Biology subjects

Lin, K.

Publications and source records attributed to Lin, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Tumor-targeted delivery of Tetanus toxoid by Listeria improves immunotherapy against ovarian cancer in mice

Ovarian cancer is known for its poor neoantigen expression and strong immunosuppression. Here, we utilized an attenuated non-pathogenic bacterium Listeria monocytogenes to deliver a highly immunogenic Tetanus Toxoid protein (Listeria-TT), as a neoantigen surrogate, into tumor cells through infection in a metastatic mouse ovarian cancer model (Id8p53-/-Luc). Gemcitabine (GEM) was added to reduce immune suppression. Listeria-TT+GEM treatments resulted in tumors expressing TT and reactivation of pre-existing CD4 and CD8 memory T cells to TT (generated early in life). These T cells were then attracted to the TT-expressing tumors now producing perforin and granzyme B. This correlated with a strong reduction in tumor burden, and significant improvement of the survival time compared to all control groups. Checkpoint inhibitors have little effect on ovarian cancer partly because of low neoantigen expression. Here we demonstrated that Listeria-TT+GEM+anti-PD1 was significantly more effective (efficacy and survival) than anti-PD1 or Listeria-TT+GEM alone. Of clinical interest, high doses of anti-PD1 (PD1H) (when added to Listeria-TT+GEM) were less effective than the low doses (PD1L). IHC and ELISPOT demonstrated that high doses of anti-PD1 inhibited T cell function in the TME. Using RNAseq, Differentially Expressed Genes (DEG) analysis and Genes Set Enrichment Analysis (GSEA) showed that gene expression levels and biological pathways were predominantly upregulated in the PD1H compared to the PD1L group, in correlation with low immune infiltration in tumors, more immune suppression, and more aggressive ovarian cancer. In summary, this study suggests that our approach may benefit ovarian cancer patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/561944v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@197942eorg.highwire.dtl.DTLVardef@8180d4org.highwire.dtl.DTLVardef@3113e6org.highwire.dtl.DTLVardef@116851_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Human concept: Childhood vaccinations with the highly immunogenic tetanus toxoid (TT) generate TT-specific memory T cells, which circulate in the blood for life. After appearance of ovarian cancer (late in life), the patients will receive one high dose with Listeria-TT to deliver TT into tumor cells, followed by multiple low doses of Listeria-TT over a period of 2 weeks to restimulate the pre-existing memory T cells to TT. MDSC are involved in the delivery of Listeria-TT to the TME. Reactivated memory T cells will in turn destroy the tumor cells expressing TT. Multiple low doses of GEM will be added after TT has been delivered at the tumor site, which reduce immune suppression by eliminating MDSC and TAM (not shown here). Since individuals have seen TT earlier in life (during childhood vaccinations) and since TT is highly immunogenic (attracting T cells to the TME) but not expressed in normal cells, TT functions here as a vaccine recall antigen and as a neoantigen surrogate, respectively. C_FIG

cancer biology↗

bioRxiv2023

Affinity hierarchies underlie the co-assembly of nucleolar and heterochromatin condensates in Drosophila embryos

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. Nucleoli are the sites of ribosome synthesis, while the repeat-rich PCH is essential for chromosome segregation, genome stability, and transcriptional silencing. How and why these two distinct condensates co-assemble is unclear. Here, using high-resolution live imaging of Drosophila embryogenesis, we find that de novo establishment of PCH around the nucleolus is highly dynamic, transitioning from the nuclear edge to surrounding the nucleolus. Eliminating the nucleolus by removing the ribosomal RNA genes (rDNA) resulted in increased PCH compaction and subsequent reorganization into a toroidal structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or neocondensates, including enrichment in the PCH toroidal hole. Combining these observations with physical modeling revealed that nucleolar-PCH associations can be mediated by a hierarchy of interaction strengths between PCH, nucleoli, and amphiphilic protein(s) that have affinities for both nucleolar and PCH components. We validated this model by identifying a candidate amphiphile, a DEAD-Box RNA Helicase called Pitchoune, whose depletion or mutation of its PCH interaction motif disrupted PCH-nucleolar associations. Together, this study unveils a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrates that nucleoli are required for normal PCH organization, and identifies Pitchoune as an amphiphilic molecular link required for PCH-nucleolar associations.

