Biology subjects

Wu, D.

Publications and source records attributed to Wu, D..

At least 217 records · Page 12Linked to original sources

bioRxiv2022

Spatiotemporal atlas of the fetal brain depicts cortical developmental gradient in Chinese population

The fetal brains experience rapid and complex development in utero during the second and third trimesters. MRI atlas of the fetal brain in this period enables us to quantify the normal fetal brain development in the spatiotemporal domain. In this study, we constructed a high quality spatiotemporal atlas between 23-38 weeks gestational age (GA) from 90 healthy Chinese fetuses using a pairwise and groupwise registration-based pipeline. We quantified the fetal cortical morphology and characterized the developmental trajectories of each morphological index. The cortical thickness exhibited a biphasic growth pattern; the developmental trend of curvature fitted well into the Gompertz model; sulcal depth increased linearly while surface area expanded exponentially. The cortical thickness and curvature trajectories consistently pointed to a characteristic time-point during development around 31 weeks of GA. The characteristic GA and growth rate obtained from individual cortical regions suggested a central-to-peripheral developmental gradient, with the earliest development in the parietal lobe, and we also observed within-lobe regional orders, which may be linked to biophysical events such as dendritic arborization and thalamocortical fibers ingrowth. The proposed atlas was also compared with an existing fetal atlas from a Caucasian/mixed population. Finally, we examined the structural asymmetry of the fetal brains and found extensive asymmetry that dynamically changed with development. The current study depicted a comprehensive profile of fetal cortical development and the established atlas could be used as a normative reference for neurodevelopmental and diagnostic purposes, especially in the Chinese population.

bioengineering↗

bioRxiv2022

RNA exosome ribonuclease DIS3 degrades Pou6f1 to promote mouse pre-implantation cell differentiation

Mammalian development is precisely controlled by cell differentiation. Discovery of new regulators and investigating their crosstalk provide insights into genetic networks defining pre-implantation development. To identify novel developmental repressors, we established a knockout mouse model of Dis3, an RNA exosome associated RNase. Homozygous Dis3 null embryos arrest at the morula-to-blastocyst transition. Using single embryo RNA-seq, we discovered persistence of Pou6f1 mRNA in homozygous null Dis3 embryos and determined that the cognate protein represses transcription of Nanog and Cdx2. The resultant defects in cell differentiation disrupted the morula-to-blastocyst transition and was embryonic lethal. To overcome the paucity of embryos, homozygous Dis3 null mouse embryonic stem cells were derived to identify additional gene targets of POU6F1. While microinjection of the Dis3 cRNA into zygotes rescued the morula-to-blastocyst block, point mutations of Dis3 in individual blastomeres transformed the cell and prevented incorporation into embryos. Our findings uncover a not heretofore reported regulatory pathway of DIS3-POU6F1 in pre-implantation mammalian embryogenesis. In BriefMammalian pre-implantation development is regulated by master transcription factors and their crosstalk. Wu and Dean report that an RNA exosome associated RNase, DIS3, degrades Pou6f1 mRNA to de-repress transcription of Nanog and Cdx2 genes. In the absence of DIS3, POU6F1 protein persists and embryos arrest as morulae unable to become blastocysts due to lack of cell differentiation. HighlightsO_LIDis3 knockout mice have morula arrest due to lack of cell differentiation. C_LIO_LIDIS3 binds and degrades Pou6f1 mRNA before the morula stage. C_LIO_LIPOU6F1 globally occupies promoters to regulate gene transcription. C_LIO_LIDIS3 mutation results in cell transformation in embryonic development. C_LI

developmental biology↗

bioRxiv2022

Jointly analyzing the association of human milk nutrients with cognition and temperament traits during the first 6 months of life

