Biology subjects

Xiang, H.

Publications and source records attributed to Xiang, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Efficient recovery of transgene-free edited tobacco using the transgene self-elimination system via de novo regeneration

Elimination of integrated editing constructs remains a major challenge for generating transgene-free progeny in plant genome editing based on Agrobacterium-mediated regeneration. Here, we adapted the Transgene Killer CRISPR (TKC) concept to tobacco and established the transgene self-elimination system, in which CRISPR/Cas9 is coupled with developmentally regulated BARNASE cassettes. Three pU3TR-derived vectors carrying DLL-, OsRP3- or PHAS-driven Barnase regenerated normally after leaf-disc transformation. The DLL construct pEB138 showed the highest T editing frequency, with 56 of 69 independent T plants (81.16%) carrying mutations at one or both NtDFR target loci. Under a stringent lineage-level screen in which up to 48 T progeny were tested per T line, 36 of 43 pEB138 lineages (83.72%) yielded uniformly marker-negative progeny for the three assayed T-DNA markers, compared with 0 of 7 lineages transformed with the parental pU3TR vector. The platform also supported recovery of dfr mutants with altered flower pigmentation and NtA622-edited lines with reduced nicotine accumulation. Moreover, introduction of DLL-iBarnase into the independent pDC45 editing backbone did not impair shoot regeneration, and representative NtBBL progeny showed markedly reduced nicotine content together with loss of the selectable cassette. These results demonstrate that BARNASE-assisted transgene elimination can be integrated with Agrobacterium-mediated de novo regeneration in a dicot, enabling one-step recovery of transgene-free edited tobacco progeny.

molecular biology↗

bioRxiv2026

CD59 Promotes SNARE Complex Assembly via Interaction with the Proline-Rich N-Terminal Domain of VAMP2

ObjectiveThe complement regulatory protein CD59 has been shown to promote SNARE complex assembly, yet its interaction with vesicle-associated membrane protein 2 (VAMP2) remains poorly characterized. This study aims to identify the key domain in VAMP2 that mediates the CD59 interaction and to evaluate whether CD59 point mutations affect SNARE complex assembly. MethodsThe interaction between CD59 and VAMP2 was examined by immunofluorescence confocal microscopy and co-immunoprecipitation (co-IP). The effect of CD59 on SNARE complex assembly was assessed by co-expressing CD59 with the three core SNARE proteins (syntaxin-1, SNAP-25, and VAMP2) and detecting complex formation by western blotting. Four CD59 single-point mutants were generated and evaluated in SNARE assembly assays. AlphaFold3 was employed to predict the interaction between CD59 and individual VAMP2 domains (confidence threshold: ipTM + pTM [≥] 0.75). Truncated VAMP2 constructs were further characterized by molecular dynamics simulations and co-IP. Results(1) CD59 directly bound VAMP2 and promoted SNARE complex assembly without altering individual SNARE protein levels. (2) All four CD59 single-point mutants retained the ability to promote SNARE assembly at a level comparable to wild-type CD59, despite showing differential effects on binding stability in molecular dynamics simulations. (3) The proline-rich (P-rich) N-terminal domain of VAMP2 was identified as the key binding interface; its deletion abolished the CD59 interaction, whereas deletion of the SNARE motif did not. ConclusionCD59 promotes SNARE complex assembly through interaction with the P-rich N-terminal domain of VAMP2. The examined point mutations do not impair this function, suggesting that these sites may tolerate substitutions or that redundant contact residues maintain the interaction.

neuroscience↗

bioRxiv2026

Reconstructing whole-organism cell phylogenies with resolved ancestral transcriptional states

Combining cell lineage tracing with single-cell RNA sequencing can reconstruct a phylogenetic tree to integrate single-cell transcriptomic atlas. However, the internal nodes of this tree - representing ancestral cells - remain transcriptionally silent, preventing along-lineage longitudinal tracing of cell state dynamics. Here, we overcome this by reconstructing high-resolution zygote-to-larva developmental cell phylogenies for 15 zebrafish larvae, with directly measured transcriptomes of the terminal nodes (i.e., sampled cells). Leveraging a set of lineage-committed upregulated genes (LUGs), we developed LUG-encoded ancestral projection (LEAP), a novel phylogeny-based computational framework, and successfully imputed the transcriptional states of internal nodes of the phylogenies. This enabled, for the first time in a non-nematode organism, lineage-informed longitudinal analysis of cell state dynamics throughout development. Our analysis revealed a major, previously unappreciated wave of fate specializations associated with hatching, distinct from the well-characterized events of gastrulation. Furthermore, we uncovered abundant incipient cell states that are already fate-determined but exhibit minimal transcriptional differentiation, revealing a hidden layer of developmental fate specializations. In sum, by resolving ancestral transcriptional states of a reconstructed cell phylogeny, this work paves the way for constructing lineage-resolved cell atlases in complex organisms to characterize comprehensive cell state dynamics.