cell biology↗

bioRxiv2023

A scalable platform for efficient CRISPR-Cas9 chemical-genetic screens of DNA damage-inducing compounds

Current approaches to define chemical-genetic interactions (CGIs) in human cell lines are resource-intensive. We designed a scalable chemical-genetic screen platform by generating a DNA damage response (DDR)-focused custom sgRNA library. We performed five proof-of-principle compound screens and found that the compounds known modes-of-action (MoA) were enriched among the compounds CGIs. These scalable screens recapitulated expected CGIs at a comparable signal-to-noise ratio (SNR) relative to genome-wide screens. Furthermore, time-resolved CGIs, captured by sequencing screens at various time points, suggested an unexpected, late time point interstrand-crosslinking (ICL) repair pathway response to camptothecin-induced DNA damage. Our approach can facilitate screening compounds at scale and produce biologically informative CGI profiles.

systems biology↗

bioRxiv2023

Genetics of skeletal proportions in two different populations

Human height can be divided into sitting height and leg length, reflecting growth of different parts of the skeleton whose relative proportions are captured by the ratio of sitting to total height (as sitting height ratio, SHR). Height is a highly heritable trait, and its genetic basis has been well-studied. However, the genetic determinants of skeletal proportion are much less well-characterized. Expanding substantially on past work, we performed a genome-wide association study (GWAS) of SHR in [~]450,000 individuals with European ancestry and [~]100,000 individuals with East Asian ancestry from the UK and China Kadoorie Biobanks. We identified 565 loci independently associated with SHR, including all genomic regions implicated in prior GWAS in these ancestries. While SHR loci largely overlap height-associated loci (P < 0.001), the fine-mapped SHR signals were often distinct from height. We additionally used fine-mapped signals to identify 36 credible sets with heterogeneous effects across ancestries. Lastly, we used SHR, sitting height, and leg length to identify genetic variation acting on specific body regions rather than on overall human height.

genetics↗

bioRxiv2023

The ENCODE Uniform Analysis Pipelines

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses. Database URL: https://www.encodeproject.org/

bioinformatics↗

bioRxiv2023

Reversing myeloid-derived suppressor cells mediated immunosuppression via p38α inhibition enhances immunotherapy efficacy in triple negative breast cancer

Infiltration of myeloid-derived suppressor cells (MDSCs) leads to Immunosuppressive tumor microenvironment (TME), which is one of the major causes for low objective response rates of immune checkpoint blockade (ICB) therapy. Here, we report that chemical inhibition of p38 reverses this MDSC-induced immunosuppressive TME and improves the immunotherapy efficacy in triple negative breast cancer (TNBC). Firstly, by combining the tumor immunological phenotype (TIP) gene signature and high throughput sequencing based high throughput screening (HTS2), we identified that ponatinib significantly inhibits the expression of "cold" tumor associated chemokines CXCL1 and CXCL2 in cancer cells. This inhibition decreases the infiltration of MDSCs and consequently increased the accumulation of "hot" tumor associated T cells and NK cells and thus reverses the immunosuppressive TME. Then, by multiple preclinical models, we found that ponatinib significantly inhibits tumor growth in a TME-dependent manner and enhances the efficacy of anti-PD-L1 immunotherapy on TNBC in vivo. Notably, ponatinib exhibits no significant inhibition on immune cells in mouse spleens. Mechanistically, ponatinib directly inhibits the kinase activity of p38, which results in the reduction of the phosphorylation of STAT1 at Ser727, and thus the decreased expression of CXCL1 and CXCL2 in cancer cells. Our study provided the therapeutic potential of combining p38 inhibition with ICB for the treatment of TNBC.

cancer biology↗

bioRxiv2023

Neutralization of SARS-CoV-2 BQ.1.1 and XBB.1.5 by Breakthrough Infection Sera from Previous and Current Waves in China

