Biology subjects

Ren, C.

Publications and source records attributed to Ren, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Label free, capillary-scale blood flow mapping in vivo reveals that low intensity focused ultrasound evokes persistent dilation in cortical microvasculature

Non-invasive, low intensity focused ultrasound (FUS) is an emerging neuromodulation technique that offers the potential for precision, personalized therapy. An increasing body of research has identified mechanosensitive ion channels that can be modulated by FUS and support acute electrical activity in neurons. However, neuromodulatory effects that persist from hours to days have also been reported. The brains ability to provide targeted blood flow to electrically active regions involve a multitude of non-neuronal cell types and signaling pathways in the cerebral vasculature; an open question is whether persistent effects can be attributed, at least partly, to vascular mechanisms. Using a novel in vivo optical approach, we found that microvascular responses, unlike larger vessels which prior investigations have explored, exhibit persistent dilation following sonication without the use of microbubbles. This finding and approach offers a heretofore unseen aspect of the effects of FUS in vivo and indicate that concurrent changes in neurovascular function may partially underly persistent neuromodulatory effects.

neuroscience↗

bioRxiv2023

DNA sensor ABCF1 phase separates with cccDNA to inhibit HBV replication

Objectives: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) contributes to viral persistence and recurrence. However, it is still less known how the host immune system responds to cccDNA and suppresses HBV replication. Therefore, it is essential to explore host proteins that interact with cccDNA and efficiently suppress HBV replication. Design: The interaction of cccDNA and ABCF1 was assessed by ChIP and pull-down assay. HBV replication was assessed in different cell models, including cccDNA surrogate-transfected and HBV-infected hepatocytes models. Phase separation of recombinant ABCF1 fusion proteins with cccDNA was observed by fluorescence microscopy. Results: Our data found that ABCF1 interacts with cccDNA to form phase-separated condensates via the poly-glutamine (PolyQ) of N-terminal intrinsically disordered low-complexity domain (LCD). Subsequently, ABCF1-cccDNA phase separation not only activates the type I/III interferon (IFN-I/III) pathway but also prevents Pol II accumulation on cccDNA to inhibit HBV transcription. Conclusions: Taken together, our study showed that ABCF1 acts as an antiviral restriction factor of HBV cccDNA by phase-separation-driven innate immune signaling and transcription inhibition. These findings shed new light on the understanding of host defense against cccDNA and provide a novel promising therapeutic strategy for HBV infection.

microbiology↗

bioRxiv2023

Two transcriptional cascades orchestrate cockroach leg regeneration

The mystery of appendage regeneration has fascinated humans for centuries, while the regulatory mechanisms remain unclear. In this study, a transcriptional landscape of regenerating leg was established in the American cockroach, Periplaneta americana, an ideal model for appendage regeneration with remarkable regeneration capacity. Through a large-scale in vivo screening, we identified multiple signaling pathways and transcription factors (TFs) controlling leg regeneration. Specifically, zfh-2 and bowl, which have not been previously implicated in appendage regeneration, contributes to blastema proliferation and morphogenesis in two novel transcriptional cascades BMP/JAK-STAT-zfh-2-bab1/B-H2/Lim1 and Notch-drm/bowl-bab1. Notably, zfh-2 was found working as a direct target of BMP signaling to promote cell proliferation in the blastema. These mechanisms might be conserved in the appendage regeneration of vertebrates from an evolutionary perspective. Overall, our findings reveal that two crucial transcriptional cascades orchestrate distinct cockroach leg regeneration processes, significantly advancing the comprehension of molecular mechanism in appendage regeneration.

developmental biology↗

bioRxiv2023

A benchmarked comparison of software packagesfor time-lapse image processing of monolayerbacterial population dynamics

