mold our minds and bodies to the hunting and gathering lifestyle. Physical activity associated with hunting animals, gathering plants, and evading predators is essential for human survival, evolution, and prosperity. Thus, physical activity (acute stress) is mostly beneficial to the body, whereas psycho-emotional stress (chronic stress) is usually detrimental at the psychological, physiological, and pathological levels. Although physical activity exerts anticancer effects and psycho-emotional stress exhibits cancer-promoting effects, both function through the same pathway involving activation of the sympathetic nervous system, release of catecholamines, and stimulation of {beta}2 adrenergic receptor ({beta}2AR) expressed on cancer cells. Here, salmeterol xinafoate was chosen to mimic physical activity. A once-weekly administration of salmeterol xinafoate by the intraperitoneal, inhalation, or transdermal route, dose-dependently extended latency, reduced cancer incidence and growth, and prolonged survival in vivo. Remarkably, the best complete response rates were 75%, 30%, and 71.43% for the therapeutic treatment of lung cancer, breast cancer, and melanoma, respectively. Notably, the salmeterol xinafoate doses used in our experiments are comparable to those used in patients. Moreover, in endogenous {beta}2AR knockout cancer cells, the exogenous {beta}2AR mutant with impaired salmeterol xinafoate binding or downstream signaling prevented the anticancer effects of salmeterol xinafoate in vivo, demonstrating the on-target action of salmeterol xinafoate on cancer cells and the requirement of the downstream signaling pathway. Finally, we showed that treatment with salmeterol xinafoate dose-dependently decreased the self-renewal capacity of lung cancer stem cells, breast cancer stem cells, and melanoma stem cells. From the perspective of human evolution into cancer biology, our study reveals that {beta}2AR represents an ideal anticancer drug target and that salmeterol xinafoate monotherapy has strong anticancer activity.

cancer biology↗

bioRxiv2024

In computer explore The neutralization mechanism of Amubarvimab and Romlusevimab against SARS-COV-2 mutants

Since the end of 2019, The coronavirus disease 2019 (COVID-19) has been endemic worldwide for three years, causing more than 6.95 million deaths and having a massive impact on the global political economy. With time, the Severe acute respiratory syndrome coronavirus 2(SARS-COV-2) is also constantly mutating. Mutations lead to stronger infectivity or virulence of the virus, and some monoclonal antibodies against wild-type SARS-COV-2 are also challenging to play a role. Amubarvimab and Romlusevimab were originally developed against wild-type SARS-COV-2; however, these monoclonal antibodies neutralizing efficacy and mechanism against these mutants are unknown. In this study, the binding ability of Amubarvimab and Romlusevimab to 7 mutant strains were tested by computer method and the interaction mechanism was explored. Our experimental data show that Amubarvimab can effectively bind most mutations and maintain the stability of the complexes mainly through hydrogen bond interaction; However, the binding efficiency of Romlusevimab was lower than that of Amubarvimab, and the stability of the complexes was maintained mainly through electrostatic interaction. Both Amubarvimab and Romlusevimab show low binding potency against E406W and Q498Y mutations, so there is a certain probability of immune escape in the face of variants carrying E406W and Q498Y mutations when Amubarvimab and Romlusevimab are used in combination.

immunology↗

bioRxiv2024

Modular Nano-Scaffold Biocatalysis for Superior PET Depolymerization and Valorization

The global crisis of polyethylene terephthalate (PET) waste demands new strategies for sustainable management. Biocatalytic recycling offers a promising route, but direct depolymerization under moderate conditions is often limited by kinetic constrains of individual enzyme. Here, we engineer and optimize modular protein scaffolds that utilize orthogonal interaction domains, including the cohesin-dockerin pairs from bacterial cellulosomes and the SH3 ligand/domain pair from mouse Crk, to assemble complementary hydrolases (PETase, MHETase, and ICCG) into a synergistic multi-enzyme cascade. This architecture overcomes intermediate inhibition, efficiently converting PET hydrolysis products into the final monomers, terephthalic acid (TPA) and ethylene glycol (EG), outperforming previous multi-enzyme systems. The systems application is further evaluated by stability enhancement via immobilization on metal-organic frameworks (MOFs), coupling depolymerization to the valorization of EG into glycolic acid (GA), and adapting the system for scalable whole-cell biocatalysis. This scaffold-based multi-enzyme approach provides a new strategy for developing integrated biocatalytic systems to advance a circular PET economy.

synthetic biology↗

bioRxiv2024

BST2 induces vascular smooth muscle cell plasticity and phenotype switching during cancer progression

