Biology subjects

Lin, Y.

Publications and source records attributed to Lin, Y..

At least 253 records · Page 14Linked to original sources

bioRxiv2022

Resilient CD8+ T cells maintain a high cytotoxic capacity by balancing ROS via ME1 upregulation

Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignances. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysisindependent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/517988v3_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@12e0f36org.highwire.dtl.DTLVardef@e8d412org.highwire.dtl.DTLVardef@1baf57eorg.highwire.dtl.DTLVardef@14b2b43_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICX3CR1+ and low Dy m identify functional resilient CD8+ T cells. C_LIO_LIResilient CD8+ T cells are highly cytotoxic and have less ROS. C_LIO_LIResilient CD8+ T cells express more ME1 that can balance extra ROS. C_LIO_LIME1 overexpression can promote CTL function of CD8+ T cells. C_LI

immunology↗

bioRxiv2022

Atomistic structure of the interlocking claw-like amyloid fibril of full-length glucagon

Glucagon is a peptide hormone which posits a significant potential as a therapeutic molecule for various human diseases. One of the major challenges hampering medicinal application of glucagon, however, is its insoluble and aggregation-prone property. Although glucagon is dissolvable, it aggregates easily and forms amyloid fibrils. To date, despite many studies to understand how glucagon aggregates and fibrillizes, its detailed amyloidogenesis mechanism is still elusive, particularly due to insufficient structural information of glucagon amyloid fibrils. Here we report the novel structure of a glucagon amyloid fibril, which was determined with cryo-electron microscopy (cryo-EM) to a 3.8-[A] resolution. Our model features with tight and extensive inter-monomer interactions, which efficiently stabilize the entire chain of full-length glucagon into the V-shape conformation, clamping the other monomer to construct a dimeric architecture orthogonal to the fibril axis. Notably, the current structure significantly differs from the previous solid-state NMR model, which gives direct evidence for multiple and dynamic amyloidogenesis mechanisms of glucagon. In addition, our results are expected to provide important insights to appreciate the molecular details of glucagon in its initial amyloidogenesis and fibril elongation processes.

molecular biology↗

bioRxiv2022

Integrated analysis of single-cell transcriptome of liver cancer and cirrhosis reveals cell lineage similarity and prognostic-associated subpopulations

Background & AimsLiver cancer is one of the most leading causes of cancer deaths. Cirrhosis is an important risk factor for liver cancer, which is the result of over-fibrosis caused by diffuse and long-term liver damage. Despite extensive research, a systematic study for characterizing similarity between liver cancer and cirrhosis at single cell resolution is still lacking. MethodsWe established a data analysis framework to elucidate cell lineage similarity between liver cancer and cirrhosis to discover prognostic-associated subpopulations. We integrated single-cell transcriptome data from liver samples at normal, cirrhotic and cancer conditions, which totally contained 78,000 cells. Gene regulation analysis, cellular interactions and trajectory analysis were performed to characterize cirrhosis-like cell subpopulations. Bulk transcriptomes were used to discover prognostic-associated subpopulations. ResultsBy aligning cellular subpopulations across different samples, we found remarkable similarity between KNG1+ hepatocytes in cirrhosis and PGA5+ hepatocytes in HCC. Furthermore, gene regulation analysis and cellular interactions implicated E2F1, FOXA2, EGF, CDH and ANGPTL signaling in maintaining cirrhosis-like ecosystem. Strikingly, subpopulations with higher expression of cirrhosis-like signatures were associated with patients worse survival. ConclusionsWe revealed a previously unexplored cirrhosis-like ecosystem of liver cancer, which could provide novel biomarkers for therapeutic interventions in HCC. Core analysis modules in this study were integrated into a user-friendly toolkit, SIMscRNA(https://github.com/xmuhuanglab/SIM-scRNA), which could facilitate the exploration of similarity and heterogeneity between precancerous diseases and solid tumors.

bioinformatics↗

bioRxiv2022

Direct activation of the Ras-Akt network mediates polarity and organizes protrusions in human neutrophil migration