developmental biology↗

bioRxiv2026

STING safeguards epithelial genome integrity and protects from carcinogenesis via mitotic checkpoint control

STING is canonically known for mediating interferon responses to cytosolic DNA, yet its cell-intrinsic role in genome maintenance beyond the immune context is unknown. Here we show that epithelial STING functions as a type I interferon-independent genome-integrity checkpoint. STING loss impairs homologous recombination repair, attenuates ATM-associated damage signaling, elevates CDK1 activity, and causes chromosomal instability revealed by single-cell Strand-seq, culminating in spontaneous intestinal adenocarcinoma. These defects arise before tumor formation and confer selective vulnerability to CDK inhibition in tumor organoids and human colorectal cancer cells. Our findings identify STING as a cell-autonomous guardian of epithelial genome stability that restrains chromosomal instability-driven tumor evolution beyond its canonical immune function.

cancer biology↗

bioRxiv2026

New insights into the evolution of spider silk proteins illuminated by long-read transcriptomes

Spider silk has long fascinated scientists because of its exceptional mechanical properties, yet the evolutionary origins and diversification of spider silk proteins (spidroins) remain incompletely understood. By analyzing long-read spidroin transcripts from spiders representing key evolutionary lineages, we identify two ancestral proteins present in basal spiders: an alanine-serine-rich (AS-type) protein and a glycine-serine-rich (GS-type) protein. These ancestral proteins likely served as primary evolutionary templates for the diversification of modern spider silks. We show that the AS-type ancestral spidroin remained relatively conserved and ultimately gave rise to the specialized tubuliform spidroin (TuSp) used in eggcase silk. In contrast, the GS-type protein underwent extensive functional radiation, evolving into differentiated minor ampullate spidroins (MiSp) through the acquisition of distinct terminal domains and {beta}-sheet-associated structural innovations. Our results further suggest that major ampullate spidroins (MaSp) may have originated from MiSp, with the GS motif representing an evolutionarily favorable genetic substrate for the emergence of high-performance silk proteins. Finally, we propose a revised evolutionary trajectory for flagelliform spidroins (Flag), suggesting that they were co-opted as components of ampullate silk during functional degeneration in modern non-web-weaving RTA clade spiders. Together, these findings provide a high-resolution framework for understanding the genetic innovations that drove the diversification of spider silk.

evolutionary biology↗

bioRxiv2025

Spatial Heterogeneity of Microbial Community Structure and Its Environmental Drivers in Surface Sediments of Erhai Lake

As a crucial plateau freshwater lake in Yunnan Province, China, Erhai Lake exhibits distinct environmental heterogeneity driven by its unique watershed characteristics and human activities, significantly influencing sediment microbial communities. This study investigated the spatial relationships between environmental factors and microbial community structures in surface sediments from the eastern, western, and northern shores using redundancy analysis (RDA) and Spearman correlation analysis. Results revealed that pH, total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), and redox potential (Eh) were key drivers of microbial community divergence. The western shore, with the highest TP, TOC, and nitrogen levels, displayed elevated microbial diversity dominated by Proteobacteria and Bacteroidetes, reflecting higher pollution loads. The northern shore exhibited severe nitrogen pollution, marked by the highest TN content and enrichment of Thiobacillus, potentially enhancing water self-purification. The eastern shore, with minimal anthropogenic disturbance, showed the highest bacterial diversity but the lowest nutrient concentrations. Fungal community structure was significantly influenced by pH, Eh, and TOC, while ecological restoration measures on the western shore enhanced fungal community stability. This study highlights how spatial heterogeneity in environmental factors regulates microbial community structure and function, ultimately affecting the stability of lake ecosystems. These findings provide a scientific basis for ecological restoration and sustainable management of plateau lakes.

