Biology subjects

Gong, P.

Publications and source records attributed to Gong, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Make it so: Rapid and affordable plasmid sequencing on ONT platforms with PICARD-seq

Plasmid construction underpins molecular biology and synthetic biology, yet validation is often limited to the inserted fragment rather than the whole plasmid, and around a third of laboratory-made plasmids carry sequence errors that can affect function. Sanger sequencing scales poorly across whole plasmids, while short-read approaches cannot resolve the repeated DNA parts, such as promoters, that are common in synthetic constructs. We present PICARD-seq, a rapid nanopore-based protocol that uses off-the-shelf Tn5 rapid barcoding reagents and a MinION to sequence pools of whole plasmids in under a day, and we systematically benchmark the computational pipelines available for analysing the resulting data. Using a curated set of 25 plasmids of known sequence spanning 3.0-20.6 kbp, including various dilution series and repetitive multi-part constructs, we ran five independent replicates of each pipeline. The ONT EPI2ME Clone Validation workflow was fast (13-18 min) but stochastic, varying between replicates for both plasmids assembled and what sequence was returned; Canu outperformed the default Flye assembler, and reducing the minimum coverage parameter from 60x to 20x substantially improved assembly of large, repetitive, and dilute samples. The ensemble assembler Autocycler was slower (81-111 min) but gave the highest and most consistent rate of recovering the expected sequence. Complementary read mapping with minimap2 distinguished genuine sequence differences from assembly artefacts. Applying PICARD-seq to problematic plasmids revealed backbone concatemers, a misincorporated promoter part, and a mixed population of rearranged molecules in a repetitive construct. PICARD-seq makes routine whole-plasmid validation practical and affordable for individual laboratories.

bioinformatics↗

bioRxiv2026

A virus co-opts vector's m⁶A machinery for adaptive compensation

Insect vectors harbor diverse viruses that threaten human health and agricultural production, yet the genetic basis of mutualistic virus-vector interactions remain elusive. This underscores a long-standing paradox in pest management: viruses that cause destructive diseases also facilitate vector adaptation in ways that benefit both parties. Here we show that the globally devastating tomato yellow leaf curl virus (TYLCV) acts as a Cooperative Partner, co-opting its vector's m6A epitranscriptomic machinery to offset the reproductive fitness costs of neonicotinoid resistance in the whitefly. In resistant vectors, TYLCV infection alleviates reproductive deficits via the m6A-dependent pathway, where the methyltransferase METTL14 and demethylase ALKBH4 coordinately stabilize vitellogenin (Vg) transcripts to boost female fecundity. Mechanistically, the viral C2 and CP proteins directly interact with METTL14 and ALKBH4, respectively, remodeling their RNA binding affinity to facilitate mA modification on Vg. Disrupting either of these viral-vector protein interactions restores a reproductive cost phenotype in viruliferous resistant vectors, offering novel targets for sustainable pest control. Our findings uncover a fundamental role of epigenetic marks in adaptive trait, reshaping beneficial virus-vector relationships and providing new insights for innovative public health and crop protection strategies to address global challenges.

microbiology↗

bioRxiv2026

RNA m6A demethylase ALKBH4 governs whitefly development

N6-methyladenosine (m6A) modification is the most predominant and ubiquitous internal modification of RNA in eukaryotes, serving as a key post-transcriptional regulator of gene expression that is dynamically modulated by methyltransferases (writers) and demethylases (erasers). However, while the functions of m6A methylases have been partially elucidated in insects, the identity of m6A erasers in arthropods and their chemical catalytic mechanisms, as well as biological functions, remains largely enigmatic. Here, we uncovered 2499 putative methylase genes and 1148 putative demethylase genes in 266 insect genomes, and demonstrated that ALKBH4 functions as an m6A demethylase in the whitefly, Bemisia tabaci, catalyzing the oxidative reversal of mRNA m6A modifications both in vitro and in vivo. Furthermore, we established that ALKBH4, in coordination with other core components of the m6A pathway, fulfills an essential function in regulating the transcript stability of Imaginal Disk Growth Factor 1 (IDGF1) during whitefly development. Collectively, our findings expand the evolutionary scope of the eukaryotic m6A modification system, and reveal a conserved yet insect-specific epitranscriptomic regulatory mechanism governing fundamental physiological processes and adaptive phenotypes. Significance statementThe addition of a methyl group to the N6-position of adenosine (m6A) is a highly abundant chemical modification of RNA. However, the functional role of m6A in insects and the key enzymes that regulate its levels remains poorly understood. In this study, we explored putative methylase genes and demethylase genes in hundreds insect genomes, and identified an m6A RNA demethylase, ALKBH4, in the whitefly, Bemisia tabaci. We demonstrate that ALKBH4 oxidatively reverses mRNA methylation in vivo and in vitro, in combination with other components of the m6A pathway, plays an important role in whitefly development. These findings provide new insight into m6A methylation system of insect.