Functions of Ras oncogenes and their downstream effectors are typically associated with cell proliferation and growth control while their role in immune cell migration has been largely unexplored. Although Ras-mediated signaling cascades have been implicated in immune response, there is no conclusive evidence to show local activation of these pathways on the plasma membrane directly regulates cell motility or polarity. Using spatiotemporally precise, cryptochrome-based optogenetic systems in human neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant-sensing receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or the guanine nucleotide exchange factor, RasGRP4, immediately increased spreading and random motility in neutrophils. Second, creating Ras activity at the cell rear generated new protrusions at the site and reversed pre-existing polarity, similar to the effects of steep chemoattractant gradients. Third, recruiting GTPase activating protein, RASAL3, at cell fronts abrogated existing protrusions and changed the direction of motility whereas dynamically inhibiting nascent fronts stopped migration completely. Fourth, combining pharmacological inhibition studies with optogenetics revealed that mTorC2 is more important than PI3K for Ras-mediated polarity and migration. Finally, local recruitment of Ras-mTorC2 effector, Akt, also generated new protrusions, rearranged pre-existing polarity, and triggered migration, even in absence of PI3K signaling. We propose that actin assembly, cell shape, and migration modes in immune cells are promptly controlled by rapid, local activities of established components of classical growth-control pathways independently of receptor activation.

immunology↗

bioRxiv2022

Network models of protein phosphorylation, acetylation, and ubiquitination connect metabolic and cell signaling pathways in lung cancer

We analyzed large-scale post-translational modification (PTM) data to outline cell signaling pathways affected by tyrosine kinase inhibitors (TKIs) in ten lung cancer cell lines. Tyrosine phosphorylated, lysine ubiquitinated, and lysine acetylated proteins were concomitantly identified using sequential enrichment of post translational modification (SEPTM) proteomics. Machine learning was used to identify PTM clusters that represent functional modules that respond to TKIs. To model lung cancer signaling at the protein level, PTM clusters were used to create a co-cluster correlation network (CCCN) and select protein-protein interactions (PPIs) from a large network of curated PPIs to create a cluster-filtered network (CFN). Next, we constructed a Pathway Crosstalk Network (PCN) by connecting pathways from NCATS BioPlanet whose member proteins have PTMs that co-cluster. Interrogating the CCCN, CFN, and PCN individually and in combination yields insights into the response of lung cancer cells to TKIs. We highlight examples where cell signaling pathways involving EGFR and ALK exhibit crosstalk with BioPlanet pathways: Transmembrane transport of small molecules; and Glycolysis and gluconeogenesis. These data identify known and previously unappreciated connections between receptor tyrosine kinase (RTK) signal transduction and oncogenic metabolic reprogramming in lung cancer. Comparison to a CFN generated from a previous multi-PTM analysis of lung cancer cell lines reveals a common core of PPIs involving heat shock/chaperone proteins, metabolic enzymes, cytoskeletal components, and RNA-binding proteins. Elucidation of points of crosstalk among signaling pathways employing different PTMs reveals new potential drug targets and candidates for synergistic attack through combination drug therapy. Author SummaryProtein post-translational modifications (PTMs), such as phosphorylation, ubiquitination, and acetylation, are extensively employed by cell signaling pathways that regulate cell division, differentiation, migration, and cancer. We used machine learning to identify PTM clusters that represent functional modules in cell signaling pathways. These clusters were used to identify protein-protein interactions, and interactions between cell signaling pathways, that were active in lung cancer cells that were treated with anti-cancer drugs. We model these interactions as networks at three levels of granularity at the pathway, protein-protein interaction, and PTM levels. Interrogation of these networks yielded insights into molecular interactions between cell signaling pathways activated by oncogenes, transmembrane transport of small molecules, and glycolysis and gluconeogenesis. These analyses identify previously unappreciated mechanisms of crosstalk among signaling pathways between oncogenic tyrosine kinase signaling and proteins that regulate metabolic reprogramming in lung cancer, revealing new potential drug targets for combination therapy.

cancer biology↗

bioRxiv2022

The protist ubiquitin ligase effector PbE3-2 targets RD21A to impede plant immunity

Clubroot caused by the soil-borne protist pathogen Plasmodiophora brassicae is one of the most devastating diseases of Brassica oil and vegetable crops worldwide. Understanding the pathogen infection strategy is crucial for the development of disease control. However, the molecular mechanism by which this pathogen promotes infection remains largely unknown. Here, we identified a P. brassicae-secreted effector PbE3-2 that impedes plant immunity by ubiquitinating the immune regulator RD21A for degradation. Overexpression of PbE3-2 in Arabidopsis thaliana resulted in higher susceptibility to P. brassicae and decreases in chitin-triggered reactive oxygen species burst and expression of marker genes in salicylic acid signaling. PbE3-2 interacted with and ubiquitinated RD21A in vitro and in vivo. Mutant plants deficient in RD21A exhibited similar susceptibility and compromsied immune responses as in PbE3-2 overexpression plants. These results suggest that P. brassicae promotes clubroot disease through RD21A degradation mediated by the effector PbE3-2. As PbE3-2 is widely conserved across different P. brassicae pathotypes, the degradation of RD21A by PbE3-2 might be a prevalent infection strategy in this pathogen.

