Biology subjects

Song, L.

Publications and source records attributed to Song, L..

At least 127 records · Page 7Linked to original sources

bioRxiv2023

A novel cell division protein critical for the assembly of the bacterial divisome

Identification of unique essential bacterial genes is important for not only the understanding of their cell biology but also the development of new antimicrobials. Here we report a previously unrecognized core component of the Acinetobacter baumannii divisome. Our results reveal that the protein, termed Aeg1 interacts with multiple cell division proteins, including FtsN, which is required for components of the divisome to localize to the midcell. We demonstrate that the FtsAE202K and FtsBE65A mutants effectively bypassed the need of Aeg1 by A. baumannii, as did the activation variants FtsWM254I and FtsWS274G. Our results suggest that Aeg1 is a cell division protein that arrives at the division site to initiate cell division by recruiting FtsN, which activates FtsQLB and FtsA to induce the septal peptidoglycan synthase FtsWI. The discovery of the new essential cell division protein has provided a new target for the development of antibacterial agents.

biochemistry↗

bioRxiv2023

Protein multiple conformations prediction using multi-objective evolution algorithm

MotivationWith the breakthrough of AlphaFold2 and the publication of AlphaFold DB, the protein structure prediction has made remarkable progress, which may further promote many potential applications of proteomics in all areas of life. However, it should be noted that AlphaFold2 models tend to represent only a single static structure, and accurately predicting multiple conformations remains a challenge. Therefore, it is essential to develop methods for predicting multiple conformations, which enable us to gain knowledge of multiple conformational states and the broader conformational landscape to better understand the mechanism of action. ResultsIn this work, we proposed a multiple conformational states folding method using the distance-based multi-objective evolutionary algorithm framework, named MultiSFold. First, a multi-objective energy landscape with multiple competing constraints generated by deep learning is constructed. Then, an iterative modal exploration and exploitation strategy based on multi-objective optimization, geometric optimization and structural similarity clustering is designed to perform conformational sampling. Finally, the final population is generated using a loop-specific perturbation strategy to adjust the spatial orientations. MultiSFold was compared with state-of-the-art methods on a developed benchmark testset containing 81 proteins with two representative conformational states. Based on the proposed metric, the success ratio of MultiSFold predicting multiple conformations was 70.4% while that of AlphaFold2 was 9.88%, which may indicate that conformational sampling combined with knowledge gained through deep learning has the potential to produce conformations spanned the range between two experimental structures. In addition, MultiSFold was tested on 244 human proteins with low structural accuracy in AlphaFold DB to test whether it could further improve the accuracy of static structures. The experimental results demonstrate that the TM-score of MultiSFold is 2.97% and 7.72% higher than that of AlphaFold2 and RoseTTAFold, respectively, supporting our hypothesis that multiple competing optimization objectives can further assist conformational search to improve prediction accuracy.

bioinformatics↗

bioRxiv2023

xTrimoGene: An Efficient and Scalable Representation Learner for Single-Cell RNA-Seq Data

AO_SCPLOWBSTRACTC_SCPLOWThe advances in high-throughput sequencing technology have led to significant progress in measuring gene expressions in single-cell level. The amount of publicly available single-cell RNA-seq (scRNA-seq) data is already surpassing 50M records for human with each record measuring 20,000 genes. This highlights the need for unsupervised representation learning to fully ingest these data, yet classical transformer architectures are prohibitive to train on such data in terms of both computation and memory. To address this challenge, we propose a novel asymmetric encoder-decoder transformer for scRNA-seq data, called xTrimoGene, which leverages the sparse characteristic of the data to scale up the pre-training. This scalable design of xTrimoGene reduces FLOPs by one to two orders of magnitude compared to classical transformers while maintaining high accuracy, enabling us to train the largest transformer models over the largest scRNA-seq dataset today. Our experiments also show that the performance of xTrimoGene improves as we increase the model sizes, and it also leads to SOTA performance over various downstream tasks, such as cell classification, perturb-seq effect prediction, and drug combination prediction.

bioinformatics↗

bioRxiv2023

Genomic analysis of cultivated infant microbiomes identifies Bifidobacterium 2'-fucosyllactose utilization can be facilitated by co-existing species