Early dietary exposure via human milk (HM) components offers a window of opportunity to support cognitive and temperamental development. While several studies have focused on associations of few pre-selected HM components with cognition and temperament, it is highly plausible that HM components synergistically and jointly support cognitive and behavioral development in early life. We aimed to discern the combined associations of a wide array of HM nutrients with cognition and temperament during the first six months of life and explore if there were persistent effects up to 18 months old, when HM is the primary source of an infants nutrition. The Mullen Scales of Early Learning and Infant Behavior Questionnaires-Revised were used to assess cognition and temperament, respectively, of fifty-four exclusively/predominantly breastfed infants in the first 6 months of life, whose follow-ups were conducted at 6-9, 9-12 and 12-18 months old. HM samples were obtained from the mothers of the participants at less than 6 months of life and analyzed for fatty acids (total monounsaturated fatty acids, polyunsaturated fatty acid, total saturated fatty acid (TSFA), arachidonic acid (ARA), docosahexaenoic acid (DHA), ARA/DHA, omega-6/omega-3 polyunsaturated fatty acids ratio (n-6/n-3)), phospholipids (phosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylinositol (PI), sphingomyelin) and choline (free choline, phosphocholine (PCho), glycerophosphocholine). Feature selection was performed to select nutrients associated with cognition and temperament, respectively. The combined effects of selected nutrients were analyzed using multiple regression. A positive association between the arachidonic acid (ARA) and surgency was observed (p = 0.024). Significant effect of DHA, n-6/n-3, PE and TSFA concentrations on receptive language (R2 = 0.39, p = 0.025), and the elevated ARA, PCho, and PI with increased surgency (R2 = 0.43, p = 0.003) was identified, suggesting that DHA and ARA may have distinct roles for temperament and language functions. Furthermore, the exploratory association analyses suggest that the effects of HM nutrients on R.L. and surgency may persist beyond the first 6 months of life, particularly surgency at 12-18 months (p = 0.002). Our studies highlighted that various HM nutrients work together to support the development of cognition and temperament traits during early infancy.

developmental biology↗

bioRxiv2022

Dissecting the conformational complexity and flipping mechanism of bacterial heme transport.

Iron-bound cyclic tetrapyrroles (hemes) are key redox-active cofactors in membrane-integrated oxygen reductases and other bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used a combination of cellular, biochemical, structural and computational methods to resolve ongoing controversies around the function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for assembly and functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryo-EM approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degrees. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward-facing states. One Sentence SummaryThe heterodimeric bacterial ABC transporter CydDC is a heme flippase essential for the functional maturation of cytochrome bd.

microbiology↗

bioRxiv2022

Persistent pervasive transcription in RNA exosome depleted oocytes results in loss of female fertility

Maturing mammalian oocytes are transcriptionally inactive and attendant RNA degradation determines the maternal transcriptome for embryonic development1. Perturbing oocyte RNA degradation can result in failure of meiosis, fertilization, or zygotic gene activation1-5. We recently reported that conditional depletion of EXOSC10, an RNA exosome associated RNase, blocks oocyte growth-to-maturation transition by interfering with ribosomal RNA processing and meiotic checkpoint genes3. Here we have established oocyte-specific knockout mice of a second RNA exosome associated RNase, Dis3. Mutant females (Dis3cKO) exhibit significantly reduced fertility because oocytes arrest at early maturation. DIS3 depletion allows persistent pervasive transcription, which blocks transcription termination and sequesters RNA polymerase II in intergenic regions. In addition, Dis3cKO oocytes gain H3K27me3 at pre-defined loci6 due to insufficient demethylases KDM6A/B. Oocyte double knockout of Dis3 and Exosc10 causes much earlier growth defects for similar persistence of pervasive transcription, suggesting the RNA exosome complex plays a critical role to ensure transcriptome integrity during oocyte development.

developmental biology↗

bioRxiv2022

Combining GWAS and TWAS to identify candidate causal genes for tocochromanol levels in maize grain

Tocochromanols (tocopherols and tocotrienols, collectively vitamin E) are lipid-soluble antioxidants important for both plant fitness and human health. The main dietary sources of vitamin E are seed oils that often accumulate high levels of tocopherol isoforms with lower vitamin E activity. The tocochromanol biosynthetic pathway is conserved across plant species but an integrated view of the genes and mechanisms underlying natural variation of tocochromanol levels in seed of most cereal crops remains limited. To address this issue, we utilized the high mapping resolution of the maize Ames panel of [~]1,500 inbred lines scored with 12.2 million single-nucleotide polymorphisms to generate metabolomic (mature grain tocochromanols) and transcriptomic (developing grain) data sets for genetic mapping. By combining results from genome- and transcriptome-wide association studies, we identified a total of 13 candidate causal gene loci, including five that had not been previously associated with maize grain tocochromanols: four biosynthetic genes (arodeH2 paralog, dxs1, vte5, and vte7) and a plastid S-adenosyl methionine transporter (samt1). Expression quantitative trait locus (eQTL) mapping of these 13 gene loci revealed that they are predominantly regulated by cis-eQTL. Through a joint statistical analysis, we implicated cis-acting variants as responsible for co-localized eQTL and GWAS association signals. Our multi-omics approach provided increased statistical power and mapping resolution to enable a detailed characterization of the genetic and regulatory architecture underlying tocochromanol accumulation in maize grain and provided insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in maize and other cereals.