ecology↗

bioRxiv2025

CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders

Transcriptional machinery often operates in the configuration of biomolecular condensates, presenting emerging opportunities to develop therapies targeting transcriptionally addicted malignancies. In this study, we discovered that cyclin-dependent kinase 12 (CDK12), a key regulator of transcription elongation, formed dynamic liquid-like droplets within nuclear speckles, a process enhanced by high CDK12 expression and CDK12 inhibitor treatment. Recognizing this unique property, we rationally designed ZSQ253, a modular cyclin K molecular glue degrader that coupled binding moieties for both CDK12 and DDB1. By promoting phase separation-driven assembly and subsequent proteasomal degradation of the CDK12-cyclin K complex, ZSQ253 interfered with oncogenic transcription and demonstrated potent antitumor capability. Further structure-guided optimization yielded HQY1428, a derivative with a multi-site gluing effect and improved drug activity. Given the frequent genomic co-amplification of CDK12 and ERBB2 across diverse human cancers, we showed that combining HQY1428 with HER2 inhibitors provided synergistic therapeutic benefits. Collectively, our findings establish CDK12 condensation within nuclear speckles as an exploitable vulnerability, and introduce a novel mechanism of action underlying cyclin K molecular glue degraders.

cancer biology↗

bioRxiv2024

MaskMol: Knowledge-guided Molecular Image Pre-Training Framework for Activity Cliffs with Pixel Masking

Activity cliffs, which refer to pairs of molecules that are structurally similar but show significant differences in their potency, can lead to model representation collapse and make the model challenging to distinguish them. Our research indicates that as molecular similarity increases, graph-based methods struggle to capture these nuances, whereas imagebased approaches effectively retain the distinctions. Thus, we developed MaskMol, a knowledge-guided molecular image selfsupervised learning framework. MaskMol accurately learns the representation of molecular images by considering multiple levels of molecular knowledge, such as atoms, bonds, and substructures. By utilizing pixel masking tasks, MaskMol extracts fine-grained information from molecular images, overcoming the limitations of existing deep learning models in identifying subtle structural changes. Experimental results demonstrate MaskMols high accuracy and transferability in activity cliff estimation and compound potency prediction across 20 different macromolecular targets, outperforming 25 state-of-the-art deep learning and machine learning approaches. Visualization analyses reveal MaskMols high biological interpretability in identifying activity cliff-relevant molecular substructures. Notably, through MaskMol, we identified candidate EP4 inhibitors that could be used to treat tumors. This study not only raises awareness about activity cliffs but also introduces a novel method for molecular image representation learning and virtual screening, advancing drug discovery and providing new insights into structure-activity relationships (SAR). Code is available at https://github.com/ZhixiangCheng/MaskMol.

bioinformatics↗

bioRxiv2024

Increased pathogenicity and transmission of SARS-CoV-2 Omicron XBB.1.9 sublineage, including HK.3 and EG.5.1

With the SARS-CoV-2 Omicron XBB.1.9 sublineage circulating worldwide, two XBB.1.9 variants, EG.5.1 and HK.3 spread rapidly and became dominant from middle 2023. However, the spike features, pathogenicity, and transmissibility of HK.3 are largely unknown. Here, we performed multiscale investigations to reveal the virological features of XBB.1.9 subvariants, especially the newly emerging HK.3. HK.3 revealed high replication efficiency in vitro. The HK.3 spike exhibited enhanced processing, although its infectivity, fusogenicity, and hACE2 binding affinity were comparable to those of the EG.5 and XBB.1 spikes. All XBB.1.9.1, EG.5.1 and HK.3 strains demonstrated efficient transmission in hamsters, although XBB.1.9.1 exhibited stronger fitness in the upper airways. HK.3 and EG.5.1 exhibited greater pathogenicity than XBB.1.9.1 and BA.2 in H11-K18-hACE2 hamsters. Our studies provide insight into the newly emerging pathogens HK.3 and EG.5.1. ImportanceIn animal models, the ongoing attenuated pathogenicity and poor transmission of Omicron subvariants seems to reach a consensus. However, our results revealed that Omicron XBB.1.9 subvariants, including one of the key variants of interest, EG.5 with its another key subvariant HK.3, universally exhibited both increased pathogenicity and highly transmission. This study reemphasized the importance of surveillance in characteristics of epidemic Omicron subvariants.