Time-lapse microscopy offers a powerful approach for analysing cellular activity. In particular, this technique is valuable for assessing the behaviour of bacterial populations, which can exhibit growth and intercellular interactions in monolayer. Such time-lapse imaging typically generates large quantities of data, limiting the options for manual investigation. Several of image processing software packages have been developed to facilitate analysis. It can thus be a challenge to identify the software package best suited to a particular research goal. Here, we compare four software packages that support analysis of 2D time-lapse images of cellular populations: CellProfiler, SuperSegger-Omnipose, DeLTA, and FAST. We compare their performance against benchmarked results on time-lapse observations of E. coli populations. Performance varies across the packages, with each of the four out-performing the others in at least one aspect of the analysis. Not surprisingly, the packages that have been in development for longer showed the strongest performance. We found that deep-learning based approaches to object segmentation outperformed traditional approaches, but the opposite was true for frame-to-frame object tracking. We offer these comparisons, together with insight on usability, computational efficiency, and feature availability, as a guide to researchers seeking image processing solutions. Significance StatementTime-lapse microscopy provides a detailed window into the world of bacterial behavior. However, the vast amount of data produced by these techniques is difficult to analyze manually. We have analysed four software tools designed to process such data and compared their performance, using populations of commonly studied bacterial species as our test subjects. Our findings offer a roadmap to scientists, helping them choose the right tool for their research. This comparison bridges a gap between microbiology and computational analysis, streamlining research efforts.

microbiology↗

bioRxiv2023

Dense Sampling of Taxa and Genomes Untangles the Phylogenetic Backbone of a Non-model Plant Lineage Rife with Deep Hybridization and Allopolyploidy

Phylogenetic networks, rather than purely bifurcating trees, more accurately depict the intricate evolutionary dynamics of most lineages, especially those characterized by extensive hybridization and allopolyploidization events. However, the challenges of achieving complete taxon sampling, and limited financial resources for studying non-model plant lineages, have hindered comprehensive and robust estimation of phylogenetic backbones with guidance from networks. The bellflower tribe, Campanuleae, characterized by a reticulate evolutionary history, serves as an ideal model to investigate how to diagnose nested ancient reticulation events. Here, by integrating multiple genomic data sources and a range of phylogenetic inference methods, we produced a robust phylogenetic backbone for the tribe Campanuleae. Our investigation of reticulate evolution indicates that hybridization and allopolyploidization were instrumental in shaping the diversity of the bellflower tribe, particularly during the initial diversification of the subtribe Phytematinae. Additionally, we ascertained that conflicting topologies resulting from distinct genomic datasets and inference methodologies significantly impact downstream estimates of divergence dating, ancestral area construction, and diversification rates. This study offers a universally relevant framework for deciphering how to use network-based phylogenetic structures using various genomic sources and inference methods. [Campanulaceae, Campanuleae, Cytonuclear discordance, paralog, phylogenomics, reticulate evolution]

plant biology↗

bioRxiv2023

Cellular Calcium Activity at Depth Predicted from Surface Potential Recordings using Ultra-high Density Transparent Graphene Arrays

