Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 451 records · Page 25Linked to original sources

bioRxiv2023

MitoSort: robust demultiplexing of pooled single-cell genomics data using endogenous germline mitochondrial variants

Multiplexing across donors has emerged as a popular strategy to increase throughput, reduce costs, overcome technical batch effects, and improve doublet detection in single-cell genomic studies. Using endogenous genetic barcodes eliminates the need for additional experimental processing steps. Among the available choices for endogenous barcodes, the unique features of mtDNA variants render them a more computationally efficient and robust option compared to genome variants. Here we present MitoSort, a method that uses mtDNA germline variants to assign cells to their donor of origin and identify cross-genotype doublets. We evaluated the performance of MitoSort by in silico pooled mtscATAC-seq libraries and experimentally multiplexed data using cell hashing method. MitoSort achieve both high accuracy and efficiency on genotype clustering and doublet detection for mtscATAC-seq data, which fills a void left by the inadequacies of current computational techniques tailored for scRNA-seq data. Moreover, MitoSort exhibits versatility and can be applied to various single-cell sequencing approaches beyond mtscATAC-seq, as long as the mtDNA variants can be reliably detected. Furthermore, through a case study, we demonstrated that demultiplexing 8 individuals assayed at the same time with MitoSort, enables the comparison of cell composition without batch effects.

bioinformatics↗

bioRxiv2023

Essential roles of the ANKRD31-REC114 interaction in meiotic recombination and mouse spermatogenesis

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine if and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31- REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31- REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114, and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction. SignificanceHomologous recombination initiated by double-strand breaks (DSBs) during meiosis is a nearly universal feature of eukaryotic lifecycles, but is also dangerous because DSBs are potentially toxic or mutagenic. The vertebrate-specific protein ANKRD31 is an important regulator of DSB formation, proposed to be a scaffold protein that coordinates the activities of multiple DSB-promoting factors, including the widely conserved REC114. We test this hypothesis here through generation of targeted Ankrd31 mutations that specifically attenuate or eliminate the ANKRD31-REC114 interaction. Analysis of this allelic series demonstrates that the ANKRD31-REC114 interaction is essential for all ANKRD31 activities in vivo, providing insight into how ANKRD31 controls DSB locations, timing, and number.

genetics↗

bioRxiv2023

Understanding the Mechanisms of Salmonella Typhimurium resistance to Cannabidiol

The emergence of multidrug resistance poses a huge risk to public health globally. Yet these recalcitrant pathogens continue to rise in incidence rate with resistance rates significantly outpacing the speed of antibiotic development. This therefore presents an aura of related health issues such as untreatable nosocomial infections arising from organ transplants, surgeries, as well as community acquired infections that are related to people with compromised immunity e.g., diabetic and HIV patients etc. There is a global effort to fight multidrug resistant pathogens spearheaded by the World Health Organization, thus calling for research into novel antimicrobials agents to fight multiple drug resistance. Previously, our laboratory demonstrated that Cannabidiol (CBD) was an effective antimicrobial against Salmonella Typhimurium (S. Typhimurium). However, we observed resistance development over time. To understand the mechanisms S. Typhimurium uses to develop resistance to Cannabidiol (CBD), we studied the abundance of bacteria lipopolysaccharide (LPS) and membrane sterols of both susceptible and resistant S. Typhimurium. Using real-time quantitative polymerase chain reaction (rt qPCR), we also analyzed the expression of selected genes known for aiding resistance development in S. Typhimurium. We discovered that there was a significantly higher expression of blaTEM, fimA, fimZ, and integrons in the CBD-resistant bacteria, and these were also accompanied by a shift in abundance in cell surface molecules such as lipopolysaccharide (LPS) and sterols.

microbiology↗

bioRxiv2023

Discovery of microRNA-derived RNAs and proteins in human cells

MicroRNAs (miRNAs) are short noncoding RNAs that can regulate gene expression through the binding of their 5-ends to mRNAs. However, the biological effects of miRNA binding via their 3-ends remain unclear. Here, we discover that the exact reverse pairing of the 3-ends of miRNAs or miRNA-like RNAs, collectively termed microsized RNAs (msRNAs), with template RNAs can initiate the production of msRNA-derived RNAs (msdRs), which can subsequently be translated into polypeptides (msdPs). Using 2,632 human msRNAs from miRBase, 11,121 msdRs and 1,239 msdPs were predicted based on a 15-nucleotide pairing threshold, and the presence of representative msdRs and msdPs was confirmed in human cells. Of clinical relevance, msdP0188 is highly expressed in human lung and breast cancers, and its corresponding msRNAs and msdRs represent novel anti-cancer targets. Notably, inhibiting telomerase reverse transcriptase, a putative RNA-dependent RNA polymerase identified by bioinformatic screening, led to reduced levels of msdP0188 in human cancer cells. Our findings reveal a novel "msRNA [->] msdR [->] msdP" axis, expanding the classical central dogma and predicting many previously unrecognized RNAs and polypeptides with potential biological functions in human cells and other systems.