microbiology↗

bioRxiv2024

Virological characteristics of SARS-CoV-2 Omicron BA.5.2.48

With the prevalence of sequentially-emerged sublineages including BA.1, BA.2 and BA.5, SARS-CoV-2 Omicron infection has transformed into a regional epidemic disease. As a sublineage of BA.5, the BA.5.2.48 outbreak and evolved into multi-subvariants in China without clearly established virological characteristics, especially the pathogenicity. Though reduced airborne transmission and pathogenicity of former Omicron sublineages have been revealed in animal models, the virological characteristics of BA.5.2.48 was unidentified. Here, we evaluated the in vitro and in vivo virological characteristics of two isolates of the prevalent BA.5.2.48 subvariant, DY.2 and DY.1.1 (a subvariant of DY.1). DY.2 replicates more efficiently than DY.1.1 in HelahACE2+ cells and Calu-3 cells. The A570S mutation (of DY.1) in a normal BA.5 spike protein (DY.2) leads to a 20% improvement in the hACE2 binding affinity, which is slightly reduced by a further K147E mutation (of DY.1.1). Compared to the normal BA.5 spike, the double-mutated protein demonstrates efficient cleavage and reduced fusogenicity. BA.5.2.48 demonstrated enhanced airborne transmission capacity in hamsters than BA.2. The pathogenicity of BA.5.2.48 is greater than BA.2, as revealed in K18-hACE2 rodents. Under immune selection pressure, DY.1.1 shows stronger fitness than DY.2 in hamster turbinates. Thus the outbreaking prevalent BA.5.2.48 multisubvariants exhibites divergent virological features. ImportanceOmicron continues to circulate and evolves novel sublineages with indistinguishable pathogenicity and transmission. Therefore humanized Omicron-sensitive animal models must be applied to evaluate the virological characteritics and antiviral therapeutics. By using multiple models including the Omicron-lethal H11-K18-hACE2 rodents, BA.5.2.48 revealed higher pathogenicity in the novel H11-K18-hACE2 rodent models than the previously epidemic BA.2, and thus the models are more adapted to Omicron studies. Moreover, the regional outbreaking of BA.5.2.48 promotes the multidirectional evolution of its subvariants, gaining either enhanced pathogenicity or a fitness in upper airways which is associated with higher transmission, highlighting the importance of surveillance and virological studies on regionally endemic sublineages which represents the short-run evolutionary direction of Omicron.

microbiology↗

bioRxiv2024

Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells

Type I CRISPR-Cas systems are widespread and have exhibited remarkable versatility and efficiency in genome editing and gene regulation in prokaryotes. However, due to the multi-subunit composition and large size, their application in eukaryotes has not been thoroughly investigated. Here, we demonstrate that the type I-F2 Cascade, the most compact among type I systems and significantly smaller than SpCas9, can be developed into programmable tools for use in human cells. For transcriptional activation, the efficiency of the tool based on the engineered I-F2 system can match or surpass that of dCas9. Besides, narrow editing windows limit the application of base editors. Although the R-loop formed by Cascade is much wider than that by Cas9 or Cas12, the potential of base editing with Cascade has not yet been explored. We successfully created a base editor with the I-F2 Cascade, which induces a considerably wide editing window ([~]30 nt) with a bimodal distribution. The wide editing window can expand the range of targetable sites and can be useful for disrupting functional sequences and genetic screening. The editing efficiency can achieve 50% in human cells. This research underscores the application potential of compact type I systems in eukaryotes and developed a new base editor with an extraordinary wide editing window.

molecular biology↗

bioRxiv2023

Respiration and growth of Paracoccus denitrificans R-1 with nitrous oxide as an electron acceptor

In the nitrogen biogeochemical cycle, the reduction of nitrous oxide (N2O) to N2 by N2O reductase, which is encoded by nos gene cluster, is the only biological pathway for N2O consumption. However, the capability and mechanisms of microbial N2O reduction are poorly understood. In this study, we investigated the ability to obtain energy for growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. This strain has strong N2O reduction capability, and the average N2O reduction rate was 5.10{+/-}0.11x10-9 mol{middle dot}h-1{middle dot}cell-1 under anaerobic condition at 30 using acetate as the electron donor in a defined medium. This reduction was accompanied by the stoichiometric consumption of acetate over time when N2O served as the sole electron acceptor and the reduction can yield energy to support microbial growth, suggesting that microbial N2O reduction is an electron transport process. Cu2+, silver nanoparticles, O2, and acidic conditions can strongly inhibit the reduction, whereas NO3- or NH4+ can promote it. Genomic analysis showed that the gene cluster encoding N2O reductase of P. denitrificans R-1 was composed of nosR, nosZ, nosD, nosF, nosY, and nosL, and nosZ, which was identified as clade I. The respiratory inhibitors test indicated that the pathway of electron transport for N2O reduction was different from that of the traditional electron transport chain for aerobic respiration. These findings suggest that modular N2O reduction by P. denitrificans R-1 is linked to the electron transport chain and energy conservation, and that dissimilatory N2O reduction is a form of microbial anaerobic respiration. IMPOETANCEIn the nitrogen biogeochemical cycle, the reduction of N2O to N2 by N2O reductase, which is encoded by nos genes, is the only biological pathway for N2O consumption. However, the capacity and mechanisms of microbial N2O reduction are poorly understood. We investigated the ability to obtain energy for growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. Our study showed that the nosZ type I bacterium, P. denitrificans R-1, can respire N2O as the sole electron donor. Thus, the modular N2O reduction process of clade I denitrifiers not only can consume N2O produced by themselves but can also consume the external N2O generated from non-denitrification biological or abiotic pathways under suitable conditions, which is critical for controlling the release of N2O from ecosystems into the atmosphere.