Recording brain activity with high spatial and high temporal resolution across deeper layers of cortex has been a long-sought methodology to study how neural information is coded, stored, and processed by neural circuits and how it leads to cognition and behavior. Electrical and optical neural recording technologies have been the key tools in neurophysiology studies toward a comprehensive understanding of the neural dynamics. The advent of optically transparent neural microelectrodes has facilitated multimodal experiments combining simultaneous electrophysiological recordings from the brain surface with optical imaging and stimulation of neural activity. A remaining challenge is to scale down electrode dimensions to single -cell size and increase the density to record neural activity with high spatial resolution across large areas to capture nonlinear neural dynamics at multiple spatial and temporal scales. Here, we developed microfabrication techniques to create transparent graphene microelectrodes with ultra-small openings and a large, completely transparent recording area. We achieved this by using long graphene microwires without any gold extensions in the field of view. To overcome the quantum capacitance limit of graphene and scale down the microelectrode diameter to 20 m, we used Pt nanoparticles. To prevent open circuit failure due to defects and disconnections in long graphene wires, we employed interlayer doped double layer graphene (id-DLG) and demonstrated cm-scale long transparent graphene wires with microscale width and low resistance. Combining these two advances, we fabricated high-density microelectrode arrays up to 256 channels. We conducted multimodal experiments, combining recordings of cortical potentials with high-density transparent arrays with two-photon calcium imaging from layer 1 (L1) and layer 2/3 (L2/3) of the V1 area of mouse visual cortex. High-density recordings showed that the visual evoked responses are more spatially localized for high-frequency bands, particularly for the multi-unit activity (MUA) band. The MUA power was found to be strongly correlated with the cellular calcium activity. Leveraging this strong correlation, we applied dimensionality reduction techniques and neural networks to demonstrate that single-cell (L2/3) and average (L1 and L2/3) calcium activities can be decoded from surface potentials recorded by high-density transparent graphene arrays. Our high-density transparent graphene electrodes, in combination with multimodal experiments and computational methods, could lead to the development of minimally invasive neural interfaces capable of recording neural activity from deeper layers without requiring depth electrodes that cause damage to the tissue. This could potentially improve brain computer interfaces and enable less invasive treatments for neurological disorders.

neuroscience↗

bioRxiv2023

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

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

plant biology↗

bioRxiv2023

Nightmare or delight: taxonomic circumscription meets reticulate evolution in the phylogenomic era

Phylogenetic studies in the phylogenomics era have demonstrated that reticulate evolution greatly impedes the accuracy of phylogenetic inference, and consequently can obscure taxonomic treatments. However, the systematics community lacks a broadly applicable strategy for taxonomic delimitation in groups identified to have pervasive reticulate evolution. The red-fruit genus, Stranvaesia, provides an ideal model for testing the effect of reticulation on generic circumscription when hybridization and allopolyploidy define a groups evolutionary history. Here, we conducted phylogenomic analyses integrating data from hundreds of single-copy nuclear (SCN) genes and plastomes, and interrogated nuclear paralogs to clarify the inter/intra-generic relationship of Stranvaesia and its allies in the framework of Maleae. Analyses of phylogenomic discord and phylogenetic networks showed that allopolyploidization and introgression promoted the origin and diversification of the Stranvaesia clade, a conclusion further bolstered by cytonuclear and gene tree discordance. The well-inferred phylogenetic backbone revealed an updated generic delimitation of Stranvaesia and a new genus, Weniomeles, characterized by purple-black fruits, trunk and/or branches with thorns, and fruit core with multilocular separated by a layer of sclereids and a cluster of sclereids at the top of the locules. Here, we highlight a broadly-applicable workflow for inferring how analyses of reticulate evolution in phylogenomic data can directly shape taxonomic revisions.

evolutionary biology↗

bioRxiv2023

Antimicrobial peptides modulate pulmonary inflammation by altering the intestinal microbiota

Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical host-derived regulators of the microbiota. However, mechanisms that support homeostasis of the microbiota in response to inflammatory stimuli such as supraphysiologic oxygen remain unclear. Here, we show that neonatal mice breathing supraphysiologic oxygen or direct exposure of intestinal organoids to supraphysiologic oxygen suppress the intestinal expression of AMPs and alters the composition of the intestinal microbiota. Oral supplementation of the prototypical AMP lysozyme to hyperoxia exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury. Together, these data support that intestinal AMPs modulate lung injury and repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/529700v2_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@800123org.highwire.dtl.DTLVardef@1caac5forg.highwire.dtl.DTLVardef@faa875org.highwire.dtl.DTLVardef@49c4e9_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefUsing a combination of murine models and organoids, Abdelgawad and Nicola et al. find that suppression of antimicrobial peptide release by the neonatal intestine in response to supra-physiological oxygen influences the progression of lung injury likely via modulation of the ileal microbiota. HighlightsO_LISupraphysiologic oxygen exposure alters intestinal antimicrobial peptides (AMPs). C_LIO_LIIntestinal AMP expression has an inverse relationship with the severity of lung injury. C_LIO_LIAMP-driven alterations in the intestinal microbiota form a gut-lung axis that modulates lung injury. C_LIO_LIAMPs may mediate a gut-lung axis that modulates lung injury. C_LI

microbiology↗

bioRxiv2023

Platelet Activating Factor Activity Modulates Hyperoxic Neonatal Lung Injury Severity