SARS-CoV-2 is continuing to evolve and diversify, with an array of various Omicron sub-lineages, including BA.5, BA.2.75, BN.1, BF.7, BQ.1, BQ.1.1, XBB and XBB.1.5, now circulating globally at recent time. In this study, we evaluated the neutralization sensitivity of a comprehensive panel of Omicron subvariants to sera from different clinical cohorts, including individuals who received homologous or heterologous booster vaccinations, vaccinated people who had Delta or BA.2 breakthrough infection in previous waves, and patients who had BA.5 or BF.7 breakthrough infection in the current wave in China. All the Omicron subvariants exhibited substantial neutralization evasion, with BQ.1, BQ.1.1, XBB.1, and XBB.1.5 being the strongest escaped subvariants. Sera from Omicron breakthrough infection, especially the recent BA.5 or BF.7 breakthrough infection, exhibited higher neutralizing activity against all Omicron sub-lineages, indicating the chance of BA.5 and BF.7 being entirely replaced by BQ or XBB subvariants in China in a short-term might be low. We also demonstrated that the BQ and XBB subvariants were the most resistant viruses to monoclonal antibodies. Continuing to monitor the immune escape of SARS-CoV-2 emerging variants and developing novel broad-spectrum vaccines and antibodies are still crucial.

immunology↗

bioRxiv2023

IMPACT: a web server for exploring immunotherapeutic predictive and cancer prognostic biomarkers

Immune checkpoint inhibitors (ICIs) are a breakthrough in oncology treatment, and studies of screening predictive biomarkers of ICIs are emerging. We developed a web server named IMPACT (http://impact.brbiotech.com/) to thoroughly explore immunotherapeutic predictive or prognostic biomarkers. IMPACT contains a large dataset of 6,276 patients treated with ICIs and integrates 11 well-designed function modules, enabling an in-depth solution for biomarkers exploration. Compared with the existing tools, IMPACT was implemented with one exclusive module for interaction analysis and several optimized conventional functions for discovering novel biomarkers. Specifically, the interaction analysis of biomarker-treatment effect is essential to determine whether a biomarker is predictive and/or prognostic for ICIs. Moreover, several optimized functions allow complicated biomarker exploration, including customized selections of variant types in more detail, automatically screening meaningful co-mutations among multiple genes, and selecting cut-off values for gene expression biomarkers. In summary, IMPACT is a comprehensive analysis resource to facilitate biomarker research of ICIs.

bioinformatics↗

bioRxiv2023

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress

Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL) but pan-Notch inhibitors were toxic in clinical trials. To find alternative ways to target Notch signals, we investigated Cell division cycle 73 (Cdc73), which is a Notch cofactor and component of transcriptional machinery, a potential target in T-ALL. While we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the lymphoid transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, Cdc73, Ets1, and Notch intersect chromatin at promoters and enhancers to activate oncogenes and genes that are important for DNA repair and oxidative phosphorylation. Consistently, Cdc73 deletion in T-ALL cells induced DNA damage and impaired mitochondrial function. Our data suggests that Cdc73 might promote a gene expression program that was eventually intersected by Notch to mitigate the genotoxic and metabolic stresses of elevated Notch signaling. We also provide mechanistic support for testing inhibitors of DNA repair, oxidative phosphorylation, and transcriptional machinery. Inhibiting pathways like Cdc73 that intersect with Notch at chromatin might constitute a strategy to weaken Notch signals without directly targeting the Notch complex.

cancer biology↗

bioRxiv2023

Integration of spatial and single-cell data across modalities with weak linkage

single-cell sequencing methods have enabled the profiling of multiple types of molecular readouts at cellular resolution, and recent developments in spatial barcoding, in situ hybridization, and in situ sequencing allow such molecular readouts to retain their spatial context. Since no technology can provide complete characterization across all layers of biological modalities within the same cell, there is pervasive need for computational cross-modal integration (also called diagonal integration) of single-cell and spatial omics data. For current methods, the feasibility of cross-modal integration relies on the existence of highly correlated, a priori "linked" features. When such linked features are few or uninformative, a scenario that we call "weak linkage", existing methods fail. We developed MaxFuse, a cross-modal data integration method that, through iterative co-embedding, data smoothing, and cell matching, leverages all information in each modality to obtain high-quality integration. MaxFuse is modality-agnostic and, through comprehensive benchmarks on single-cell and spatial ground-truth multiome datasets, demonstrates high robustness and accuracy in the weak linkage scenario. A prototypical example of weak linkage is the integration of spatial proteomic data with single-cell sequencing data. On two example analyses of this type, we demonstrate how MaxFuse enables the spatial consolidation of proteomic, transcriptomic and epigenomic information at single-cell resolution on the same tissue section.

bioinformatics↗

bioRxiv2023

High-throughput single molecule tracking identifies drug interactions and cellular mechanisms