cell biology↗

bioRxiv2023

Mechanistic characterization of a Drosophila model of paraneoplastic nephrotic syndrome

Paraneoplastic syndromes occur in cancer patients and originate from dysfunction of organs at a distance from the tumor or its metastasis. A wide range of organs can be affected in paraneoplastic syndromes; however, the pathological mechanisms by which tumors influence host organs are poorly understood. Recent studies in the fly uncovered that tumor secreted factors target host organs, leading to pathological effects. In this study, using a Drosophila gut tumor model, we characterized a mechanism of tumor-induced kidney dysfunction. Specifically, we found that Pvf1, a PDGF/VEGF signaling ligand, secreted by gut tumors activates the PvR/JNK/Jra signaling pathway in the principal cells of the kidney, leading to mis-expression of renal genes and paraneoplastic renal syndrome-like phenotypes. Our study describes a novel mechanism by which gut tumors perturb the function of the kidney, which might be of clinical relevance for the treatment of paraneoplastic syndromes.

zoology↗

bioRxiv2023

A parabrachial-hypothalamic parallel circuit governs cold defense in mice

Thermal homeostasis is vital for mammals and is controlled by brain neurocircuits. Remarkable advances have been made in understanding how neurocircuits centered in the hypothalamic preoptic area (POA), the brains thermoregulation center, control warm defense, whereas mechanisms by which the POA regulates cold defense remain unclear. Here, we confirmed that the pathway from the lateral parabrachial nucleus (LPB) to the POA, is critical for cold defense. Parallel to this pathway, we uncovered that a pathway from the LPB to the dorsomedial hypothalamus (DMH), namely the LPB[->]DMH pathway, is also essential for cold defense. Projection-specific blockings revealed that both pathways provide an equivalent and cumulative contribution to cold defense, forming a parallel circuit. Specifically, activation of the LPB[->]DMH pathway induced strong cold-defense responses, including increases in thermogenesis of brown adipose tissue (BAT), muscle shivering, heart rate, and physical activity. Further, we identified a subpopulation of somatostatin+ neurons in the LPB that target the DMH to promote BAT thermogenesis. Therefore, we reveal a parabrachial-hypothalamic parallel circuit in governing cold defense in mice. This not only enables resilience to hypothermia but also provides a scalable and robust network in heat production, reshaping our understanding of how neural circuits regulate essential homeostatic behaviors.

neuroscience↗

bioRxiv2023

GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

How cell metabolism regulates DNA repair is incompletely understood. Here, we define a GTP-mediated signaling cascade that links metabolism to DNA repair and has significant therapeutic implications. GTP, but not other nucleotides, regulates the activity of Rac1, a G protein, that promotes the dephosphorylation of serine 323 on Abl-interactor 1 (Abi-1) by protein phosphatase 5 (PP5). Dephosphorylated Abi-1, a protein previously not known to activate DNA repair, promotes non-homologous end joining. In patients and mouse models of glioblastoma, Rac1 and dephosphorylated Abi-1 mediate DNA repair and resistance to standard of care genotoxic treatments. The GTP-Rac1-PP5-Abi-1 signaling axis is not limited to brain cancer, as GTP supplementation promotes DNA repair and Abi-1-S323 dephosphorylation in non-malignant cells and protects mouse tissues from genotoxic insult. This unexpected ability of GTP to regulate DNA repair independently of deoxynucleotide pools has important implications for normal physiology and cancer treatment.

cancer biology↗

bioRxiv2023

Engrailed-1 Promotes Pancreatic Cancer Metastasis

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, we report that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating a role in the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. Our findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

cancer biology↗

bioRxiv2023

An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping

Proximity labeling (PL) through biotinylation coupled with mass spectrometry (MS) has emerged as a powerful technique for capturing spatial proteomes within living cells. Large-scale sample processing for proximity proteomics requires a workflow that minimizes hands-on time while enhancing quantitative reproducibility. Here, we present a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. By combining this pipeline with an optimized quantitative MS acquisition method based on data-independent acquisition (DIA), we not only significantly increased sample throughput but also improved the reproducibility of protein identification and quantification. We applied this pipeline to delineate subcellular proteomes across various cellular compartments, including endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, employing 5HT2A serotonin receptor as a model, we investigated temporal changes of proximal interaction networks induced by the receptors activation with serotonin. Finally, to demonstrate the applicability of our PL pipeline across multiple experimental conditions, we further modified the PL pipeline for reduced sample input amounts to accommodate CRISPR-based gene knockout, and assessed the dynamics of the 5HT2A network in response to the perturbation of selected proximal interactors. Importantly, the presented PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