Hyperoxia-induced inflammation contributes significantly to developmental lung injury and bronchopulmonary dysplasia (BPD) in preterm infants. Platelet activating factor (PAF) is known to be a major driver of inflammation in lung diseases such as asthma and pulmonary fibrosis, but its role in BPD has not been previously investigated. Therefore, to determine whether PAF signaling independently modulates neonatal hyperoxic lung injury and BPD pathogenesis, lung structure was assessed in 14 day-old C57BL/6 wild-type (WT) and PAF receptor knockout (PTAFR KO) mice that were exposed to 21% (normoxia) or 85% O2 (hyperoxia) from postnatal day 4. Lung morphometry showed that PTAFR KO mice had attenuated hyperoxia-induced alveolar simplification when compared to WT mice. Functional analysis of gene expression data from hyperoxia-exposed vs. normoxia-exposed lungs of WT and PTAFR KO showed that the most upregulated pathways were the hypercytokinemia/hyperchemokinemia pathway in WT mice, NAD signaling pathway in PTAFR KO mice, and agranulocyte adhesion and diapedesis as well as other pro-fibrotic pathways such as tumor microenvironment and oncostatin-M signaling in both mice strains, indicating that PAF signaling may contribute to inflammation but may not be a significant mediator of fibrotic processes during hyperoxic neonatal lung injury. Gene expression analysis also indicated increased expression of pro-inflammatory genes such as CXCL1, CCL2 and IL-6 in the lungs of hyperoxia-exposed WT mice and metabolic regulators such as HMGCS2 and SIRT3 in the lungs of PTAFR KO mice, suggesting that PAF signaling may modulate BPD risk through changes in pulmonary inflammation and/or metabolic reprogramming in preterm infants.

pathology↗

bioRxiv2023

Three-dimensional imaging and quantification of mouse ovarian follicles via optical coherence tomography

Ovarian tissue cryopreservation has been successfully applied worldwide for fertility preservation. Correctly selecting the ovarian tissue with high follicle loading for freezing and reimplantation increases the likelihood of restoring ovarian function, but it is a challenging process. In this work, we explore the use of three-dimensional spectral-domain optical coherence tomography (SD-OCT) to identify different follicular stages, especially primary follicles, compare the identifications with H&E images, and measure the size and age-related follicular density distribution differences in mice ovaries. We use the thickness of the layers of granulosa cells to differentiate primordial and primary follicles from secondary follicles. The measured dimensions and age-related follicular distribution agree well with histological images and physiological aging. Finally, we apply attenuation coefficient map analyses to significantly improve the image contrast and the contrast-to-noise ratio (p < 0.001), facilitating follicle identification and quantification. We conclude that SD-OCT is a promising method to noninvasively evaluate ovarian follicles.