microbiology↗

bioRxiv2022

Comparison of DNA targeting CRISPR editors in human cells

Profiling and comparing the performance of current widely used DNA targeting CRISPR systems provide the basic information for the gene-editing toolkit and can be a useful resource for this field. Herein we made a parallel comparison between the recently reported miniature Cas12f1 and the widely used Cas12a and Cas9 nucleases in mammalian cells. The results revealed that as a CRISPRa activator, Un1Cas12f1 could induce gene expression with a comparable level to these of Cas12a and Cas9, while as a cleaving editor, Cas12f1 exhibited similar properties to Cas12a, like high specificity and dominantly induced deletions over insertions, but with less activity. In contrast, wild-type SpCas9 showed the highest activity, lowest specificity, and induced balanced deletions over insertions. Thus, Cas12f1 is recommended for gene-activation-based applications, Cas12a is for therapy applications, and wild-type Cas9 is for in vitro and animal investigations. The comparison provided the editing properties of current widely used DNA-targeting CRISPR systems for the gene-editing field.

synthetic biology↗

bioRxiv2022

VStrains: De Novo Reconstruction of Viral Strains via Iterative Path Extraction From Assembly Graphs

With the high mutation rate in viruses, a mixture of closely related viral strains (called viral quasispecies) often co-infect an individual host. Reconstructing individual strains from viral quasispecies is a key step to characterizing the viral population, revealing strain-level genetic variability, and providing insights into biomedical and clinical studies. Reference-based approaches of reconstructing viral strains suffer from the lack of high-quality references due to high mutation rates and biased variant calling introduced by a selected reference. De novo methods require no references but face challenges due to errors in reads, the high similarity of quasispecies, and uneven abundance of strains. In this paper, we propose VStrains, a de novo approach for reconstructing strains from viral quasispecies. VStrains incorporates contigs, paired-end reads, and coverage information to iteratively extract the strain-specific paths from assembly graphs. We benchmark VStrains against multiple state-of-the-art de novo and reference-based approaches on both simulated and real datasets. Experimental results demonstrate that VStrains achieves the best overall performance on both simulated and real datasets under a comprehensive set of metrics such as genome fraction, duplication ratio, NGA50, error rate, etc. AvailabilityVStrains is freely available at https://github.com/MetaGenTools/VStrains.

bioinformatics↗

bioRxiv2022

Development of an affinity-enhanced clinical candidate TCR targeting NY-ESO-1 with optimal potency and high specificity

The clinical success of T-cell receptor (TCR)-based immunotherapy depends on the efficacy and specificity of TCRs. Naturally occurring TCRs have limited anti-tumor potency due to their low affinity for tumor antigens. Affinity enhancement is a promising strategy to generate highly potent TCRs. However, it is concerned that affinity-enhanced TCRs are prone to lose specificity. We isolated low affinity TCRs specific for NY-ESO-1157-165/HLA-A*02:01 from peripheral blood mononuclear cells of healthy donors. An affinity-enhanced TCR candidate with optimal affinity and specificity was generated using phage display and an extensive set of in vitro and in vivo assays. Alanine scanning mutagenesis showed that the TCR candidate retained specificity by making extensive contacts to the side chains of NY-ESO-1157-165 peptide. Adoptive transfer of T cells engineered with this candidate (termed TAEST16001) significantly inhibited tumor growth in subcutaneous, metastatic, and patient-derived xenograft (PDX) mouse tumor models. This study demonstrates that sophisticated engineering and screening techniques can be utilized to generate a clinical candidate TCR with potent anti-tumor activity without losing specificity. TAEST16001 was approved by the Center for Drug Evaluation (CDE) as the first TCR-based immunotherapy clinical trial in China (ClinicalTrials.gov Identifier: NCT03159585).

immunology↗

bioRxiv2022

Not just with the natives, but phylogenetic relationship between stages of the invasion process determines invasion success of alien plant species

Darwin proposed two alternate hypotheses on the invasion success of alien plant species based on their close or distant phylogenetic relationship with the natives. Here we tested these hypotheses along the invasion process at large spatio-temporal scale. Using two abundance-based phylogenetic metrics for the native and alien flora of China, our study showed that invasion success of alien plant species is influenced by the phylogenetic relationship with native and alien species of different invasion stages. Phylogenetic similarity with the native species helps the alien ones to introduce and naturalize, but phylogenetic dissimilarity with the natives facilitates invasion success. The co-occurrence of invasive and naturalized aliens also formed more clustered assemblages, showed specialized responses to environmental stress, and provided temporal stability to the phylogenetic measures. The native-alien phylogenetic relationship is dynamic across spatial, environmental, and taxonomic scales. Therefore, assessing Darwins naturalization conundrum at different gradients of community assembly process is important.