genetics↗

bioRxiv2022

Development of visual cortex in human neonates are selectively modified by postnatal experience

Experience-dependent cortical plasticity is a pivotal process of human brain development and essential for the formation of most cognitive functions. Although studies found that early visual experience could influence the endogenous development of visual cortex in animals, little is known about such impact on human infants. Using the multi-modal MRI data from developing human connectome project, we revealed the early structural and functional maps in the ventral visual cortex and their development across the first month of age. Particularly, we found the postnatal experience could modulate both the cortical morphology in ventral visual cortex and the functional circuit between bilateral primary visual cortices. But the cortical myelination and overall functional circuits of ventral cortex, particularly that of the high-order visual cortex, developed without significant influence of postnatal experience in such early period. These experience-dependent cortical properties were further validated in the preterm-born infants who have longer postnatal time but immature cortical development at birth. The results confirmed that the development of cortical thickness was dominated by the postnatal experience but the functional circuit might be determined by the overall maturity of visual cortex. These data suggest in human newborns that early postnatal experience shapes the structural and functional development of the visual cortex in selective and organized pattern.

neuroscience↗

bioRxiv2022

Structure and mechanism of a mitochondrial AAA+ disaggregase CLBP

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space and functions to promote the solubilization of various mitochondrial proteins. CLPB deficiency by mutations is associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. The mechanism of CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo- tetradecamers in apo-state and is remodeled into homo-dodecamers upon binding to substrates. Conserved pore- loops on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of two amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

molecular biology↗

bioRxiv2022

Deciphering the developmental patterns of white matter tracts in a diffusion MRI based fetal brain atlas

White matter of the fetal brain undergoes rapid development to form the early structural connections. Diffusion MRI has shown to be a useful tool to depict the fetal brain white matter in utero, and many studies have observed increasing fractional anisotropy and decreasing mean diffusivity in the fetal brain during the second-to-third trimester, whereas others reported non-monotonic changes. Unbiased diffusion MRI atlases of the fetal brain are important for characterizing developmental trajectories of white matter and providing normative references for in-utero diagnosis of prenatal abnormalities. To date, the sole fetal brain diffusion MRI atlas was collected from a Caucasian/mixed population, and was constructed based on the diffusion tensor model with limited spatial resolution. In this work, we proposed a fiber orientation distribution (FOD) based pipeline for the generation of fetal brain diffusion MRI atlases, which showed better registration accuracy than diffusion tensor-based pipeline. Based on the FOD pipeline, we constructed the first Chinese fetal brain diffusion MRI atlas using 89 normal fetal diffusion MRI scans at GA between 24 and 38 weeks. Complex non-monotonic trends of tensor- and FOD-derived microstructural parameters in eight white matter tracts were observed, which jointly pointed to different phases of microstructural development. Specifically, we speculated that the turning point of the diffusivity trajectory may correspond to the starting point of pre-myelination, based on which, the developmental order of white matter tracts can be mapped and the order was in agreement with order of myelination from histological studies. The normative atlas also provided a reference for detection of abnormal white matter development, such as that in congenital heart disease. Therefore, the high-order fetal brain diffusion MRI atlas established in this study depicted the spatiotemporal pattern of early white matter development, and findings may help decipher the distinct microstructural events in utero.

neuroscience↗

bioRxiv2022

Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by 3-4 different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced co-assembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

biophysics↗

bioRxiv2022

Generative pretraining from large-scale transcriptomes: Implications for single-cell deciphering and clinical translation

Exponential accumulation of single-cell transcriptomes poses great challenge for efficient assimilation. Here, we present an approach entitled tGPT towards integration of 22.3 million single-cell transcriptomes by modeling gene expression rankings as generative pretraining task. tGPT is conceptually simple in that it autoregressively models the ranking of a gene in the context of its preceding neighbors. We demonstrated the high performance of tGPT on a range of fundamental single-cell analysis tasks and novel applications on bulk tissues. The single-cell clusters and cell lineage trajectories derived from tGPT are highly aligned with known cell labels and states. The feature patterns of tumor bulk tissues learned by tGPT are associated with a wide range of genomic alteration events, prognosis and treatment outcome of immunotherapy. tGPT represents a new analytical paradigm for integrating and deciphering massive amount of transcriptome data and it will facilitate the interpretation and clinical translation of single-cell transcriptomes.

