Biology subjects

Zhou, S.

Publications and source records attributed to Zhou, S..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Temperature, but not plant cultivar, influence the efficacy of insecticidal dsRNA in Colorado potato beetle

Environmental RNAi (eRNAi) is a recent innovation in insect pest control, and comprehensive risk assessment is needed to ensure the environmental safety and longevity of this technology. As eRNAi relies on the insects cellular machinery for its mode of action, environmentally mediated plasticity in the activity of cellular processes required for RNAi could influence efficacy and the development of resistance. Here, we investigated the extent to which plant cultivar and temperature influence the efficacy of insecticidal double-stranded RNA (dsRNA) targeting actin in larvae of the Colorado potato beetle (CPB; Leptinotarsa decemlineata). Potato cultivar did not significantly affect survival or gene silencing in dsRNA-treated larvae, indicating that efficacy is consistent across potato varieties, at least under laboratory conditions. However, the amount of leaf tissue consumed by larvae varied across cultivars, with consumption being inversely proportional to protein content. Thus, the ingested dose of dsRNA may vary across crop varieties. Temperature did influence RNAi efficacy, with both gene silencing and mortality being reduced when dsRNA treatment occurred at lower temperatures. After three days of feeding with dsRNA, gene silencing occurred at all temperatures, but knockdown efficiency was 62% at 30{degrees}C and 35% at 18{degrees}C. Beetles consumed significantly less leaf tissue (and thus dsRNA) at 18{degrees}C, but the observed temperature-dependent effects could not be fully explained by the quantity of dsRNA ingested. Further, efficacy at different temperatures was not related to transcript levels of core RNAi genes, indicating that other mechanisms are responsible for the observed effects. Overall, these results indicate that environmental conditions can influence the efficacy of insecticidal eRNAi and may affect the rate at which insects develop resistance to these technologies.

molecular biology↗

bioRxiv2024

Abnormalities of the liver have big impacts on orthotopic liver transplantation

BackgroundOrthotopic rat liver transplantation (OLT) is widely used in basic research; normal liver anatomy and structures are attributable to its success while its deformities complicate to have a negative on OLT. MethodsFor tolerance induction project, we performed OLT from Lewis to Brown Norway (BN) rat as chronic rejection model and encountered two anatomical deformities of recipient rats: of 47 liver transplantations, accessory liver lobe occurred to 4 cases and bifurcations of liver outflow to 5 cases in BN rats. ResultsFor the accessory liver lobe, we discontinued OLT for one case with a big accessory liver lobe; two rats died from pneumothorax upon separation; and succeed in one case with the small lobe. For two vein outflows of the liver, we succeeded in OLT due to its reconstruction in one case but the recipient died one week later, and succeeded in 1 case after one small orifice was sutured, we failed in 3 cases due to thrombosis following OLT. For 38 rats with normal livers, only 4 rats failed to survive LT. There were significant differences in OLT success (p<0.01) ConclusionLiver abnormal anatomy has negative impact on OLT, giving a clue to that pre-transplant comprehensive screening is significant and beneficial clinically.

physiology↗

bioRxiv2024

Proteolysis-targeting vaccines (PROTAVs) for robust tumor combination immunotherapy

Protein/peptide subunit vaccines are promising to promote the tumor therapeutic efficacy of immune checkpoint blockade (ICB). However, current protein/peptide vaccines elicit limited antitumor T cell responses, leading to suboptimal therapeutic efficacy. Here, we present proteolysis-targeting vaccines (PROTAVs) that facilitate antigen proteolytic processing and cross-presentation to potentiate T cell responses for robust ICB combination immunotherapy of melanoma. PROTAVs are modular conjugates of protein/peptide antigens, E3 ligase-binding ligands, and linkers. In antigen-presenting cells (APCs), PROTAVs bind to E3 ligases to rapidly ubiquitinate PROTAV antigens, facilitating antigen proteolytic processing by proteasome, and thereby promoting antigen cross-presentation to T cells and potentiating CD8+ T cell responses. We developed a melanoma PROTAV using a tandem peptide of trivalent melanoma-associated antigens. Co-delivered by lipid nanoparticles (LNPs) with bivalent immunostimulant adjuvants, this PROTAV promotes the quantity and quality of melanoma-specific CD8+ T cells in mice. Further, combining PROTAV and ICB ameliorates the immunosuppressive melanoma microenvironment. As a result, PROTAV and ICB combination enhances melanoma complete regression rates and eradicated 100% large BrafV600E melanoma without recurrence in syngeneic mice. PROTAVs hold the potential for robust tumor combination immunotherapy.