The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis. As a result, studies using super-resolution imaging are typically drawing conclusions from tens of cells and a few experimental conditions tested. We addressed these limitations by developing a high-throughput single-molecule tracking (htSMT) platform for pharmacologic dissection of protein dynamics in living cells at an unprecedented scale (capable of imaging > 106 cells/day and screening > 104 compounds). We applied htSMT to measure the cellular dynamics of fluorescently tagged estrogen receptor (ER) and screened a diverse library to identify small molecules that perturbed ER function in real time. With this one experimental modality, we determined the potency, pathway selectivity, target engagement, and mechanism of action for identified hits. Kinetic htSMT experiments were capable of distinguishing between on-target and on-pathway modulators of ER signaling. Integrated pathway analysis recapitulated the network of known ER interaction partners and suggested potentially novel, kinase-mediated regulatory mechanisms. The sensitivity of htSMT revealed a new correlation between ER dynamics and the ability of ER antagonists to suppress cancer cell growth. Therefore, measuring protein motion at scale is a powerful method to investigate dynamic interactions among proteins and may facilitate the identification and characterization of novel therapeutics.

cell biology↗

bioRxiv2022

Heterologous boost with mRNA vaccines against SARS-CoV-2 Delta/Omicron variants following an inactivated whole-virus vaccine

The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants following an order of BA.1>BA.2>BA.4/5. In naive animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccines targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection across all VOCs. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by "recall" and "reshape" the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine- primed populations.

molecular biology↗

bioRxiv2022

Multi-ancestry GWAS of major depression aids locus discovery, fine-mapping, gene prioritisation, and causal inference

Most genome-wide association studies (GWAS) of major depression (MD) have been conducted in samples of European ancestry. Here we report a multi-ancestry GWAS of MD, adding data from 21 studies with 88,316 MD cases and 902,757 controls to previously reported data from individuals of European ancestry. This includes samples of African (36% of effective sample size), East Asian (26%) and South Asian (6%) ancestry and Hispanic/Latinx participants (32%). The multi-ancestry GWAS identified 190 significantly associated loci, 53 of them novel. For previously reported loci from GWAS in European ancestry the power-adjusted transferability ratio was 0.6 in the Hispanic/Latinx group and 0.3 in each of the other groups. Fine-mapping benefited from additional sample diversity: the number of credible sets with [≤]5 variants increased from 3 to 12. A transcriptome-wide association study identified 354 significantly associated genes, 205 of them novel. Mendelian Randomisation showed a bidirectional relationship with BMI exclusively in samples of European ancestry. This first multi-ancestry GWAS of MD demonstrates the importance of large diverse samples for the identification of target genes and putative mechanisms.

genetics↗

bioRxiv2022

Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

RNA transport and local translation provide spatial control of gene expression, and RNA binding proteins (RBPs) act as critical adapters in this multi-step process. Muscleblind-like (MBNL) RNA binding proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and distal neurites through unknown mechanisms. We found that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1b and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs also associate with these kinesins, implicating a motor-RBP specificity code. Live cell imaging and fractionation revealed that membrane anchoring is mediated through the unstructured carboxy-terminal tail of MBNL1. Both kinesin- and membrane-recruitment functions were reconstituted using MBNL-MS2 coat protein fusions. This approach, termed RBP Module Recruitment and Imaging (RBP-MRI), decouples RNA binding, kinesin recruitment, and membrane anchoring functions, while also establishing general strategies for studying multi-functional, modular domains of RBPs.

cell biology↗

bioRxiv2022

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease,, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.

neuroscience↗

bioRxiv2022

Commercial ChIP-Seq library preparation kits performed differently for different classes of protein targets