bioinformatics↗

bioRxiv2022

In vivo correction of cystic fibrosis mediated by PNA nanoparticles

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. This is the first report of systemic gene editing for CF. Our data suggest that systemic delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate the disease in multiple affected organs.

bioengineering↗

bioRxiv2022

Spatially resolved single-cell deconvolution of bulk RNA-seq using Bulk2Space

Uncovering the tissue molecular architecture at single-cell resolution could help better understand organisms biological and pathological processes. However, bulk RNA-seq can only measure gene expression in cell mixtures, without revealing the transcriptional heterogeneity and spatial patterns of single cells. Herein, we introduce Bulk2Space (https://github.com/ZJUFanLab/bulk2space), a deep learning framework-based spatial deconvolution algorithm that can simultaneously disclose the spatial and cellular heterogeneity of bulk RNA-seq data using existing single-cell and spatial transcriptomics references. The use of bulk transcriptomics to validate Bulk2Space unveils, in particular, the spatial variance of immune cells in different tumor regions, the molecular and spatial heterogeneity of tissues during inflammation-induced tumorigenesis, and spatial patterns of novel genes in different cell types. Moreover, Bulk2Space is utilized to perform spatial deconvolution analysis on bulk transcriptome data from two different mouse brain regions derived from our in-house developed sequencing approach termed Spatial-seq. We have not only reconstructed the hierarchical structure of the mouse isocortex but also further annotated cell types that were not identified by original methods in the mouse hypothalamus.

bioinformatics↗

bioRxiv2022

Conserved Untranslated Regions of Multipartite Viruses: Natural Markers of Novel Viral Genomic Components and Tags of Viral Evolution

Viruses with split genomes are categorized as being either segmented or multipartite according to whether their genomic segments occur in single or different virions. Some complexity will exist, in that inherited "core" vital segments viruses may renew the others once host and environmental alterations keep driving viral evolution. Despite this uncertainty, empirical observations have been made across the split genomes in the untranslated regions (UTRs) on the short or long stretches of conserved or identical sequences. In this study, we describe a methodology that combines RNA and small RNA sequencing, conventional BLASTx, and iterative BLASTn of UTRs to detect viral genomic components even if they encode orphan genes (ORFans). Within the phylum Kitrinoviricota, novel putative multipartite viruses and viral genomic components were annotated using data obtained from our sampling or publicly available sources. The novel viruses, as extensions or intermediate nodes, enriched the information of the evolutionary networks. Furthermore, the diversity of novel genomic components emphasized the evolutionary roles of reassortment and recombination, as well as genetic deletion, strongly supporting the genomic complexity. These data also suggest insufficient knowledge of these genomic components for categorizing some extant viral taxa. The relative conservation of UTRs at the genome level may explain the relationships between monopartite and multipartite viruses and how the multipartite viruses can have a life strategy involving multiple host cells. Author summaryThe current workflows for virus identification are largely based on high-throughput sequencing and coupled protein sequence homology-dependent analysis methods and tools. However, for viruses with split genomes, the identification of genomic components whose deduced protein sequences are not homologous to known sequences is inadequate. Furthermore, many plant-infecting multipartite viruses contain conserved UTRs across their genomic components. Based on this, we propose a practical method of UTR-backed iterative BLASTn (UTR-iBLASTn) to explore the components with ORFans and study virus evolution using the UTRs as signals. These shed light on viral "dark matter"--unknown/omitted genomic components of segmented/multipartite viruses from different kingdoms and hosts, and the origins of these components.

microbiology↗

bioRxiv2022

Horizontal Transfers Lead to the Birth of Momilactone Biosynthetic Gene Clusters in Grass