systems biology↗

bioRxiv2023

Dimerisation of European robin cryptochrome 4a

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerisation of avian cryptochromes and the role it could play in the mechanism of magnetic sensing in migratory birds. Here we present a combined experimental and computational investigation of the dimerisation of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulphide bonds, chemical cross-linking and photometric measurements show that disulphide-linked dimers are routinely formed, the most likely cysteines being C317 and C412. Computational modelling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

biochemistry↗

bioRxiv2023

Deep Learning for Flexible and Site-Specific Protein Docking and Design

Protein complexes are vital to many biological processes and their understanding can lead to the development of new drugs and therapies. Although the structure of individual protein chains can now be predicted with high accuracy, determining the three-dimensional structure of a complex remains a challenge. Protein docking, the task of computationally determining the structure of a protein complex given the unbound structures of its components (and optionally binding site information), provides a way to predict protein complex structure. Traditional docking methods rely on empirical scoring functions and rigid body simulations to predict the binding poses of two or more proteins. However, they often make unrealistic assumptions about input structures, and are not effective at accommodating conformational flexibility or binding site information. In this work, we present DockGPT (Generative Protein Transformer for Docking), an end-to-end deep learning method for flexible and site-specific protein docking that allows conformational flexibility and can effectively make use of binding site information. Tested on multiple benchmarks with unbound and predicted monomer structures as input, we significantly outperform existing methods in both accuracy and running time. Our performance is especially pronounced for antibody-antigen complexes, where we predict binding poses with high accuracy even in the absence of binding site information. Finally, we highlight our methods generality by extending it to simultaneously dock and co-design the sequence and structure of antibody complementarity determining regions targeting a specified epitope.

bioinformatics↗

bioRxiv2023

Steroid Receptor Coactivator-3 is a Key Modulator of Regulatory T Cell-Mediated Tumor Evasion

AbstractsSteroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were permanently eradicated in a genetically engineered tamoxifen-inducible Treg-cell specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the Chemokine (C-C motif) ligand (Ccl) 19/Ccl21/ Chemokine (C-C motif) Receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-{gamma}/C-X-C Motif Chemokine Ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and Natural Killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish pre-established breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3 deleted Tregs represents a novel approach to completely block tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators. Significance statementTregs are essential in restraining immune responses for immune homeostasis. SRC-3 is a pleiotropic coactivator, the second-most highly expressed transcriptional coactivator in Tregs, and a suspect in Treg function. The disruption of SRC-3 expression in Tregs leads to a complete lifetime eradication of tumors in aggressive syngeneic breast cancer mouse models because deletion of SRC-3 alters the expression of a wide range of key genes involved in efferent and afferent Treg signaling. SRC-3KO Tregs confer this long-lasting protection against cancer recurrence in mice without an apparent systemic autoimmune pathological phenotype. Therefore, treatment with SRC-3 deleted Tregs could represent a novel and efficient future target for eliminating tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators.

cancer biology↗

bioRxiv2023

A Single-Nucleus Atlas of Seed-to-Seed Development in Arabidopsis

Extensive studies of the reference plant Arabidopsis have enabled a deep understanding of tissues throughout development, yet a census of cell types and states throughout development is lacking. Here, we present a single-nucleus transcriptome atlas of seed-to-seed development employing over 800,000 nuclei, encompassing a diverse set of tissues across ten developmental stages, with spatial transcriptomic validation of the dynamic seed and silique. Cross-organ analyses revealed transcriptional conservation of cell types throughout development and heterogeneity within individual cell types influenced by organ-of-origin and developmental timing, including groups of transcription factors, suggesting gatekeeping by transcription factor activation. This atlas provides a resource for the study of cell type specification throughout the development continuum and a reference for stimulus-response and genetic perturbations at the single-cell resolution.