BackgroundSmooth muscle cell (SMC) plasticity and phenotypic switching play prominent roles in the pathogenesis of multiple diseases, but their role in tumorigenesis is unknown. We investigated whether and how SMC diversity and plasticity plays a role in tumor angiogenesis and the tumor microenvironment. Methods and ResultsWe use SMC-specific lineage-tracing mouse models and single cell RNA sequencing to observe the phenotypic diversity of SMCs participating in tumor vascularization. We find that a significant proportion of SMCs adopt a phenotype traditionally associated with macrophage-like cells. These cells are transcriptionally similar to resolution phase M2b macrophages, which have been described to have a role in inflammation resolution. Computationally predicted by the ligand-receptor algorithm CellChat, signaling from BST2 on the surface of tumor cells to PIRA2 on SMCs promote this phenotypic transition; in vitro SMC assays demonstrate upregulation of macrophage transcriptional programs, and increased proliferation, migration, and phagocytic ability when exposed to BST2. Knockdown of BST2 in the tumor significantly decreases the transition towards a macrophage-like phenotype, and cells that do transition have a comparatively higher inflammatory signal typically associated with anti-tumor effect. ConclusionAs BST2 is known to be a poor prognostic marker in multiple cancers where it is associated with an M2 macrophage-skewed TME, these studies suggest that phenotypically switched SMCs may have a previously unidentified role in this immunosuppressive milieu. Further translational work is needed to understand how this phenotypic switch could influence the response to anti-cancer agents and if targeted inhibition of SMC plasticity would be therapeutically beneficial.

cancer biology↗

bioRxiv2024

Integrated Single-Cell Multiomic Profiling of Caudate Nucleus Suggests Key Mechanisms in Alcohol Use Disorder

Alcohol use disorder (AUD) induces complex transcriptional and regulatory changes across multiple brain regions including the caudate nucleus, which remains understudied. Using paired single-nucleus RNA-seq and ATAC-seq on caudate samples from 143 human postmortem brains, including 74 with AUD, we identified 17 distinct cell types. We found that a significant portion of the alcohol-induced changes in gene expression occurred through altered chromatin accessibility. Notably, we identified novel transcriptional and chromatin accessibility differences in medium spiny neurons, impacting pathways such as RNA metabolism and immune response. A small cluster of D1/D2 hybrid neurons showed distinct differences, suggesting a unique role in AUD. Microglia exhibited distinct activation states deviating from classical M1/M2 designations, and astrocytes entered a reactive state partially regulated by JUND, affecting glutamatergic synapse pathways. Oligodendrocyte dysregulation, driven in part by OLIG2, was linked to demyelination and increased TGF-{beta}1 signaling from microglia and astrocytes. We also observed increased microglia-astrocyte communication via the IL-1{beta} pathway. Leveraging our multiomic data, we performed cell type-specific expression quantitative trait loci analysis, integrating that with public genome-wide association studies to identify AUD risk genes such as ADAL and PPP2R3C, providing a direct link between genetic variants, chromatin accessibility, and gene expression in AUD. These findings not only provide new insights into the genetic and cellular mechanisms in the caudate related to AUD but also demonstrate the broader utility of large-scale multiomic studies in uncovering complex gene regulation across diverse cell types, which has implications beyond the substance use field.

neuroscience↗

bioRxiv2024

A Tough Biointerface in Human Knee Empowered by Dynamic Phase-transforming Minerals in Collagenous Matrix

Joining heterogeneous materials in engineered structures remains a daunting challenge because of stress concentration, often resulting in unexpected failures1,2. Studying the structures in organisms that evolved for centuries provides valuable insights that can be instrumental in addressing this mechanical challenge3-5. The human meniscus root-bone interface is a remarkable example known for its exceptional fatigue resistance, toughness and interfacial adhesion properties throughout its lifespan6-8. We studied the multiscale graded mineralization structure designs within the 30-micron soft-hard interface at the root-bone junction and examined its toughening mechanisms. This graded interface with interdigitated structures and exponential modulus increase exhibits a phase transition from amorphous calcium phosphate (ACP) to gradually matured hydroxyapatite (HAP) crystals, mediated by location-specific distributed biomolecules. In coordination with collagen fibril deformation and reorientation, ACP particles debond with collagen and slide to new positions which enable frictional energy dissipation, and HAP particles arrest cracks. The mineral in transforming phases work synergistically to provide interfacial toughening. The presented biointerface model exemplifies human musculoskeletal systems adaptations to mechanical requirements, offering a blueprint for developing tough interfaces in broad applications.

bioengineering↗

bioRxiv2024

The connectional diaschisis and normalization of cortical language network dynamics after basal ganglia and thalamus stroke