Momilactone A, an important plant labdane-related diterpenoid, functions as a phytoalexin against pathogens and an allelochemical against neighboring plants. The genes involved in biosynthesis of momilactone A are found in clusters, i.e., MABGCs (Momilactone A biosynthetic gene clusters), in the rice and barnyardgrass genomes. How MABGCs originate and evolve is still not clear. Here, we integrated results from comprehensive phylogeny and comparative genomic analyses of the core genes of MABGC-like clusters and MABGCs in 40 monocot plant genomes, providing convincing evidence for the birth and evolution of MABGCs in grass species. The MABGCs found in the PACMAD clade of the core grass lineage (including Panicoideae and Chloridoideae) originated from a MABGC-like cluster in Triticeae (BOP clade) via horizontal gene transfer (HGT) and followed by recruitment of MAS and CYP76L1 genes. The MABGCs in Oryzoideae originated from PACMAD through another HGT event and lost CYP76L1 afterwards. The Oryza MABGC and another Oryza diterpenoid cluster c2BGC are two distinct clusters, with the latter being originated from gene duplication and relocation within Oryzoideae. Further comparison of the expression patterns of the MABGC genes between rice and barnyardgrass in response to pathogen infection and allelopathy provides novel insights into the functional innovation of MABGCs in plants. Our results demonstrate HGT-mediated origination of MABGCs in grass and shed lights into the evolutionary innovation and optimization of plant biosynthetic pathways.

evolutionary biology↗

bioRxiv2021

Transcriptomic Profiling Reveals Cancer-Associated Fibroblasts as Potential Targets for the Prognosis and Treatment of Cervical Squamous Cell Carcinoma

Effective treatment of advanced invasive cervical cancer remains challenging nowadays. Herein, single-nucleus RNA sequencing (snRNA-seq) and SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq) technology are used to investigate the immunological microenvironment of cervical squamous cell carcinoma (CSCC), a major type of cervical cancers. The expression levels of most immune checkpoint genes in tumor and inflammation areas of CSCC were not significantly higher than those in the non-cancer samples except for LGALS9 and IDO1. Stronger signals of CD56+ NK cells and immature dendritic cells are found in the hypermetabolic tumor areas, while more eosinophils, immature B cells, and Treg cells are found in the hypometabolic tumor areas. Moreover, a cluster of cancer-associated fibroblasts (CAFs) are identified around some tumors, which highly expressed ACTA2, POSTN, ITGB4, and FAP. The CAFs might support the growth and metastasis of tumors by inhibiting lymphocyte infiltration and remodeling the tumor extracellular matrix. Furthermore, CAFs are associated with poorer survival probability in CSCC patients and might be present in a small fraction ([~]20%) of advanced cancer patients. Collectively, these findings might enhance understanding of the CSCC immunological microenvironment and shed some light on the treatment of advanced CSCC.

cancer biology↗

bioRxiv2021

Horizontal Transfer and Evolution of the Biosynthetic Gene Cluster for Benzoxazinoid

Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites, first identified in maize (Zea mays) and subsequently shown to be encoded by a biosynthetic gene cluster (BGC), the Bx cluster. Data mining of mining 40 high-quality grass genomes identified complete Bx clusters (containing genes Bx1 to Bx5 and Bx8) in three genera (Zea, Echinochloa and Dichanthelium) in the Panicoideae and partial clusters in the Triticeae. The Bx cluster originated from gene duplication of native analogues of Bx genes and chromosomal translocation. An ancient Bx cluster including additional Bx genes (e.g., Bx6) is found in ancestral Panicoideae. The ancient Bx cluster was gained by the Triticeae ancestor via a horizontal transfer (HT) event from the ancestral Panicoideae and later separated into three parts on different chromosomes. Bx6 appears to have been under less constrained selection during evolution of the Panicoideae as evidenced by the fact that was translocated [~]1.31-Mb away from the Bx cluster in Z. mays, moved to other chromosomes in Echinochloa, and even lost in Dichanthelium. Further investigation indicated that intense selection and polyploidization shaped the evolutionary trajectory of the Bx cluster in the grass family. This study provides the first case of HT of BGCs among plants and sheds new insights on the evolution of BGCs. SignificanceBiosynthetic gene clustering and horizontal gene transfer are two evolutionary inventions for rapid adaption by organisms. Horizontal transfer of a gene cluster has been reported in fungi and bacteria, but not in plants up to now. By mining the genomes of 40 monocot species, we deciphered the organization of Bx gene cluster, a biosynthetic gene cluster for benzoxazinoids in grasses. We found that the Bx cluster was formed by gene duplication of native analogues of individual Bx genes and directional translocation. More importantly, the Bx cluster in Triticeae was inherited from the Panicoideae via horizontal transfer. Compared with the native analogues, Bx clusters in grasses show constrained purifying selection underscoring their significance in environmental adaption.

evolutionary biology↗

bioRxiv2021

Directed Evolution of Aptamer Discovery Technologies

ConspectusAlthough antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of "non-nattural" aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

bioengineering↗