zoology↗

bioRxiv2025

Engineered translation release factor 1 suppresses disease-causing nonsense mutations and enables multi-site nonstandard amino acids incorporation

Nonsense mutations convert sense codons into premature termination codons (PTC), resulting in early termination of translation of mRNAs, underlie [~]11% of human genetic diseases. Restoring translation of genes carrying nonsense mutations remains a major therapeutic challenge. Here we present OPENER (Overexpression of Protein-engineered eRF1 for Nonsense Elision and Readthrough), a strategy to efficiently suppress nonsense mutations based on engineered variants of the eukaryotic translation termination factor eRF1. Through saturation mutagenesis of the human eRF1 N-domain, we identified variants, notably eRF1S77E, that promote efficient readthrough of all three PTCs (UAG, UGA, UAA) and rescue 12 pathogenic PTCs reporters in multiple disease contexts. Systemic delivery of OPENER-S77E via AAV8 in a Duchenne muscular dystrophy mouse model (DmdQ995X) restored dystrophin expression, improved muscle function, and ameliorated pathology without detected toxicity. Furthermore, when combined with orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs, OPENER-S77E enabled efficient, multi-site incorporation of nonstandard amino acids (BocK and AzF) into a single protein in mammalian cells, achieving efficiency of 27.77% and 19.83%, respectively. The incorporation efficiency of BocK was further enhanced to 32.80% in conditional eRF1-knockout cells. OPENER thus provides a platform for treating nonsense-mediated diseases and expands the synthetic biology toolkit for precise protein engineering.

synthetic biology↗

bioRxiv2025

CREation of an expanded plant memory gene circuit toolkit

Genetic circuits rely on modular, well-characterized genetic parts to achieve predictable cellular behaviour. Despite their widespread adoption, biological parts are complex and when used in a new molecular context can result in unexpected outcomes. Site-specific recombinases have been widely used due to their unique ability to induce precise, irreversible changes to a DNA sequence, making them ideal tools for memory logic operations. However, unexpected context-specific failures of even the most widely used recombinase, Cre, has limited the expansion and complexity of synthetic gene circuits in diverse species. Here, through a systematic analysis of Cre failure in plant gene circuits, we uncovered multiple unexpected post-recombination interactions within the transcriptional unit between the promoter region, recombinase, and their cognate recognition sites. These significantly inhibit transcriptional activity, preventing circuit functionality. Specifically, post-recombination Cre recombinase exhibits an inhibitory property by binding to lox sites, with the remaining lox site repressing transcription. By thoroughly characterizing these dynamics, we restored Cre functionality and expanded the plant-based recombinase toolkit for Cre-related recombinases, developing combinations of recombinases and cognate sites with different activation levels for constructing logic gates with stable memory functions. We further developed multiple functional split-recombinase systems for simultaneous logic using a range of dimerization domains and constructed complex logic gates, including 3-input and 6-input AND gates. Together, this exploration of component context dependency in genetic circuits, even with commonly used parts like the Cre/lox system, has implications extending beyond plant systems, and significantly expands plant memory circuit capabilities.

synthetic biology↗

bioRxiv2025

A Rapid and Highly Sensitive CRISPR Assay Utilizing Cas12a Orthologs for the Detection of Novel Duck Reovirus