Human milk oligosaccharides (HMOs) ensure proper infant gut microbiome establishment. Isolate studies have revealed the genetic basis for HMO metabolism, but they exclude the possibility of HMO assimilation via synergistic interactions involving multiple organisms. Here, we investigated microbiome responses to 2-fucosyllactose (2FL), a prevalent HMO and infant formula additive, by establishing individualized microbiomes using fecal samples from three different infants as the inocula. Bifidobacterium breve, a prominent member of infant microbiomes, typically cannot metabolize 2FL. Using metagenomic data, we predicted that extracellular fucosidases encoded by co-existing members such as Ruminococcus gnavus initiate 2FL breakdown, thus critical for B. breves growth. Using both targeted co-cultures and by supplementation of R. gnavus into one microbiome, we show that R. gnavus can promote extensive growth of B. breve through the release of lactose from 2FL. Overall, microbiome cultivation combined with genome-resolved metagenomics demonstrated that HMO utilization can vary with an individuals microbiome.

microbiology↗

bioRxiv2023

Transcription Factor Dynamics in Cross-Regulation of Plant Hormone Signaling Pathways

Cross-regulation between hormone signaling pathways is indispensable for plant growth and development. However, the molecular mechanisms by which multiple hormones interact and co-ordinate activity need to be understood. Here, we generated a cross-regulation network explaining how hormone signals are integrated from multiple pathways in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. To do so we comprehensively characterized transcription factor activity during plant hormone responses and reconstructed dynamic transcriptional regulatory models for six hormones; abscisic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and strigolactone/karrikin. These models incorporated target data for hundreds of transcription factors and thousands of protein-protein interactions. Each hormone recruited different combinations of transcription factors, a subset of which were shared between hormones. Hub target genes existed within hormone transcriptional networks, exhibiting transcription factor activity themselves. In addition, a group of MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) were identified as potential key points of cross-regulation between multiple hormones. Accordingly, the loss of function of one of these (MPK6) disrupted the global proteome, phosphoproteome and transcriptome during hormone responses. Lastly, we determined that all hormones drive substantial alternative splicing that has distinct effects on the transcriptome compared with differential gene expression, acting in early hormone responses. These results provide a comprehensive understanding of the common features of plant transcriptional regulatory pathways and how cross-regulation between hormones acts upon gene expression.

plant biology↗

bioRxiv2023

Defective mechanosensory transduction of the new inner hair cells prevents hearing recover in the damaged cochlea

ABSTRACTHearing loss is a major health problem worldwide. Numerous attempts at regenerating functional hair cells (HCs) have been unsuccessful, but little is known about the main barrier that prevents us from achieving it and improving the hearing ability after damage. Here, we developed an in vivo genetic mouse model, by which the inner HCs (IHCs), the primary sound receptors innervated by the auditory neurons, were specifically damaged and the neighboring nonsensory supporting cells (SCs) were transformed into IHCs by ectopic expression of transient Atoh1 and permanent Tbx2. Despite [~]477 new IHCs were regenerated per cochlea and their differentiation status was more advanced than reported previously, no significant hearing improvement was achieved. By taking advantage of this unique model, we further found that the new IHCs expressed the functional marker vGlut3, harbored the similar transcriptomic profiles and electrophysiological properties as the endogenous IHCs. However, the mechanosensory transduction (MET) current could not be recorded in the new IHCs. Thus, our study indicated that the defective MET should be the main barrier that stops us from restoring the hearing capacity in the damaged cochlea and would pave the way for regenerating IHCs in vivo.

neuroscience↗

bioRxiv2023

xTrimoDock: Rigid Protein Docking via Cross-Modal Representation Learning and Spectral Algorithm

Protein-protein interactions are the basis for the formation of protein complexes which are essential for almost all cellular processes. Knowledge of the structures of protein complexes is of major importance for understanding the biological function of these protein-protein interactions and designing protein drugs. Here we address the problem of rigid protein docking which assumes no deformation of the involved proteins during interactions. We develop a method called, xTrimoDock, which leverages a cross-modal representation learning to predict the protein distance map, and then uses a spectral initialization and gradient descent to obtain the roto-translation transformation for docking. We show that, on antibody heavy-chain and light-chain docking, and antibody-antigen docking, xTrimoDock consistently outperforms the state-of-the-art such as AlphaFold-Multimer and HDock, and can lead to as much as a 10% improvement in DockQ metric. xTrimoDock has been applied as a useful tool in protein drug design at BioMap.