Stroke affecting the basal ganglia and thalamus can lead to language deficits. In addition to the lesions direct impact on language processing, connectional diaschisis involving cortical-subcortical interactions also plays a critical role. This study investigated connectional diaschisis using the "dynamic meta-networking framework of language" in patients with basal ganglia and thalamus stroke, analyzing longitudinal resting-state fMRI data collected at 2 weeks (n = 32), 3 months (n = 19), and one year post-stroke (n = 23). As expected, we observed dynamic cortico-subcortical interactions between cortical language regions and subcortical regions in healthy controls (HC, n = 25). The cortical language network exhibited dynamic domain-segregation patterns in HCs, severely disrupted in the acute phase following stroke. The connectional diaschisis manifested as dual effects characterized by both hypo- and hyper-connectivity, which positively and negatively correlated with language deficits, respectively. State-specific changes in nodal and topological properties were also identified. Throughout language recovery, cortical language network dynamics gradually normalized toward sub-optimal domain-segregation patterns, accompanied by the normalization of nodal and topological properties. These findings underscore the crucial role of cortico-subcortical interactions in language processing.

neuroscience↗

bioRxiv2024

Pre-Assembly NGS Correction of ONT Reads Achieves HiFi-Level Assembly Quality

BackgroundRecently developed hybrid assemblies can achieve Telomere-to-Telomere (T2T) completeness of some chromosomes. However, such approaches involve sequencing a large volume of both Pacific Biosciences high-fidelity (HiFi) and Oxford Nanopore Technologies (ONT) sequencing reads. Along with this, third-generation sequencing techniques are rapidly advancing, increasing the available length and accuracy. To reduce the final cost of genome assembly, here we investigated the possibility of assembly from low-coverage samples and with only ONT corrected by Next-Generation Sequencing (NGS) sequencing reads. We demonstrated that ONT-based assembly approaches corrected by NGS can achieve performance metrics comparable to more expensive hybrid approaches based on HiFi sequencing. ResultsWe investigated the assembly of different chromosomes of HG002 and the low-coverage performance of state-of-the-art hybrid assembly tools, including Verkko and Hifiasm, and ONT-based assemblers Shasta and Flye. Our study shows that even with the best NG50, Hifiasm has many misassemblies in the centromere region, raising doubts about its T2T capabilities. Additionally, ONT-based assemblies perform well at low coverage, offering a cost-effective solution. To achieve better assembly quality, we evaluated the performance of different NGS technologies across various aspects of hybrid genome assembly, including pre-assembly correction, haplotype phasing, and polishing, and found them to be similarly effective. For the NGS technology stLFR, we proposed two-round assembly methods that utilize stLFR linked-read data to achieve assembly phasing performance comparable to that obtained with trio data. ConclusionWe found that assemblies based on ONT corrected by NGS can match or exceed HiFi-based assemblies in accuracy. It appears that no assembly tool investigated here can achieve T2T completeness, even with 50X coverage. Therefore, we recommend using a combination of regular R9 or R10 simplex ONT reads and accurate NGS reads for assembly without aiming for T2T completeness. These ONT-based assemblies perform well even at low coverage, offering a cost-effective solution.

genomics↗

bioRxiv2024

Pharmacological rescue of motor circuit dysfunction in a Drosophila model of paroxysmal dyskinesia

BackgroundParoxysmal dyskinesias (PxDs) are characterised by bouts of involuntary dystonic and choreiform movements. The patho-mechanisms underlying these debilitating disorders remain poorly understood, and drug treatments are often limited. We recently generated a Drosophila model of a PxD-linked mutation causing BK potassium channel gain- of-function (BK GOF), and showed that BK GOF perturbs movement in Drosophila by disrupting neurodevelopment. However, whether locomotor capacity in BK GOF flies can be pharmacologically restored following neurodevelopmental insults has remained unclear. ObjectiveTo identify pharmacological suppressors of motor defects caused by BK GOF. MethodsUsing adult BK GOF flies, we performed an unbiased, in vivo, locomotion-based screen of 370 FDA-approved drugs. To test the impact of positive hits from this screen on motor circuit activity, we used optical imaging to record the intrinsic rhythmic activity of Drosophila larval motor circuits driving peristalsis and turning behaviors. ResultsWe found that inhibitors of acetylcholinesterase - a protein that degrades acetylcholine in cholinergic synapses - partially rescued movement defects caused by BK GOF. Inhibition of acetylcholinesterase also partially restored intrinsic activity of motor circuits controlling forward movement and turning in BK GOF larvae. ConclusionsOur findings indicate that elevating cholinergic tone can reverse motor circuit dysfunction in an animal model of PxD caused by BK GOF. Furthermore, our study provides proof-of-principle that Drosophila can be utilised for screens to uncover putative drug treatments for involuntary movement disorders.

neuroscience↗