The novel duck reovirus (NDRV) disease presents a significant threat to the poultry industry due to the absence of effective therapeutic measures. As a result, there is an urgent need to develop innovative rapid diagnostic methods for early virus detection. In this study, we developed a Rapid Visual CRISPR Assay to detect the NDRV S3 gene using novel Cas12a orthologs. Specifically, we compared the performance of two candidates, Gs12-16 and Gs12-18, in detecting the NDRV S3 gene to identify a highly sensitive and efficient CRISPR-based diagnostic method. Our results demonstrated that both Gs12-16 and Gs12-18 exhibited strong cis- and trans-cleavage activities for classical "TTTV" protospacer adjacent motif (PAM)-containing targets in vitro, although they required different reaction temperatures. Notably, Gs12-18 showed relatively higher activity for dsDNA targets compared to Gs12-16, indicating that Gs12-18 is more suitable for CRISPR-based nucleic acid detection applications. To leverage these properties, we integrated Gs12-18 with loop-mediated isothermal amplification (LAMP) technology to establish a LAMP-CRISPR/Gs12-18-mediated method for detecting the NDRV S3 gene. This approach enables highly sensitive and visually detectable on-site identification of the NDRV S3 gene, achieving a sensitivity of 38 copies per reaction. Our LAMP-CRISPR/Gs12-18-based method can be utilized for highly sensitive detection of NDRV nucleic acids.

synthetic biology↗

bioRxiv2024

Unexpected Activities of CYP152 Peroxygenases towards Non-carboxylic Substrates Reveal Novel Substrate Recognition Mechanism and Catalytic Versatility

Exploring and exploiting the catalytic promiscuity of enzymes is a central topic and captivating challenge in enzymology. CYP152 peroxygenases are attractive biocatalysts for diverse reactions under mild conditions using H2O2 as cofactor. However, their substrate scope is limited by a carboxyl group required for acid-base catalysis, following the well-accepted principle that heme-dependent H2O2-utilizing enzymes employ a carboxyl group within their active sites to facilitate H2O2 activation. Herein, we reveal for the first time that several CYP152 family members can directly degrade various aromatic pollutants without any carboxyl group, exhibiting novel aromatic hydroxylation and dehalogenation activities. Through crystal structure analysis, isotope tracing experiments, and QM/MM calculations, we elucidate that the phenolic hydroxyl group activated by electron-withdrawing substituent(s) functionally replaces the carboxyl group, forming hydrogen bonds with the conserved arginine leading to Compound I formation. The oxygen atom of the newly formed hydroxyl group originates from water, bypassing the conventional oxygen rebound step. These findings provide first insights into the mechanisms of P450 peroxygenases towards non-carboxylic substrates, expanding our knowledge of biological C-H activation and C-halogen bond cleavage beyond canonical P450 reactions. This discovery holds immense potential for harnessing these enzymes in innovative strategies for industrial biocatalysis and environmental remediation.

biochemistry↗

bioRxiv2024

Synfire Chain Dynamics Unravelling Theta-nested Gamma Oscillations for Balancing Prediction and Dodge in Navigation

Theta-nested gamma oscillations, widely observed in experiments, play a crucial role in navigation, yet their functional roles and the origin of the positive correlation between theta frequency and motion velocity remain unclear. We propose that the objects survival relies on both prediction and dodge - predicting future events and staying alert to unpredictable ones, the latter of which has seldom been considered in goal-navigation tasks. By building a biologically plausible spiking neuronal network model and reproducing experimental results, we leverage synfire chain properties - length and separation - to elucidate the functional roles of theta-nested gamma oscillations: theta oscillations for self-location awareness, gamma oscillations for predictive capabilities and their coupling for enhancing functionality. The positive correlation between theta frequency and motion velocity is demonstrated to optimally balance representing predictable events for planning and staying alert to unexpected events. Our study offers a new avenue for unravelling the neural mechanisms of navigation.

neuroscience↗

bioRxiv2023

Interferon-Induced Bone Marrow Stromal Antigen 2 (BST2) Is A Functional Tumor-Initiating Cell Marker In Triple-Negative Breast Cancer