bioinformatics↗

bioRxiv2022

Broader functions of TIR domains in Arabidopsis immunity

TIR domains are NAD-degrading enzymes that function during immune signaling in prokaryotes, plants, and animals. In plants, most TIR domains are incorporated into intracellular immune receptors. In Arabidopsis, TIR-derived small molecules bind and activate EDS1 heterodimers, which in turn activate RNLs, a class of cation channel-forming immune receptors. RNL activation drives cytoplasmic Ca2+ influx, transcriptional reprogramming, pathogen resistance and host cell death. We screened for mutants that suppress an RNL activation mimic allele and identified a TIR-containing immune receptor, SADR1. Despite functioning downstream of an auto-activated RNL, SADR1 is not required for defense signaling triggered by other tested TIR-containing immune receptors. SADR1 is required for defense signaling initiated by some trans-membrane pattern recognition receptors and contributes to the unbridled spread of cell death in lesion simulating disease 1. Together with RNLs, SADR1 regulates defense gene expression at infection site borders, likely in a non-autonomous manner. RNL mutants that cannot sustain this pattern of gene expression are unable to prevent disease spread beyond localized infection sites, suggesting that this pattern corresponds to a pathogen containment mechanism. SADR1 potentiates RNL-driven immune signaling partially through the activation of EDS1, but also partially independently of EDS1. We studied EDS1-independent TIR function using nicotinamide, an NADase inhibitor. We observed decreased defense induction from trans-membrane pattern recognition receptors and decreased calcium influx, pathogen growth restriction and host cell death following intracellular immune receptor activation. We demonstrate that TIR domains can potentiate calcium influx and defense and are thus broadly required for Arabidopsis immunity.

plant biology↗

bioRxiv2022

Using Haplotype and QTL Analysis to Fix Favorable Alleles in Diploid Potato Breeding

At present, the potato of international commerce is autotetraploid, and the complexity of this genetic system creates limitations for breeding. Diploid potato breeding has long been used for population improvement, and thanks to improved understanding of the genetics of gametophytic self-incompatibility, there is now sustained interest in the development of uniform F1 hybrid varieties based on inbred parents. We report here on the use of haplotype and QTL analysis in a modified backcrossing (BC) scheme, using primary dihaploids of S.tuberosum as the recurrent parental background. In Cycle 1 we selected XD3-36, a self-fertile F2 clone homozygous for the self-compatibility gene Sli. Signatures of gametic and zygotic selection were observed at multiple loci in the F2 generation, including Sli. In the BC1 cycle, an F1 population derived from XD3-36 showed a bimodal response for vine maturity, which led to the identification of late vs. early alleles in XD3-36 for the gene StCDF1 (Cycling DOF Factor 1). Greenhouse phenotypes and haplotype analysis were used to select a vigorous and self-fertile F2 individual with 43% homozygosity, including for Sli and the early-maturing allele StCDF1.3. Partially inbred lines from the BC1 and BC2 cycles have been used to initiate new cycles of selection, with the goal of reaching higher homozygosity while maintaining plant vigor, fertility, and yield. Core IdeasO_LIPartially inbred, diploid potato lines were developed for transitioning to an inbred-hybrid breeding system. C_LIO_LIMulti-generational linkage analysis was used to track and fix favorable alleles without haplotype-specific markers. C_LIO_LISignatures of gametic and zygotic selection were detected by maximum likelihood. C_LI

genetics↗

bioRxiv2022

ALiCE: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as ALiCE(R), engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 L microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer(R) CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

biochemistry↗

bioRxiv2022

Chromosome-level genome assembly of Torreya grandis provides insights into the origin and evolution of gymnosperm-specific sciadonic acid biosynthesis

Species in genus Torreya are nut trees that produce dry fruits with a wide assortment of functions. Here, we report the 19-Gb chromosome-level genome assembly of T. grandis. The genome is shaped by an ancient whole genome duplication and recurrent LTR retrotransposon bursts. Comparative genomic analyses reveal key genes involved in reproductive organ development, cell wall biosynthesis and seed storage. Two genes encoding a C18 {Delta}9-elongase and a C20 {Delta}5-desaturase are identified in T. grandis to be responsible for sciadonic acid biosynthesis and both are present in diverse plant lineages except angiosperms. We demonstrate that the histidine-rich boxes of the {Delta}5-desaturase are crucial for its catalytic activity. Methylome analysis reveals that methylation valleys of the T. grandis seed genome harbor genes associated with important seed activities, including cell wall and lipid biosynthesis. Moreover, seed development is accompanied by DNA methylation changes that possibly fuel energy production. This study provides important genomic resource for gymnosperms and unravels key enzymes for biosynthesis of sciadonic acid as a hallmark metabolite of gymnosperms.