bioengineering↗

bioRxiv2024

FragLlama: Next-fragment prediction for molecular design

The emergence of ChatGPT has drawn significant attention to Large Language Models (LLMs) due to their impressive performance. While LLMs primarily focus on next token/word prediction, we apply this principle to molecular design by reframing the task as predicting the next token/fragment. We present FragLlama, a large language model trained for molecular design, featuring custom tokens that represent molecular fragments and functional groups. The model is for generating molecules given one or two fragments, for application scenarios like general hit-to-lead and lead optimization stage drug design, PROTAC linker design; mapping to commonly used drug design strategies like fragment growing and scaffold hopping. In the pre-training stage, we adapted the Llama 3 architecture to create FragLlama, training it to learn conditional probabilities of these fragment-level tokens. The subsequent alignment stage employed fine-tuning to guide the model towards generating molecules with desired properties. The effectiveness of FragLlama is demonstrated through its applications in designing molecular glue libraries, PROTAC linkers and EGFR binders. FragLlama demonstrates proficiency in reproducing expert-level designs while also exploring novel and promising chemical spaces, highlighting its potential to augment the capabilities of medicinal chemists in drug design.

bioinformatics↗

bioRxiv2024

Microbiome and climate: Skin microbial diversity and community functions of Polypedates megacephalus (Anura: Rhacophoridae) associated with bioclimate

The microbiome inhabiting animal skin plays a crucial role in host fitness by influencing both the composition and function of microbial communities. Environmental factors, including climate, significantly impact microbial diversity and the functional attributes of these communities. However, it remains unclear how specific climatic factors affect amphibian skin microbial composition, community function, and the relationship between these two aspects. Given that amphibians are poikilotherms, and thus more susceptible to temperature fluctuations, understanding these effects is particularly important. Here, we investigated the skin microbiome of the rhacophorid tree frog Polypedates megacephalus across different climatic regimes using 16S rRNA gene sequencing. Skin swab samples were collected from nine populations of P. megacephalus adults in the Guangxi region, China. The majority of the core microbiota were found to belong to the genus Pseudomonas. Our findings indicate that microbial community diversity, composition, and function are associated with changes in climatic conditions. Specifically, the taxonomic and functional diversity of the skin microbiome increased in response to greater climate variability, particularly in temperature fluctuations. Additionally, the functional attributes of microbial communities changed in parallel with shifts in community diversity and composition, suggesting that environmental filtering driven by climate changes negatively impacts microbial community functional redundancy. These results highlight the critical influence of climatic factors on amphibian skin microbiomes and offer new insights into how microbial composition and function contribute to host adaptation in varying environmental conditions. IMPORTANCEThis study is important in understanding the association between climate variability, microbial diversity, and host adaptation in amphibians, particularly vulnerable to environmental changes due to their poikilothermic nature. Amphibians rely on their skin microbiome for key functions like disease resistance, yet little is known about how climate fluctuations impact these microbial communities. By analyzing the microbiome of Polypedates megacephalus across different climatic regimes, our analysis reveals that while climate variability enhances microbial diversity, it reduces functional redundancy. These findings highlight the potential ecological consequences of climate change and emphasize the need to integrate microbiome health into amphibian conservation strategies.

microbiology↗

bioRxiv2024

Caves as species pumps: key innovations, isolation, and periodic introgression drive the world's largest cavefish radiation in a dynamic karstic landscape