A tumor cell subpopulation of tumor-initiating cells (TIC), or "cancer stem cells", are associated with therapeutic resistance, as well as both local and distant recurrence. Enriched populations of TIC are identified by markers including aldehyde dehydrogenase (ALDH1) activity, the cell surface marker combination CD44+/CD24-, or fluorescent reporters for signaling pathways that regulate TIC function. We showed previously that Signal Transducer and Activator of Transcription (STAT)-mediated transcription allows enrichment for TIC in claudin-low models of human triple-negative breast cancer using a STAT-responsive reporter. However, the molecular phenotypes of STAT TIC are not well understood, and there is no existing method to lineage-trace TIC as they undergo cell state changes. Using a new STAT-responsive lineage-tracing (LT) system in conjunction with our original reporter, we enriched for cells with enhanced mammosphere-forming potential in some, but not all, basal-like triple-negative breast cancer (TNBC) xenograft models (TNBC) indicating TIC-related and TIC-independent functions for STAT signaling. Single-cell RNA sequencing (scRNAseq) of reporter-tagged xenografts and clinical samples identified a common interferon (IFN)/STAT1-associated transcriptional state, previously linked to inflammation and macrophage differentiation, in TIC. Surprisingly, most of the genes we identified are not present in previously published TIC signatures derived using bulk RNA sequencing. Finally, we demonstrated that bone marrow stromal cell antigen 2 (BST2), is a cell surface marker of this state, and that it functionally regulates TIC frequency. These results suggest TIC may exploit the IFN/STAT1 signaling axis to promote their activity, and that targeting this pathway may help eliminate TIC. SignificanceTIC differentially express interferon response genes, which were not previously reported in bulk RNA sequencing-derived TIC signatures, highlighting the importance of coupling single-cell transcriptomics with enrichment to derive TIC signatures.

cancer biology↗

bioRxiv2023

Migratory herbivorous waterfowl couple the seed wave and the green wave during spring migration

East Asian herbivorous waterfowl intensively use farmland in spring, next to their natural habitat. Accordingly, they might have expanded their migration strategy from merely tracking the green wave of newly emerging vegetation to also incorporating the availability of post-harvest agricultural seeds (here dubbed the seed wave). However, if and how waterfowl use multiple food resources to time their seasonal migration is still unknown. We test this migration strategy using 167 spring migration tracks of five East Asian herbivorous waterfowl species and mixed-effect resource selection function models. We find all study species arrive at their core stopover sites in the Northeast China Plain after agricultural seeds become available, extend their stay after spring vegetation emerges, and arrive at breeding sites around the emergence of vegetation. Moreover, the cue used in exploiting the availability of both food resources varies in different regions and among species. At the core stopover sites, larger-bodied species use the seed wave and smaller-bodied ones adopt the green wave. At the breeding sites, the former follow the green wave, and the latter track the snow-free time. Our findings suggest that waterfowl track multiple resource waves to finetune their migration, highlighting new opportunities for conservation.

ecology↗

bioRxiv2023

A cataract-causing Y204X mutation of CRYβB1 promotes C-terminal degradation and higher-order oligomerization

Crystallin (Cry) proteins are a class of main structural proteins of vertebrate eye lens, and their solubility and stability directly determine transparency and refractive power of the lens. Mutation in genes that encode for these Cry proteins is the common cause for congenital cataract. Despite extensive studies, the pathogenic and molecular mechanisms remain unclear. In this study, we identified a novel mutation in CRYBB1 from a congenital cataract family, and demonstrated that this mutation led to an earlier termination of protein translation, resulting in a 49-residue truncation at the CRY{beta}B1 C-terminus. This mutant is susceptible to proteolysis and allows us to determine a 1.2- [A] resolution crystal structure of CRY{beta}B1 without the entire C-terminal domain. In this crystal lattice, two N-terminal domain monomers form a dimer that structurally resembles a wild-type (WT) monomer, but with different surface characteristics. Biochemical analyses suggest that this mutant is significantly more liable to aggregate and degrade, when compared to WT CRY{beta}B1. All our results provide an insight into the mechanism regarding how a mutant Cry contributes to the development of congenital cataract possibly through alteration of inter-protein interactions that result in the opacity of eye lens.