microbiology↗

bioRxiv2023

An interface-based molecular generative framework for protein-protein interaction inhibitors

Protein-protein interactions (PPIs) play a crucial role in numerous biochemical and biological processes. Although several structure-based molecular generative models have been developed, PPI interfaces and compounds targeting PPIs exhibit distinct physicochemical properties compared to traditional binding pockets and small-molecule drugs. As a result, generating compounds that effectively target PPIs, particularly by considering PPI complexes or interface hotspot residues, remains a significant challenge. In this work, we constructed a comprehensive dataset of PPI interfaces with active and inactive compound pairs. Based on this, we propose a novel molecular generative framework tailored to PPI interfaces, named GENiPPI. Our evaluation demonstrates that GENiPPI captures the implicit relationships between the PPI interfaces and the active molecules, and can generate novel compounds that target these interfaces. Moreover, GENiPPI can generate structurally diverse novel compounds with limited PPI interface modulators. To the best of our knowledge, this is the first exploration of a structure-based molecular generative model focused on PPI interfaces, which could facilitate the design of PPI modulators. The PPI interface-based molecular generative model enriches the existing landscape of structure-based (pocket/interface) molecular generative model.

bioinformatics↗

bioRxiv2023

Genomic insights into post-domestication expansion and selection of body size in ponies

Horses domestication revolutionized human civilization by changing transportation, farming, and warfare patterns. Despite extensive studies on modern domestic horse origins, the intricate demographic history and genetic signatures of pony size demand further exploration. Here, we present a high-quality genome of the Chinese Debao pony and extensively analyzed 385 individuals from 49 horse breeds. We reveal the conservation of ancient components in East Asian horses and close relationships between Asian horses and specific European pony lineages. Genetic analysis uncovers Asian paternal origin for European pony breeds, and these pony-sized horses share a close genetic affinity due to the presence of a potential ancestral ghost pony population. Additionally, we identify promising cis-regulatory elements influencing horse withers height by regulating genes like RFLNA and FOXO1. Overall, our study provides insightful perspectives into the development history and genetic determinants underlying body size in ponies and offers broader implications for horse population management and improvement. TeaserDecoding pony genetics: exploring origins and size determinants sheds light on their historical and biological impacts.

genomics↗

bioRxiv2023

Global Marine Cold Seep Metagenomes Reveal Diversity of Taxonomy, Metabolic Function, and Natural Products

Cold seeps in the deep sea are closely linked to energy exploration as well as global climate change. The alkane-dominated chemical energy-driven model makes cold seeps an oasis of deep-sea life, showcasing an unparalleled reservoir of microbial genetic diversity. By analyzing 113 metagenomes collected from 14 global sites across 5 cold seep types, we present a comprehensive Cold Seep Microbiomic Database (CSMD) to archive the genomic and functional diversity of cold seep microbiome. The CSMD includes over 49 million non-redundant genes and 3175 metagenome-assembled genomes (MAGs), which represent 1897 species spanning 106 phyla. In addition, beta diversity analysis indicates that both sampling site and cold seep type have substantial impact on the prokaryotic microbiome community composition. Heterotrophic and anaerobic metabolisms are prevalent in microbial communities, accompanied by considerable mixotrophs and facultative anaerobes, indicating the versatile metabolic potential in cold seeps. Furthermore, secondary metabolic gene cluster analysis indicates that at least 98.81% of the sequences encode potentially novel natural products. These natural products are dominated by ribosomal processing peptides, which are widely distributed in archaea and bacteria. Overall, the CSMD represents a valuable resource which would enhance the understanding and utilization of global cold seep microbiomes.