BackgroundChIP-Seq is a powerful method commonly used to study global protein-DNA interactions including both transcription factors and histone modifications. We have found that the choice of ChIP-Seq library preparation protocol plays an important role in overall ChIP-Seq data quality. However, very few studies have compared ChIP-Seq libraries prepared by different protocols using multiple targets and a broad range of input DNA levels. ResultsIn this study, we evaluated the performance of four ChIP-Seq library preparation protocols [NEB NEBNext Ultra II, Roche KAPA HyperPrep, Diagenode MicroPlex, and Bioo (now PerkinElmer) NEXTflex] on three target proteins, chosen to represent the three typical signal enrichment patterns in ChIP-Seq experiments: sharp peaks (H3K4me3), broad domains (H3K27me3) and punctate peaks with a protein binding motif (CTCF). We also tested a broad range of different input DNA levels from 0.10 to 10 ng for H3K4me3 and H3K27me3 experiments. ConclusionsOur results suggest that the NEB protocol may be better for preparing H3K4me3 (and potentially other histone modifications with sharp peak enrichment) libraries; the Bioo protocol may be better for preparing H3K27me3 (and potentially other histone modifications with broad domain enrichment) libraries, and the Diagenode protocol may be better for preparing CTCF (and potentially other transcription factors with well-defined binding motifs) libraries. For ChIP-Seq experiments using novel targets without a known signal enrichment pattern, the NEB protocol might be the best choice as it performed well for each of the three targets we tested across a wide array of input DNA levels.

bioinformatics↗

bioRxiv2022

Dopamine D1-like receptor blockade and stimulation decreases operant responding for nicotine and food in male and female rats

Dopamine has been implicated in smoking, but there remains a need for a better understanding of the effects of dopamine D1-like receptor agonists on nicotine intake and the role of sex in the effects of dopaminergic drugs on nicotine intake. This work studied the effects of D1-like receptor stimulation and blockade on operant responding for nicotine and food and locomotor activity in male and female rats. The effects of the D1-like receptor antagonist SCH 23390 (0.003, 0.01, 0.03 mg/kg) and the D1-like receptor agonist A77636 (0.1, 0.3, 1 mg/kg) on responding for nicotine and food, and locomotor activity were investigated. The effects of SCH 23390 were investigated 15 min and 24 h after treatment, and the effects of the long-acting drug A77636 were investigated 15 min, 24 h, and 48 h later. Operant responding for nicotine and food was decreased 15 min, but not 24 h, after treatment with SCH 23390. Operant responding for nicotine was decreased 15 min, 24 h, and 48 h after treatment with A77636, and food responding was decreased 15 min and 24 h later. Locomotor activity was decreased 15 min, but not 24 h, after treatment with SCH 23390. A77636 only decreased locomotor activity 48 h after treatment. There were no sex differences in the effects of SCH 23390 or A77636. In conclusions, the D1-like receptor antagonist SCH 23390 reduces nicotine intake and causes sedation in rats. Stimulation of D1-like receptors with A77636 decreases nicotine intake at time points that the drug is not sedative.

pharmacology and toxicology↗

bioRxiv2022

Improved production and expanded application of CVS-N2c-ΔG virus for retrograde tracing

Neurotropic virus tracers, particularly those with low toxicity and high efficient tracing, are powerful tools for structural and functional dissections of neural circuits. The retrograde trans-mono-synaptic technology based on rabies virus CVS-N2c strain has reduced cytotoxicity and enhanced efficiency, attains long-term gene manipulation for functional studies, but suffers from difficult preparation and low yield. To overcome these shortcomings, an improved production system was established for rapid rescue and preparation of CVS-N2c-{Delta}G virus, CVS-N2c-{Delta}G with the same titer as SAD-B19-{Delta}G can be prepared within a short time. Meanwhile, we found that N2cG coated CVS-N2c-{Delta}G allows efficient retrograde access to projection neurons, and further expand its application in VTA/SNc to DLS pathway that unaddressed by rAAV9-Retro, and the efficiency is 6 folds higher than that of rAAV9-Retro. Then the trans-synaptic efficiency of CVS-N2c-{Delta}G virus was evaluated. Results showed that the trans-mono-synaptic efficiency of oG-mediated CVS-N2c-{Delta}G was 2-3 folds higher than that of oG-mediated SAD-B19-{Delta}G, but there was no difference between oG-mediated and N2cG-mediated CVS-N2c-{Delta}G system. In addition, codon modified N2cG (optiG) did not increase the efficiency of CVS-N2c-{Delta}G tracing. Finally, we found that the CVS-N2c-{Delta}G produced by the improved method can be used for monitoring neural activity of projection neurons, and the time window can be maintained for 3 weeks, and it can also express sufficient recombinases for efficient transgene recombination. That is, the virus produced by the improved production system does not affect its own function, paving the way for its further optimization, popularization and application in structural and functional studies of neural circuits.

neuroscience↗