bioengineering↗

bioRxiv2022

Targeted genome editing in grape using multiple CRISPR-guided editing systems

The CRISPR/Cas9 system, together with newly developed CRISPR technologies such as CRISPR/LbCpf1 and base editors, have expanded the scope of targeted genome editing in plants. However, in grape, applications of these novel CRISPR-guided tools have not been reported. Here, we employed EGFP (enhanced green fluorescent protein) and RUBY to help to screen transformed grape cells based on fluorescence and red betalain and tested the activities of CRISPR/LbCpf1, CRISPR/xCas9 and cytidine base editor (CBE) in grape, respectively. The grape TMT1 (tonoplastic monosaccharide transporter1) and TMT2 genes were simultaneously edited by using LbCpf1, resulting in an efficiency of 16-48%. Furthermore, high temperature (34{degrees}C) could enhance editing efficiencies at most of the designed targets. The CRISPR/xCas9 could induce targeted mutagenesis at the target with NGG PAM, but the efficiencies were very low (< 1.9%). The targets with GAA and GAT PAMs that are reported in mammalian cells and rice were not recognized by xCas9 in our study. Moreover, successful C-to-T substitutions were achieved in GAI1 (gibberellin insensitive1) gene by using CBE. The editing efficiencies ranged from 2.4 to 15% at the two targets in GAI1 in grape cells. Analysis of independent embryos revealed a C-to-T efficiency of 12.5% at the first target of GAI1. Taken together, our results demonstrate the efficacy of these CRISPR-guided tools in grape and provide evidence for further application of these editing tools in this economically important species.

plant biology↗

bioRxiv2022

Rewiring Dynamics of Functional Connectome in Motor Cortex during Motor Skill Learning

The brains connectome continually rewires throughout the life of an organism. In this study, we sought to elucidate the operational principles of such rewiring by analyzing the functional connectomes in mouse primary motor cortex (M1) during a 14-session (day) lever-press task learning in response to an auditory cue. Specifically, we employed Calcium imaging recordings of L2/3 and L5 of M1 in awake mice to reconstruct and analyze functional connectomes across learning sessions. Our results show that functional connectomes in L2/3 and L5 follow a similar learning-induced rewiring trajectory. More specifically, the connectomes rewire in a biphasic manner, where functional connectivity increases over the first few learning sessions, and then, it is gradually pruned to return to a homeostatic level of network density. We demonstrated that the increase of network connectivity in L2/3 connectomes, but not in L5, generates neuronal co-firing activity that correlates with higher motor performance (shorter cue-to-reward time), while motor performance remains relatively stable throughout the pruning phase. The results show a biphasic rewiring principle that involves the maximization of reward / performance and maintenance of network density. Finally, we demonstrated that the connectome rewiring in L2/3 is clustered around a core set of movement-associated neurons that form a highly interconnected hub in the connectomes, and that the activity of these core neurons stably encodes movement throughout learning. Significance StatementConnectomes in the motor cortex rewire during motor skill learning, but the operational principle behind this rewiring is yet to be determined. Here, we characterized the rewiring dynamics of functional connectomes in L2/3 and L5 of M1 in mice engaging in a lever-press learning, using two-photon fluorescence microscopy data. We identified a universal biphasic rewiring trajectory across animals and layers in the motor cortex that reflects two objectives: an exploratory phase that increases functional connectivity and optimizes motor performance, and a pruning phase that brings connectivity back to a homeostatic level while maintaining motor performance. We found further that connectome rewiring during motor skill learning concentrates around a core set of highly interconnected neurons in L2/3 that reliably encode movements.

neuroscience↗

bioRxiv2022

JQ-1 ameliorates schistosomiasis liver granuloma in mice by suppressing male and female reproductive systems and egg development of Schistosoma japonicum