microbiology↗

bioRxiv2023

Degenerated CRISPRs widely regulate Cas expression to balance immunity and cost

CRISPR RNAs (crRNAs) and Cas proteins together provide prokaryotes with adaptive immunity against genetic invaders. How Cas expression is fine-tuned to avoid energy burden while satisfying the dynamic need of crRNAs remains poorly understood. Here we experimentally demonstrated widespread degenerated mini-CRISPRs encode CreR (Cas-regulating) RNAs to mediate autorepression of type I-B, I-E and V-A Cas proteins, based on their partial complementarity to cas promoters. This autorepression decreases energy burden and autoimmune risks, thus mitigating the fitness cost on host cell, and remarkably, senses and responds to alterations in the volume of canonical crRNAs, which compete with CreR for Cas proteins. Moreover, CreR-guided Cas autorepression can be subverted by diverse anti-CRISPR (Acr) proteins that destruct Cas proteins, which in turn replenishes the weapon depot. Our data unveil a general degenerated crRNA-guided autorepression paradigm for diverse Cas effectors, which highlights the intricate (self-)regulation of CRISPR-Cas and its transcriptional counterstrategy against Acr attack.

microbiology↗

bioRxiv2023

Tat-fimbriae ("tafi") - novel type of haloarchaeal surface structures

In present study, we describe "tat-fimbriae (tafi)" - a novel type of archaeal surface appendages isolated from haloarchaeon Haloarcula hispanica. These filamental structures are unique because they are formed of protein subunits secreted through the twin-arginine translocation pathway (Tat-pathway), in contrast to well-known archaeal surface filamentous structures secreted by the general secretory pathway (Sec-pathway). No cases of the role of Tat-pathway in the assembly of archaeal and bacterial filamentous structures have been described to date. "Tafi" are the first example of such structures. The precursor of the major tafi protein subunit TafA contains the N-terminal signal peptide carrying a twin-arginine consensus motif and fimbria-forming mature TafA lacks this signal peptide. We analyzed the gene neighborhood of the tafA homologues in the known haloarchaeal genomes and found a conservative cluster of seven associated genes tafA, B, C, D, E, F, G. We assume that all of them take part in the tafi synthesis. TafC and TafE proteins, whose precursor sequences also contain twin-arginine motifs, were detected as minor components of tafi. TafE protein is structurally similar to TafA, while TafC contains a TafA-like N-terminal domain and a C-terminal "laminin G-like" domain capable of functioning as an adhesin. TafD is annotated as a signal peptidase I. The functions of TafB, TafF and TafG are not known yet. This study demonstrated that {Delta}tafA and {Delta}tafD deletion mutant strains synthesized archaella and not tafi, and only tafi were detected in {Delta}arlK (gene of common archaellin/pilin signal peptidase) deletion strain. It was shown that the expression of complete Har. hispanica taf-gene cluster in a heterologous host Haloferax volcanii that does not have similar genes leads to synthesis of recombinant tafi structures similar to the native ones. The tafi function remains elusive, but our preliminary data suggest that these structures may be involved in cell adhesion to different surfaces or substrates.

microbiology↗

bioRxiv2023

High-throughput screening of functional neo-antigens and their specific TCRs via the Jurkat reporter system combine with droplet microfluidics

T-cell receptor (TCR)-engineered T cells can precisely recognize a broad repertoire of targets derived from both intracellular and surface proteins of tumor cells. TCR-T adoptive cell therapy has shown safety and promising efficacy in solid tumor immunotherapy. However, antigen-specific functional TCR screening is time-consuming and expensive, which limits its application clinically. Here, we developed a novel integrated antigen-TCR screening platform based on droplet microfluidics technology, enabling high-throughput peptide-major histocompatibility complex (pMHC) library-to-TCR library screening with high sensitivity and low background signal. We introduced DNA barcoding technology to label peptide antigen candidate-loaded antigen-presenting cells (APCs) and Jurkat reporter cells to check the specificity of pMHC-TCR candidates. Coupled with the next-generation sequencing pipeline, interpretation of the DNA barcodes and the gene expression level of the Jurkat T-cell activation pathway provided a clear peptide-MHC-TCR recognition relationship. Our proof-of-principle study demonstrates that the platform could achieve unbiased pMHC-TCR library-on-library screening, which is expected to be used in the cross-reactivity and off-target testing of candidate pMHC-TCR libraries in clinical applications.

bioengineering↗