ecology↗

bioRxiv2022

Prioritizing prognostic-associated subpopulations and individualized recurrence risk signatures from single-cell transcriptomes of colorectal cancer

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies. There are few recurrence risk signatures for CRC patients. Single-cell RNA-sequencing (scRNA-seq) provides a high resolution platform for prognostic signature detection. However, scRNA-seq is not practical in large cohorts due to its high cost and most single-cell experiments lack clinical phenotype information. Few studies have been reported to use external bulk transcriptome with survival time to guide the detection of key cell subtypes in scRNA-seq data. We proposed a data analysis framework to prioritize prognostic-associated subpopulations based on relative expression orderings (REOs). Cell type specific gene pairs (C-GPs) were identified to evaluate prognostic value for each cell type. We found REOs-based signatures could accurately classify most cell subtypes. C-GPs achieves higher precision compared with four current methods. Moreover, we developed single-cell gene pair signatures to predict recurrence risk for patients individually. Fibro_SGK1 cells and IgA+ IGLC2+ B cells were novel prognostic-associated subpopulations. A user-friendly toolkit, scRankXMBD (https://github.com/xmuyulab/scRank-XMBD), was developed to enable implementation of this framework. Our work facilitate the application of the rank-based method in scRNA-seq data for prognostic biomarker discovery and precision oncology.

bioinformatics↗

bioRxiv2022

Pathway elucidation of pharmaceutical cucurbitacin IIa in Hemsleya chinensis and high-level production of precursor cucurbitadienol in engineered Saccharomyces cerevisiae and Nicotiana benthamiana

Cucurbitacin IIa is a triterpene isolated exclusively from Hemsleya plants, which is non-steroidal anti-inflammatory drug that function as the main ingredient of Hemslecin capsules and Supplemental Tablets in China. In this study, the biosynthetic pathway of cucurbitacin IIa was elucidated by characterization of squalene epoxidases (HcSE1, HcSE2), cucurbitenol synthases (HcOSC6) and acyltransferases (HcAT1) in Hemsleya chinensis. Meanwhile, cycloartenol synthases (HcOSC1), isomultiflorenol synthases (HcOSC5) and {beta}-amyrin synthase (HcOSC2-4) involved in sterol and triterpenes biosynthesis were functionally illustrated. The high-level production of yeast the key cucurbitacin precursor, cucurbitadienol, was constructed to produce 296.37 mg/L cucurbitadienol and 722.99 mg/L total triterpenoid which is the highest yield cucurbitadienol from known engineered microbes. Moreover, production of cucurbitenol in transient expression of tobacco was employed to achieve 94.8 mg/g dry weight (dw) cucurbitenol from leaves. In this study, the key genes involved in cucurbitacin IIa biosynthesis were identified to facilitate its medical applications via biosynthetic strategy. Meanwhile, the high-level production of cucurbitadienol chassis yeast and tobacco transient expression offered a robust and sSupplemental Table substrate for pharmaceutical cucurbitacin production and screening platform for candidate genes involved in cucurbitacin biosynthesis.

synthetic biology↗

bioRxiv2022

Enrichment and application of bacterial sialic acids containing polymers from the extracellular polymeric substances of Candidatus Accumulibacter

Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that "Candidatus Accumulibacter" can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a "Ca. Accumulibacter" enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of "Ca. Accumulibacter" contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with "Ca. Accumulibcater" and used size-exclusion chromatography to separate them into different molecular weight fractions. This separation resulted in two high molecular weight (> 5,500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This suggest that they can serve as source for sepsis treatment drugs, although further purification needs to be evaluated. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/508216v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1f59ff7org.highwire.dtl.DTLVardef@d933fcorg.highwire.dtl.DTLVardef@1e3ff78org.highwire.dtl.DTLVardef@1994948_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. C_LIO_LISize exclusion chromatography allows to obtain high molecular weight polysaccharide-rich fractions enriched with NulOs. C_LIO_LIEPS and the NulOs-rich fractions can serve as source for sepsis treatment drugs. C_LI