Schistosomiasis is a serious and widespread parasitic disease caused by infection with Schistosoma japonicum. Because the parasites eggs are primarily responsible for schistosomiasis dissemination and pathogenesis, inhibiting egg production is a potential approach to control the spread and severity of the disease. The bromodomain and extra-terminal (BET) proteins represent promising targets for the development of epigenetic drugs against Schistosoma. JQ-1 is a selective inhibitor of the BET protein family. In the present study, JQ-1 was applied S. japonicum in vitro. By using laser confocal scanning microscopy and EdU incorporation assays, we showed that application of JQ-1 to worms in vitro affected egg laying and the development of both the male and female reproductive systems. JQ-1 also inhibited the expression of the reproductive-related genes SjPlk1 and SjNanos1 in S. japonicum. Mice infected with S. japonicum were treated with JQ-1 during egg granuloma formation. JQ-1 treatment significantly reduced the size of the liver granulomas and levels of serum alanine aminotransferase and aspartate aminotransferase in mice and suppressed both egg laying and the development of male and female S. japonicum reproductive systems in vivo. Moreover, the mRNA expression levels of some proinflammatory cytokines were decreased in the parasites. Our findings suggest that JQ-1 treatment attenuates S. japonicum egg- induced hepatic granuloma due at least in part to suppressing the development of the reproductive system and egg production of S. japonicum. These findings further suggest that JQ-1 or other BET inhibitors warrant additional study as a new approach for the treatment or prevention of schistosomiasis. Author summaryAmong neglected tropical diseases, schistosomiasis is a serious disease caused by infection with the parasite Schistosomiasis japonicum. Treatment of schistosomiasis is currently almost exclusively with praziquantel, which kills mainly adult parasites, with minimal effectiveness against immature schistosomes and eggs. However, the parasites eggs are primarily responsible for schistosomiasis dissemination and pathology. In addition, overuse of praziquantel in epidemic areas has led to drug resistance and a reduced cure rate. Thus, new parasite targets for the development of novel therapeutics are crucial. Here, we evaluated the potential of JQ-1, a bromodomain and extra-terminal protein inhibitor, to suppress the production of S. japonicum eggs. Application of JQ-1 to S. japonicum in vitro decreased the number of mature germ cells, the rates of oviposition, and the number of eggs produced in each male-female pairing. JQ-1 treatment of mice infected with S. japonicum ameliorated hepatic granuloma and decreased serum liver enzymes, suggesting improved liver function. These results indicate that JQ-1 inhibits reproductive development and egg production in S. japonicum, providing supporting evidence that JQ-1 warrants additional study for use as a novel approach in the prevention or treatment of schistosomiasis.

microbiology↗

bioRxiv2021

Decoding of cortex-wide brain activity from local recordings of neural potentials

ObjectiveElectrical recordings of neural activity from brain surface have been widely employed in basic neuroscience research and clinical practice for investigations of neural circuit functions, brain-computer interfaces, and treatments for neurological disorders. Traditionally, these surface potentials have been believed to mainly reflect local neural activity. It is not known how informative the locally recorded surface potentials are for the neural activities across multiple cortical regions. ApproachTo investigate that, we perform simultaneous local electrical recording and wide-field calcium imaging in awake head-fixed mice. Using a recurrent neural network model, we try to decode the calcium fluorescence activity of multiple cortical regions from local electrical recordings. Main resultsThe mean activity of different cortical regions could be decoded from locally recorded surface potentials. Also, each frequency band of surface potentials differentially encodes activities from multiple cortical regions so that including all the frequency bands in the decoding model gives the highest decoding performance. Despite the close spacing between recording channels, surface potentials from different channels provide complementary information about the large-scale cortical activity and the decoding performance continues to improve as more channels are included. Finally, we demonstrate the successful decoding of whole dorsal cortex activity at pixel-level using locally recorded surface potentials. SignificanceThese results show that the locally recorded surface potentials indeed contain rich information of the large-scale neural activities, which could be further demixed to recover the neural activity across individual cortical regions. In the future, our cross-modality inference approach could be adapted to virtually reconstruct cortex-wide brain activity, greatly expanding the spatial reach of surface electrical recordings without increasing invasiveness. Furthermore, it could be used to facilitate imaging neural activity across the whole cortex in freely moving animals, without requirement of head-fixed microscopy configurations.

neuroscience↗

bioRxiv2021

Phylogenomic analyses in the apple genus Malus s.l. reveal widespread hybridization and allopolyploidy driving the diversifications, with insights into the complex biogeographic history in the Northern Hemisphere