plant biology↗

bioRxiv2022

T1K: efficient and accurate KIR and HLA genotyping with next-generation sequencing data

Killer immunoglobulin-like receptor (KIR) genes and human leukocyte antigen (HLA) genes are highly polymorphic in a population and play important roles in innate and adaptive immunity. We have developed a novel computational method T1K that can efficiently and accurately infer the KIR or HLA alleles from next-generation sequencing data. T1K is flexible and is compatible with various sequencing platforms including RNA-seq and genomic sequencing data. We applied T1K on CD8+ T cell single-cell RNA-seq data, and identified that KIR2DL4 allele expression levels were enriched in tumor-specific CD8+ T cells.

bioinformatics↗

bioRxiv2022

Quartet protein reference materials and datasets for multi-platform assessment of label-free proteomics

Quantitative proteomics is an indispensable tool in life science research. However, there is a lack of reference materials for evaluating the reproducibility of label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based measurements among different instruments and laboratories. We developed the Quartet as a proteome reference material with built-in truths, and distributed the same aliquots to 15 laboratories with nine conventional LC-MS/MS platforms across six cities in China. Relative abundance of over 12,000 proteins on 816 MS files were obtained and compared for reproducibility among the instruments and laboratories to ultimately generate proteomics benchmark datasets. There was a wide dynamic range of proteomes spanning ~7 orders of magnitude (101-108 copies/cell), and the injection order had marked effects on quantitative instead of qualitative. Overall, the Quartet offers valuable standard materials and data resources for improving the quality control of proteomic analyses as well as the reproducibility and reliability of research findings.

molecular biology↗

bioRxiv2022

Plant and prokaryotic TIR domains generate distinct cyclic ADPR NADase products

Toll/interleukin-1 receptor (TIR) domain proteins function in cell death and immunity. In plants and bacteria, TIR domains are enzymes that produce isomers of cyclic ADPR (cADPR) as putative immune signaling molecules. The identity and functional conservation of cADPR isomer signals is unclear. A previous report found that a plant TIR could cross-activate the prokaryotic Thoeris TIR-immune system, suggesting the conservation of plant and prokaryotic TIR-immune signals. Here, we generate auto-active Thoeris TIRs and test the converse hypothesis: do prokaryotic Thoeris TIRs also cross-activate plant TIR-immunity? Using in planta and in vitro assays, we find that Thoeris and plant TIRs generate overlapping sets of cADPR isomers, and further clarify how plant and Thoeris TIRs activate the Thoeris system via producing 3cADPR. This study demonstrates that the TIR-signaling requirements for plant and prokaryotic immune systems are distinct and that TIRs across kingdoms generate a diversity of small molecule products.

plant biology↗

bioRxiv2022

Crossmodal reorganization in deaf auditory cortices compensates for the impaired body-centered visuomotor transformation after early deafness

Early deafness leads to the reorganization of large-scale brain networks, involving and extending beyond the auditory system. Body-centered visuomotor transformation is impaired after early auditory deprivation, associated with a hyper-crosstalk between the task-critical frontoparietal network (FPN) and the default-mode network (DMN). It remains to be elucidated, how the reorganized functional connectivity between the auditory system, the FPN, and the DMN contributes to the impaired visuomotor transformation after early deafness. In this fMRI study, we asked early deaf participants and hearing controls to judge the spatial location of a visual target, either about the middle-sagittal line of their own body (the egocentric task) or another background object (the allocentric task). The bilateral superior temporal gyrus (STG) in the deaf group exhibited cross-modal reorganization, with generally enhanced neural activity during the visual tasks, compared to hearing controls. Moreover, the STG showed significantly increased functional connectivity with both the FPN and the DMN in the deaf group compared to hearing controls, specifically during the egocentric task. The increased STG-FPN and STG-DMN coupling, however, showed antagonistic effects on the egocentric performance of the deaf participants. The increased STG-FPN connectivity was associated with improved (i.e., a beneficial role) while the increased STG-DMN with deteriorated (i.e., a detrimental role) egocentric performance in the deaf participants. No such effect was observed in hearing controls. Therefore, the auditory cortex is reorganized to functionally resemble the FPN in the deaf brain, representing compensatory neuroplasticity to mitigate the impaired visuomotor transformation after early deafness. Significance StatementOur brain constantly plans vision-guided actions, transforming visuospatial representations of external visual targets into visuomotor representations. The frontoparietal network (FPN) critically supports this visuomotor transformation process, which is impaired after early deafness. To mitigate the impaired visuomotor transformation, the deaf auditory cortex in the bilateral superior temporal gyrus (STG) shows compensatory cross-modal reorganization that functionally resembles the FPN regions. Specifically, the deaf auditory cortex becomes functionally coupled with the dorsal FPN regions. The stronger the STG-FPN coupling, the more improved the deaf adults visuomotor transformation performance, indicating the reorganized STG as a critical node of the task-critical network. Correspondingly, increased coupling between the task-critical deaf STG and the default-mode network impairs the visuomotor transformation.