biochemistry↗

bioRxiv2023

Duck pan-genome reveals two transposon-derived structural variations caused bodyweight enlarging and white plumage phenotype formation during evolution

Structural variations (SVs) are a major source of domestication and improvement traits, however SV profiles of duck and their phenotypic impacts largely hidden. We present the first duck pan-genome constructed using five genome assemblies capturing [~]40.98 Mb new sequences. This pan-genome together with high-depth sequencing data ([~]46.5X) identified 101,041 SVs, of which substantial proportions were derived from transposable element (TE) activity. Many TE-derived SVs anchoring in a gene body or regulatory region are linked to ducks domestication and improvement. By combining quantitative genetics with molecular experiments, we dissect how TE-derived SVs change gene expression of IGF2BP1 and generate a novel transcript of MITF, shaping bodyweight and white plumage. In the IGF2BP1 locus, the TE-derived SV explains the largest effect on bodyweight among avian species (27.61% of phenotypic variation). Our findings highlight the importance of using a pan-genome as a reference in genomics studies and explore the roles of TE-derived SVs in trait formation and in livestock breeding.

genetics↗

bioRxiv2023

Identification and Characterization of novel long non-coding RNAs in vascular smooth cells

A significant portion of the RNA produced from the human genome consists of long non-coding RNAs (lncRNAs). These molecules tend to have lower levels of expression, are more specific to certain tissues, and show greater variation in expression between individuals compared to protein-coding messenger RNAs (mRNAs). LncRNAs have been linked with regulatory roles in gene expression and genome architecture. There is growing evidence that lncRNAs play important roles in many biological processes and diseases, and a number of lncRNAs have been identified as potential therapeutic targets. Here, we report the identification and characterization of the lncRNA landscape of vascular smooth muscle cells (VSMC). We used an ensemble of bioinformatics tools to identify 329 novel lncRNAs from a large VSMC RNA-Seq dataset. We found that majority of the novel lncRNAs are natural antisense transcripts of protein-coding genes. In addition, we predicted cellular localization and potential miRNAs that targets the novel lncRNAs and found that most localize in the cytoplasm and that miRNA target site ranged from 2-889 sites on each novel lncRNA. Furthermore, we identified co-expressed lncRNAs that correlate with the proliferation, migration and apoptosis of vascular smooth muscle cells. These results suggest that we have identified a diverse set of previously unknown lncRNAs that may be involved in important regulatory pathways in vascular smooth muscle cells.

bioinformatics↗

bioRxiv2022

Segmented MS/MS acquisition of a1 ion-based strategy for in-depth proteome quantitation

In-depth proteome quantitation is of great significance for understanding protein functions, advancing biological, medical, environmental and metabolic engineering research. Herein, benefiting from the high formation efficiencies and intensities of dimethyl-labeled a1 ions for accurate quantitation, we developed an in-depth a1 ion-based proteome quantitation method, named deep-APQ, by a sequential MS/MS acquisition of the high mass range for identification and the low mass range for a1 ion intensity extraction to increase quantitative protein number and sequence coverage. By the analysis of HeLa protein digests, our developed method showed deeper quantitative coverage than our previously reported a1 ion-based quantitation method without mass range segmentation and lower missing values than widely-used label-free quantitation method. It also exhibited excellent accuracy and precision within a 20-fold dynamic range. We further integrated a workflow combining the deep-APQ method with highly efficient sample preparation, high-pH and low-pH reversed-phase separation and high-field asymmetric waveform ion mobility spectrometry (FAIMS) to study E. coli proteome responses under the nutritional conditions of glucose and acetate. A total of 3447 proteins were quantified, representing 82% of protein-coding genes, with the average sequence coverage up to 40%, demonstrating the high coverage of quantitation results. We found that most of the quantified proteins related to chemotaxis were differentially expressed, including the low-abundance proteins such as trg, fliL, and cheA, indicating that chemotaxis would play an important role for E. coli cell to survive from acetate toxicity. The above results demonstrated that the deep-APQ method is of great promising to achieve the deep-coverage proteome quantitation with high confidence. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/501662v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1457bdeorg.highwire.dtl.DTLVardef@c74e53org.highwire.dtl.DTLVardef@e72e9forg.highwire.dtl.DTLVardef@1a5cc43_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Synthetic memory circuits for programmable cell reconfiguration in plants