Species diversification is shaped by intricate interactions among biotic drivers, including gene flow, hybridization, and key innovations, and abiotic drivers, such as historical climate change, geological events, and ecological opportunity. However, the relative contributions of these drivers in large radiations remain poorly understood. We investigate the interplay among these factors in the diversification of Sinocyclocheilus, a cavefish radiation comprising 79 species. Sinocyclocheilus include typical surface-dwelling forms, with well-developed eyes and pigmentation, to cave-dwelling forms with regressed eyes, reduced pigmentation, and the presence of a horn and a hump. Using reduced representation genomic data (RADseq), we show extensive gene flow events across different species, with introgression playing a key role compared to incomplete lineage sorting in creating phylogenetic discordance and contributing genetic variation for cave adaptation and diversification in this group. Key traits such as eye degeneration, reduced pigmentation, and horn evolved independently multiple times, as adaptations for effectively exploiting cave environments. Furthermore, the uplift of the Tibetan plateau and the late Miocene cooling also significantly impacted speciation rates. Demographic analyses suggest population expansions during the Gonghe Movement and stability during the Last Glacial Maximum, possibly due to cave refugia. Periodic events of introgression promoted by isolation and reconnections due to the changing climate and geological activity, combined with the repeated evolution of key cave-adapted traits, are the primary drivers of this radiation. Our findings underscore the complex interplay of biotic and abiotic factors in the evolution of Sinocyclocheilus fish, offering new insights into the mechanisms of cave adaptation and diversification.

evolutionary biology↗

bioRxiv2024

Sociability genetically separable from social hierarchy in amniotes

The emergence of social structure and organization is essential for the evolution of amniotes, including human beings. Sociability and social hierarchy are two key features to form a social group. However, it remains unknown whether sociability and social hierarchy are genetically separable. In this study, we examined the social hierarchy, social and social novelty preference of PAS1 (placental-accelerated sequence 1) knock-out and knock-in mice. PAS1 is a social enhancer that modulates social hierarchy. We found that PAS1 knock-out mice lack social hierarchy while wallaby/chicken PAS1 knock-in mice establish stable social ranks. Moreover, social and social novelty preference was observed in all PAS1-mutated mice. PAS1 knock-in mice have stronger preference to interact with other mice than wild-type mice (C57BL/6). No aggressive alteration was found in PAS1-mutated mice. Overall, our results showed that PAS1 is an indispensable regulatory element in the formation of social hierarchy while PAS1 regulates one of pathways modulating sociability. Therefore, sociability is genetically separable from social hierarchy in amniotes, providing insights into how social structure and organization evolved. HighlightsPAS1, a social enhancer, is indispensable for amniotes to establish social hierarchy. PAS1 modulates sociability in amniotes. Sociability and social hierarchy are regulated differently in amniotes. The frequency of PAS1 allele was not rare when social hierarchy first appeared.

animal behavior and cognition↗

bioRxiv2024

Design of diverse, functional mitochondrial targeting sequences across eukaryotic organisms using variational autoencoder

Mitochondria play a key role in energy production and cellular metabolism, making them a promising target for metabolic engineering and disease treatment. However, despite the known influence of passenger proteins on localization efficiency, only a few protein-localization tags have been characterized for mitochondrial targeting. To address this limitation, we exploited Variational Autoencoder (VAE), an unsupervised deep learning framework, to design novel mitochondrial targeting sequences (MTSs). In silico analysis revealed that a high fraction of generated peptides are functional and possess features important for mitochondrial targeting. Additionally, we devised a sampling scheme to indirectly address biases arising from the differences in mitochondrial protein import machinery and characterized artificial MTSs in four eukaryotic organisms. These sequences displayed significant diversity, sharing less than 60% sequence identity with MTSs in the UniProt database. Moreover, we trained a separate VAE and employed latent space interpolation to design dual targeting sequences capable of targeting both mitochondria and chloroplasts, shedding light on their evolutionary origins. As a proof-of-concept, we demonstrate the application of these artificial MTSs in increasing titers of 3-hydroxypropionic acid through pathway compartmentalization and improving 5-aminolevulinate synthase delivery by 1.62-fold and 4.76-fold, respectively. Overall, our work not only demonstrates the potential of generative artificial intelligence in designing novel, functional mitochondrial targeting sequences but also highlights their utility in engineering mitochondria for both fundamental research and practical applications in biology.

synthetic biology↗

bioRxiv2024

Low-cost and highly efficient generation of near-complete bacterial pathogen genomes by TELL-Seq