neuroscience↗

bioRxiv2022

AAV Vector Transduction Restriction and Attenuated Toxicity in hESCs via a Rationally Designed Inverted Terminal Repeat

Recombinant adeno-associated virus (rAAV) inverted terminal repeats (ITRs) induce p53-dependent apoptosis in human embryonic stem cells (hESCs). To interrogate this phenomenon, a rationally designed ITR (SynITR), deleted for p53 binding sites was evaluated for vector production and gene delivery. While SynITR genomes were decreased for transgenic genome replication compared to wtITRs, similar production titers indicated that replication is not rate-limiting. Packaged in the AAV2 capsid, wtITR and SynITR vectors demonstrated similar transduction efficiencies of human cell lines with no differences in reporter kinetics. Following rAAV2-wtITR infection of hESCs, rapid apoptosis was observed, in contrast, rAAV2-SynITR infection resulted in attenuated hESC toxicity with cells retaining their differentiation potential. While hESC particle entry and double stranded circular episomes was similar for the ITR contexts, reporter expression was significantly inhibited from transduced SynITR genomes. Infection of hESCs induced {gamma}H2AX in an ITR-independent manner, however, canonical activation of p53 was uncoupled using rAAV-SynITR. Further hESC investigations revealed 2 additional novel findings: i) p53{beta} is uniquely and constitutively active, and ii) rAAV infection, independent of the ITR sequence, induces activation of p53{psi}. The data herein reveal an ITR-dependent rAAV transduction restriction specific to hESCs and manipulation of the DNA damage response via ITR engineering.

molecular biology↗

bioRxiv2022

In vivo base editing rescues photoreceptors in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of retinal diseases that cause the progressive death of retinal photoreceptor cells and eventually blindness. Mutations in the {beta}-domain of the phosphodiesterase 6 (Pde6b) gene are among the most identified causes of autosomal recessive RP. Here, we report a base editing approach in which adeno-associated virus (AAV)-mediated adenine base editor (ABE) delivery to postmitotic photoreceptors is used to correct the Pde6b mutation in a retinal degeneration 10 (rd10) mouse model of RP. Subretinal delivery of AAV8-ABE corrects Pde6b mutation with up to 37.41% efficiency at the DNA level and up to 91.95% efficiency at the cDNA level, restores PDE6B expression, preserves photoreceptors and rescues visual function. RNA-seq reveals upregulation of genes associated with phototransduction and photoreceptor survival. Our data demonstrate that base editing is a potential gene therapy that could provide durable protection against RP.

genetics↗

bioRxiv2022

Multiplex CRISPR strategy targeting viral genome for agriculture and clinical use

African Swine Fever (ASF) is a viral disease with exceptionally high lethality in domestic pigs and wild boar worldwide1, 2, without any effective vaccine or drug to prevent its spread. In this study, we established a multiplexable CRISPR-Cas-gRNA system targeting 13 genomic loci in the ASF virus genome that could prevent viral replication by cutting its genome. Furthermore, we generated pig strains expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing and demonstrated that the gene-edited pigs were more resistant to ASFV infection and less likely to spread the virus upon infection. As far as we know, our study presents the first living organism generated via germline editing to demonstrate resistance to viral infection via CRISPR-Cas. We anticipate our work to be helpful for both agricultural and biomedical applications, such as xenotransplantation.

bioengineering↗