Plant biotechnology predominantly relies on a restricted set of genetic parts with limited capability to customize spatiotemporal and conditional expression patterns. Synthetic gene circuits have the ability to integrate multiple customizable input signals through a processing unit constructed from biological parts, to produce a predictable and programmable output. Here, we present a suite of functional recombinase-based gene circuits for use in plants. We first established a range of key gene circuit components compatible with plant cell functionality. We then used these to develop a range of operational logic gates using the identify function (activation) and negation function (repression) in Arabidopsis protoplasts and in vivo, demonstrating their utility for programmable manipulation of transcriptional activity in a complex multicellular organism. Through utilization of genetic recombination these circuits create stable long-term changes in expression and recording of past stimuli. This highly-compact programmable gene circuit platform provides new capabilities for engineering sophisticated transcriptional programs and previously unrealised traits into plants.

synthetic biology↗

bioRxiv2022

SpotLink enables sensitive and precise identification of site non-specific cross-links at the proteome scale

We developed SpotLink software for identifying site non-specific cross-links at the proteome scale. Contributed by the dual pointer dynamic pruning (DPDP) algorithm and the quality control of cross-linking sites, SpotLink identified more than 3000 cross-links from human proteome database with rich site information in a few days. We demonstrated that SpotLink outperformed other approaches in terms of sensitivity and precision on a simulated dataset and a protein complexes dataset with known structures. Additionally, we discovered some valuable protein-protein interaction (PPI) information contained in the protein complexes dataset and HeLa dataset, indicating the unique identification advantages of site non-specific cross-linking. The excellent performance of SpotLink will increase the usage of site non-specific cross-linking in the near future. SpotLink is publicly available on GitHub [https://github.com/DICP1810/SpotLink].

bioinformatics↗

bioRxiv2021

pGlycoQuant with a deep residual network for precise and minuscule-missing-value quantitative glycoproteomics enabling the functional exploration of site-specific glycosylation

Interpreting large-scale glycoproteomic data for intact glycopeptide identification has been tremendously advanced by software tools. However, software tools for quantitative analysis of intact glycopeptides remain lagging behind, which greatly hinders exploring the differential expression and functions of site-specific glycosylation in organisms. Here, we report pGlycoQuant, a generic software tool for accurate and convenient quantitative intact glycopeptide analysis, supporting both primary and tandem mass spectrometry quantitation for multiple quantitative strategies. pGlycoQuant enables intact glycopeptide quantitation with very low missing values via a deep residual network, thus greatly expanding the quantitative function of several powerful search engines, currently including pGlyco 2.0, pGlyco3, Byonic and MSFragger-Glyco. The pGlycoQuant-based site-specific N-glycoproteomic study conducted here quantifies 6435 intact N-glycopeptides in three hepatocellular carcinoma cell lines with different metastatic potentials and, together with in vitro molecular biology experiments, illustrates core fucosylation at site 979 of the L1 cell adhesion molecule (L1CAM) as a potential regulator of HCC metastasis. pGlycoQuant is freely available at https://github.com/expellir-arma/pGlycoQuant/releases/. We have demonstrated pGlycoQuant to be a powerful tool for the quantitative analysis of site-specific glycosylation and the exploration of potential glycosylation-related biomarker candidates, and we expect further applications in glycoproteomic studies.

systems biology↗

bioRxiv2021

SAMBA controls the rate of cell division in maize development through APC/C interaction

SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex (APC/C) which controls unidirectional cell cycle progression in Arabidopsis, but so far its role was not studied in monocots. Here, the association of SAMBA with APC/C was shown to be conserved in maize. Two samba CRISPR alleles showed growth defects that aggravated with plant age such as dwarfed plants due to shortened upper leaf length, erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion. Despite the fact that in both alleles the frameshift occurred at the same position, the two alleles differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knock-out allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity or efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal.

plant biology↗