microbiology↗

bioRxiv2022

Study on the interactions between cisplatin and cadherin

Cisplatin is an important platinum drug in the chemotherapy of many malignant tumors in clinical application. It is well accepted that its main target is DNA molecule. However, recent studies have shown that platinum drugs may also act on some important functional proteins in the human body and have complicate effects on their efficacy. E-cadherin is newly discovered glycoproteins which have been regarded as the important signs of the occurrence and development of some tumors. In this study, the interactions between cisplatin and E-cadherin were explored by fluorescence spectroscopy and atomic force microscopy (AFM). The fluorescence spectroscopy results indicated that cisplatin can efficiently quench the fluorescence of E-cadherin, and the calculated binding constant Kb was 3.20x106 (25{degrees}C), 1.36 x106(31{degrees}C) and 8.22x105 L mol-1 (37{degrees}C). The thermodynamic parameters {Delta}H <0, {Delta}S <0, {Delta}G <0, reveal that the fluorescence quenching effect of cisplatin on E-cadherin is static quenching, which is dominated by hydrogen bond and van der Waals interaction. The AFM results revealed that there was a staggered interaction between the intermolecular domains of E-cadherin to form a long spherical chain structure. The addition of cisplatin could significantly affect the staggered effect of the E-cadherin molecules. All these results confirmed that there are strong interactions between cisplatin and cadherin.

biochemistry↗

bioRxiv2022

A Meta-learning based Graph-Hierarchical Clustering Method for Single Cell RNA-Seq Data

Single cell sequencing techniques enable researchers view complex bio-tissues from a more precise perspective to identify cell types. However, more and more recent works have been done to find more detailed subtypes within already known cell types. Here, we present MeHi-SCC, a method which utilized meta-learning protocol and brought in multi scRNA-seq datasets information in order to assist graph-based hierarchical sub-clustering process. In result, MeHi-SCC outperformed current-prevailing scRNA clustering methods and successfully identified cell subtypes in two large scale cell atlas. Our codes and datasets are available online at https://github.com/biomed-AI/MeHi-SCC

bioinformatics↗

bioRxiv2022

Single molecule co-occupancy of RNA-binding proteins with an evolved RNA deaminase

RNA-protein interactions broadly regulate gene expression. To understand RNA regulation, it is critical to measure RNA-protein interactions in cells. Current approaches to measure RNA-protein interactions often rely on crosslinking and shortread RNA sequencing, which has considerably advanced the understanding of gene expression but also suffers from some limitations. We present REMORA (RNA Encoded Molecular Recording in Adenosines), a new strategy to measure RNA-binding events on single RNA molecules in cells. In REMORA, adenosine deamination serves as a molecular record of RNA-protein interactions that are identified by mutations by sequencing. We performed RNA-based directed evolution to identify an RNA deaminase variant with high activity on arbitrary adenosine residues in RNA. We show that this RNA deaminase has high activity, low local sequence or structure bias, low background, and is generally applicable to diverse RNA-binding proteins. By combining our improved A-to-I RNA deaminase with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, our approach enables simultaneous recording of the locations two RNA binding proteins on single mRNA molecules. Orthogonal RNA molecular recording of two Pumilio family proteins, PUM1 and PUM2, reveals that PUM1 competes with PUM2 for some but not all Pumilio binding sites in cells, despite having the same in vitro binding preferences. Our work thus measures competition between RNA-binding proteins for RNA sites in cells, and our genetically encodable RNA deaminase enables single-molecule identification of RNA-protein interactions with cell type specificity.

molecular biology↗

bioRxiv2022

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

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

plant biology↗

bioRxiv2022

Genetically encoded sensors for measuring histamine release both in vitro and in vivo

Histamine (HA) is a key biogenic monoamine involved in a wide range of physiological and pathological processes in both the central nervous system and the periphery. Because the ability to directly measure extracellular HA in real-time will provide important insights into the functional role of HA in complex circuits under a variety of conditions, we developed a series of genetically encoded G protein-coupled receptor activation-based (GRAB) HA (GRABHA) sensors. These sensors produce a robust increase in fluorescence upon HA application, with good photostability, sub-second kinetics, nanomolar affinity, and high specificity. Using these GRABHA sensors, we measured electrical stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. Moreover, we recorded HA release in the preoptic area of the hypothalamus and in the medial prefrontal cortex during the sleep-wake cycle in freely moving mice, finding distinct patterns of HA release in these specific brain regions. Together, these in vitro and in vivo results show that our GRABHA sensors have high sensitivity and specificity for measuring extracellular HA, thus providing a robust new set of tools for examining the role of HA signaling in both health and disease.

neuroscience↗