Phylogenomic evidence from an increasing number of studies has demonstrated that different data sets and analytical approaches often reconstruct strongly supported but conflicting relationships. In this study, hundreds of single-copy nuclear (SCN) genes (785) and complete plastomes (75) were used to infer the phylogenetic relationships and estimate the historical biogeography of the apple genus Malus sensu lato, an economically important lineage disjunctly distributed in the Northern Hemisphere involved in known and suspected hybridization and allopolyploidy events. The nuclear phylogeny recovered the monophyly of Malus s.l. (including Docynia); however, it was supported to be biphyletic in the plastid phylogeny. An ancient chloroplast capture event best explains the cytonuclear discordance that occurred in the Eocene in western North America. Our conflict analysis demonstrated that ILS, hybridization, and allopolyploidy could explain the widespread nuclear gene tree discordance. We detected one deep hybridization event (Malus doumeri) involving the ancestor of pome-bearing species and Docynia delavayi, and one recent hybridization event (Malus coronaria) between M. sieversii and a combined clade of M. ioensis and M. angustifolia. Furthermore, our historical biogeographic analysis combining living and fossil species supported a widespread East Asian-western North American origin of Malus s.l., followed by a series of extinction events in the Eocene in northern East Aisa and western North America. This study provides a valuable evolutionary framework for the breeding and crop improvement of apples and their close relatives.

evolutionary biology↗

bioRxiv2021

A high throughput bispecific antibody discovery pipeline

Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy but inefficiency in the current BsAb discovery paradigm has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based BsAb functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification with single-cell PCR and sequencing and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model system, we demonstrate that our single cell platform possesses a high throughput screening efficiency of up to one and half million variant library cells per run and can isolate rare functional clones at low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones including extremely rare ones ([~] 0.001% abundance). We also discovered BiTEs that exhibit novel properties contradictory to conventional wisdom, including harboring rigid scFv connecting peptide linkers yet with in vitro cytotoxicity comparable to that of clinically approved Blinatumomab. Through sequencing analyses on sorted BiTE clones, we discovered multiple design variable preferences for functionality including the CD19VL-VH- CD3VH-VL and CD19VH-VL-CD3VH-VL arrangements being the most favored orientation. Sequence analysis further interrogated the sequence composition of the CDRH3 domain in scFvs and identified amino acid residues conserved for function. We expect our single cell platform to not only significantly increase the development speed of high quality of new BsAb therapeutics for cancer and other disorders, but also enable identifying generalizable design principles for new BsAbs and other immunotherapeutics based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

bioengineering↗

bioRxiv2021

Capturing single-copy nuclear genes, organellar genomes, and nuclear ribosomal DNA from deep genome skimming data for plant phylogenetics: A case study in Vitaceae

With the decreasing cost and availability of many newly developed bioinformatics pipelines, next-generation sequencing (NGS) has revolutionized plant systematics in recent years. Genome skimming has been widely used to obtain high-copy fractions of the genomes, including plastomes, mitochondrial DNA (mtDNA), and nuclear ribosomal DNA (nrDNA). In this study, through simulations, we evaluated optimal (minimum) sequencing depth and performance for recovering single-copy nuclear genes (SCNs) from genome skimming data, by subsampling genome resequencing data and generating 10 datasets with different sequencing coverage in silico. We tested the performance of the four datasets (plastome, nrDNA, mtDNA, and SCNs) obtained from genome skimming based on phylogenetic analyses of the Vitis clade at the genus-level and Vitaceae at the family-level, respectively. Our results showed that optimal minimum sequencing depth for high-quality SCNs assembly via genome skimming was about 10x coverage. Without the steps of synthesizing baits and enrichment experiments, we showcase that deep genome skimming (DGS) is effective for capturing large datasets of SCNs, in addition to plastomes, mtDNA, and entire nrDNA repeats, and may serve as an economical alternative to the widely used target enrichment Hyb-Seq approach.

evolutionary biology↗