Recent evidences suggest that de novo genome assembly can provide additional insights into genomic variation landscape beyond short-read NGS analyses. Despite the advent of longread sequencing technologies, generating high-quality bacterial genome assemblies remains expensive and requires high-quality and large quantities of DNA. TELL-Seq, an emerging linked-read technology, has been reported as a low-cost alternative for producing nearcomplete genome assembly in certain bacterial species. However, a systematic assessment of its performance and characteristics in de novo bacterial genome assembly has not yet been conducted. To address this, we benchmarked TELL-Seq using a set of clinical and standard bacterial pathogens with a wide range of genome size (2.0 6.4 Mbp), GC-content (32% - 68%), genome complexity (mappability from 0.15 to 0.60) and Gram types. Our findings indicate that, with 1/6 cost and 1/15,000 of DNA compared to PacBio HiFi, TELL-Seq could generate as high quality near-complete bacterial genome assemblies. The optimal depth for assembly is around 200x, as increased depth improves contiguity and completeness, though quality plateaus at 250x. In general, genome complexity was significantly correlated with assembly quality, but GC-content was not. Nevertheless, genomes with extreme GC-content may require higher depths for accurate assembly. Our study suggests that TELL-Seq could be a scalable method for large-scale bacterial genomic surveys. The data generated from this study could serve as a benchmark dataset for further algorithm development. Impact StatementAn increasing body of literature have shown that de novo genome assembles could provide additional insights into genomic variation beyond standard short-read technologies. Its particularly useful in clinical and epidemiological studies of bacteria, due to high variable nature of their genomes. Although great advances have been made in long-read sequencing technology, its still expensive with high requirement over sample DNAs quality and quantity, limiting its adoption in large-scale studies or surveys. Linked-read technology is an inexpensive alternative which used single molecule barcoding to mark origin of reads for better scaffolding of contigs assembled from normal short reads. TELL-Seq, as one of these promising technologies, was reported to be able to inexpensively and efficiently generate near-complete bacterial de novo genomic assemblies with low DNA inputs. Theres not yet a systematic benchmarking of this technology in bacterial pathogens. In this study, a set of clinically important bacterial pathogens with a wide spectrum of GC-content, genome size, genome complexity, and Gram Type were used to investigate the performance of TELL-Seq technology in de novo genome assembly. We found that, with about 1/6 of cost and 1/15000 of DNA quantity, it could generate comparable de novo assemblies compared to standard reference genomes or PacBio HiFi assemblies, while beating conventional short-read assemblies. We found several key factors, such as genome complexity, and sequencing depth, which would impact the quality of the assembly. Our study suggests that a sequencing depth of 200x is sufficient to achieve satisfactory results. Our study has revealed characteristics of this technology in bacterial genome assembly and paved the way for its large-scale application in clinical or epidemiological surveys. Data SummaryO_LIRaw sequencing data for all isolates have been deposited to SRA, accessible through these NCBI BioProject PRJNA1133244. C_LIO_LIA full list of SRA BioSample accession numbers are available in Tab S5. C_LIO_LIAssemblies and analyses companion this paper is available at: https://github.com/x-lab/tellseq_bacteria C_LI

bioinformatics↗

bioRxiv2024

RIPK4 driven by TP53 mutations promotes resistance to redox stress of CRC by phosphorylating MTHFD1

This study investigates advanced colorectal cancer (CRC), focusing on its tendency for distant metastasis and chemotherapy resistance. It highlights the importance of PANoptosis, a cell death pathway, and the role of the Receptor-interacting serine/threonine-protein kinase (RIPK) family in tumor progression. RIPK4s tissue-specific functions in cancer cell behavior are emphasized, including its influence on invasion, migration, and oxidative stress resistance. The study reveals the critical balance of reactive oxygen species (ROS) in cancer cells, linked to antioxidant defenses and NADPH production for survival. A key finding is the connection between TP53 mutations in CRC and increased RIPK4 expression, which enhances MTHFD1 phosphorylation, boosts NADPH production, reduces ROS, and promotes resistance to PANoptosis, leading to metastasis. The research identifies the molecular basis of CRC metastasis, showing how RIPK4 regulates MTHFD1 to resist PANoptosis, offering new therapeutic targets for metastatic CRC and potential improvements in patient outcomes.

molecular biology↗

bioRxiv2024

PDS5s control the Arabidopsis 3D genome by suppressing the formation of TAD-like domains

One key structural element in the three-dimensional (3D) chromatin organization is the Topologically Associating Domain (TAD), which facilitates the formation of distinct chromatin compartments and fosters specific chromatin interactions. Similar chromatin compartments, known as TAD-like domains, have been identified in many plant species. However, the model plant Arabidopsis thaliana has been an exception. In this study, we address this long-standing issue by presenting evidence that Arabidopsis PDS5 proteins play a crucial role in preventing the formation of TAD-like domains.

molecular biology↗

bioRxiv2024

A unique mechanism of snRNP core assembly

The assembly of spliceosomal snRNP cores involves seven Sm proteins (D1/D2/F/E/G/D3/B) forming a ring around each snRNA and typically requires several essential assembly chaperones, particularly the SMN-Gemins complex implicated in spinal muscular atrophy (SMA). Strikingly, in budding yeast, snRNP core assembly only involves Brr1, a nonessential homolog of Gemin2. To unravel this enigma, we systematically investigated snRNP core assembly in budding yeast using biochemical and genetic approaches. We discovered two distinct pathways in budding yeast: chaperone-mediated and direct. The chaperone-mediated pathway involves two proteins, Brr1 and a novel protein, Lot5, but is inefficient. Lot5 binds D1/D2/F/E/G to form a heterohexameric ring (6S). Brr1 binds D1/D2/F/E/G and 6S but cannot displace Lot5 to facilitate assembly. Disruption of both BRR1 and LOT5 genes caused mild growth retardation, but LOT5 overexpression substantially impeded growth. The direct pathway uniquely involves F/E/G as a trimer and a stable D1/D2/F/E/G intermediate complex, explaining the non-essentiality of assembly chaperones. These findings unveil a unique assembly mechanism of snRNP cores, illuminate the evolution of assembly chaperones, and suggest avenues for studying SMA pathophysiology.

molecular biology↗

bioRxiv2024

A safe and effective method for thin cornea cross-linking

The mechanical properties of the collagen matrix determine the corneal regularity. Thus, corneal thinning, cone-like protrusion, and loss of corneal astigmatism characterize corneal ecstatic diseases, including keratoconus and post-LASIK ectasia. Currently, riboflavin (RF)/ultraviolet A (UVA) based corneal collagen cross-linking (CXL) is commonly used in clinics to stiffen cornea and halt disease progression in patients. However, due to tissue damage, especially irreversible endothelium loss, standard RF/UVA protocol is only allowed in eyes with corneal thickness over 400 m after epithelium debridement. Thus, lots of patients with thin cornea have limited therapeutic options. Here, we introduced a new strategy to cross-link cornea collagens using synthesized photo-initiators lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and visible blue light (BL). The LAP/BL protocol could effectively increase corneal stiffness, similar to the RF/UVA protocol in both porcine and rabbit cornea. When the LAP/BL protocol was used, both epithelial wound healing, stromal cell repopulation, and corneal edema recovery rates were accelerated compared with the RF/UVA protocol. More importantly, as BL is used instead of UVA, corneal endothelium was mostly preserved in both rabbit and mouse models even when cornea thickness was as thin as 100 m. In summary, we propose that the LAP/BL protocol is a safe and effective choice for thin cornea CXL.

bioengineering↗

bioRxiv2024

Cancer-associated snaR-A noncoding RNA interacts with core splicing machinery and disrupts processing of mRNA subpopulations

RNA polymerase III (Pol III) activity in cancer is linked to the production of small noncoding (nc)RNAs that are otherwise silent in most tissues. snaR-A (small NF90-associated RNA isoform A) - a hominid-specific ncRNA shown to enhance cell proliferation, migration, and invasion - is a cancer-emergent Pol III product that remains largely uncharacterized despite promoting growth phenotypes. Here, we applied a combination of genomic and biochemical approaches to study the biogenesis and subsequent protein interactions of snaR-A and to better understand its role as a putative driver of cancer progression. By profiling the chromatin landscapes across a multitude of primary tumor types, we show that predicted snaR-A upregulation is broadly linked with unfavorable outcomes among cancer patients. At the molecular level, we unexpectedly discover widespread interactions between snaR-A and mRNA splicing factors, including SF3B2 - a core component of the U2 small nuclear ribonucleoprotein (snRNP). We find that SF3B2 levels are sensitive to high snaR-A abundance and that depletion of snaR-A alone is sufficient to decrease intron retention levels across subpopulations of mRNA enriched for U2 snRNP occupancy. snaR-A sensitive genes are characterized by high GC content, close spatial proximity to nuclear bodies concentrated in pre-mRNA splicing factors, and functional enrichment for proteins involved in deacetylation and autophagy. We highlight examples of splicing misregulation and increased protein levels following snaR-A depletion for a wide-ranging set of factors, suggesting snaR-A-driven splicing defects may have far-reaching effects that re-shape the cellular proteome. These findings clarify the molecular activities and consequences of snaR-A in cancer, and altogether establish a novel mechanism through which Pol III overactivity may promote tumorigenesis.

molecular biology↗

bioRxiv2024

Comparative analysis of culture- and ddPCR-based wastewater surveillance for carbapenem-resistant bacteria

With the widespread use of last-resort antibiotics, carbapenems, clinical reports of infections associated with carbapenem-resistant Enterobacterales (CRE) have increased. Clinical surveillance for CRE involves susceptibility testing and/or whole genome sequencing of resistant isolates, which is laborious, resource intensive, and requires expertise. Wastewater surveillance can potentially complement clinical surveillance of CRE, and population-level antibiotic resistance (AR) surveillance more broadly. In this study, we quantitatively and qualitatively compared two widely used methods for AR wastewater surveillance: (1) a culture-based approach for quantifying carbapenem-resistant bacteria and (2) a digital droplet PCR (ddPCR) assay targeting five major carbapenemase genes. We developed a multiplexed ddPCR assay to detect five carbapenemase genes and applied it to wastewater samples from three sites over 12 weeks. In parallel, we quantified carbapenem resistant bacteria and carbapenemase-producing bacteria using culture-based methods. We assessed associations between the concentrations of carbapenemase genes and resistant bacteria. Although both approaches showed similar trends in the overall abundance of dominant carbapenem-resistant bacteria and genes, there were weak correlations between the quantitative levels of resistance. Nanopore sequencing of the resistome of the carbapenem-resistant bacteria revealed that discrepancies arose from differences in the sensitivity and specificity of the methods. This study enhances our understanding of the application of wastewater surveillance in tracking carbapenem resistance and highlights how method choice impacts the results from AR wastewater surveillance.

microbiology↗

bioRxiv2024

Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters

RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occupancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings reshape our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.

genomics↗

bioRxiv2024

Enhancing lipid production in plant cells through high-throughput genome editing and phenotyping via a scalable automated pipeline

Plant bioengineering is a time-consuming and labor-intensive process, with no guarantee of achieving the desired trait. Here we report a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB). FAST-PB achieves gene cloning, genome editing, and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrate that FAST-PB can streamline the Golden Gate cloning process, with the capacity to construct 96 vectors in parallel. To prove the concept, using FAST-PB, we first found that PEG2050 significantly increases transfection efficiency by over 45%. To validate the pipeline, we established a reporter-gene-free method for CRISPR editing via mutation of HCF136, affecting cellular chlorophyll fluorescence. Next, we applied this pipeline for lipid production and found that diverse lipids were significantly enhanced up to sixfold through introducing multi-gene cassettes via CRISPR activation, and regenerated plant using this platform. Lastly, we harnessed FAST-PB to achieve high-throughput single-cell lipid profiling through the integration of MALDI-MS with the biofoundry, and differentiated engineered and unengineered cells using the single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering, and change what is possibly using single-cell metabolomics in plants.

synthetic biology↗

bioRxiv2024

A longitudinal atlas of post-viral lung regeneration reveals persistent injury-associated cell states

Functional regeneration of the lungs gas exchange surface following injury requires the coordination of a complex series of cell behaviors within the alveolar niche. Using a multi-modal approach, we have mapped the temporal sequencing of mouse lung regeneration after acute viral injury, demonstrating that this response is asynchronously phased across different cellular compartments. This longitudinal atlas of regeneration has produced a catalogue of new cell states that reflect transient and persistent transcriptional alterations in daughter cells as they transit across axes of differentiation. These new cell states include an injury-induced capillary endothelial cell (iCAP) that arises after injury, persists indefinitely, and shares transcriptional hallmarks with both developing lung endothelium and the endothelial aberrations found in degenerative human lung diseases. This comprehensive atlas of lung regeneration provides a foundational resource to understand the complexity of the cellular and molecular responses to injury, reveals the critical importance of capillary endothelium in maintaining and rebuilding the alveolar niche after injury, and correlates these responses to those found in development and human lung diseases.

cell biology↗