plant biology↗

bioRxiv2023

Split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80

The split-Gal4 system allows for intersectional genetic labeling of highly specific cell-types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a new split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is fully repressible by Gal80. We demonstrate the potent inducibility of "split-intein Gal4" in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell-type specific genetic drivers based on in silico predictions generated by single cell RNAseq (scRNAseq) datasets, and we describe a new algorithm ("Two Against Background" or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible. Significance statementThe split-Gal4 system allows Drosophila researchers to drive transgene expression with extraordinary cell type specificity. However, the existing split-Gal4 system cannot be controlled temporally, and therefore cannot be applied to many important areas of research. Here, we present a new split-Gal4 system based on a self-excising split-intein, which is fully controllable by Gal80, as well as a related drug-inducible split GeneSwitch system. This approach can both leverage and inform single-cell RNAseq datasets, and we introduce an algorithm to identify pairs of genes that precisely and narrowly mark a desired cell cluster. Our split-intein Gal4 system will be of value to the Drosophila research community, and allow for the creation of highly specific genetic drivers that are also inducible/repressible.

genetics↗

bioRxiv2023

Quantifying peripheral modulation of olfaction by trigeminal agonists

In the mammalian nose, two chemosensory systems, the trigeminal and the olfactory mediate the detection of volatile chemicals. Most odorants in fact are able to activate the trigeminal system, and vice versa, most trigeminal agonists activate the olfactory system as well. Although these two systems constitute two separate sensory modalities, trigeminal activation modulates the neural representation of an odor. The mechanisms behind the modulation of olfactory response by trigeminal activation are still poorly understood. In this study, we addressed this question by looking at the olfactory epithelium, where olfactory sensory neurons and trigeminal sensory fibers co-localize and where the olfactory signal is generated. We characterize the trigeminal activation in response to five different odorants by measuring intracellular Ca2+ changes from primary cultures of trigeminal neurons (TGNs). We also measured responses from mice lacking TRPA1 and TRPV1 channels known to mediate some trigeminal responses. Next, we tested how trigeminal activation affects the olfactory response in the olfactory epithelium using electro-olfactogram (EOG) recordings from WT and TRPA1/V1-KO mice. The trigeminal modulation of the olfactory response was determined by measuring responses to the odorant, 2-phenylethanol (PEA), an odorant with little trigeminal potency after stimulation with a trigeminal agonist. Trigeminal agonists induced a decrease in the EOG response to PEA, which depended on the level of TRPA1 and TRPV1 activation induced by the trigeminal agonist. This suggests that trigeminal activation can alter odorant responses even at the earliest stage of the olfactory sensory transduction. Significance StatementMost odorants reaching the olfactory epithelium can simultaneously activate olfactory and trigeminal systems. Although these two systems constitute two separate sensory modalities, trigeminal activation can alter odor perception. Here, we analyzed the trigeminal activity induced by different odorants proposing an objective quantification of their trigeminal potency independent from human perception. We show that trigeminal activation by odorants reduces the olfactory response in the olfactory epithelium and that such modulation correlates with the trigeminal potency of the trigeminal agonist. These results show that the trigeminal system impacts the olfactory response from its earliest stage.

neuroscience↗

bioRxiv2023

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an -ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the -ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

biochemistry↗

bioRxiv2023

Small change, big difference: A novel praziquantel derivative P96 with multistage antischistosomal activity for chemotherapy of schistosomiasis japonica

BackgroundPraziquantel (PZQ) has been the first line antischistosomal drug for all species of Schistosoma, and the only available drug for schistosomiasis japonica, without any alternative drugs since the 1980s. However, PZQ cannot prevent reinfection, and cannot cure schistosomiasis thoroughly because of its poor activity against juvenile schistosomes. In addition, reliance on a single drug is extremely dangerous, the development and spread of resistance to PZQ is becoming a great concern. Therefore, development of novel drug candidates for treatment and control of schistosomiasis is urgently needed. Methodologys/Principal findingsA novel PZQ derivative P96 was synthesized with the substitution of cyclohexyl by cyclopentyl. We investigated the in vitro and in vivo activities of our drug candidate P96 against different developmental stages of S. japonicum. Parasitological studies and scanning electron microscopy were used to study the primary action characteristics of P96 in vitro. Both mouse and rabbit models were employed to evaluate schistosomicidal efficacy of P96 in vivo. Besides calculation of worm reduction rate and egg reduction rate, quantitative real-time PCR was used to evaluate the in vivo antischistosomal activity of P96 at molecular level. In vitro, after 24h exposure, P96 demonstrated the highest activities against both juvenile and adult worm of S. japonicum in comparison to PZQ. The antischistosomal efficacy was concentration-dependent, with P96 at 50M demonstrating the most evident schistosomicidal effect. Scanning electron microscopy demonstrated that P96 caused more severe damages to schistosomula and adult worm tegument compared to PZQ. In vivo, our results showed that P96 was effective against S. japonicum at all developmental stages. Notably, its efficacy against young stage worms was significantly improved compared to PZQ. Moreover, P96 retained the high activity comparable to PZQ against the adult worm of S. japonicum. ConclusionsP96 is a promising drug candidate for chemotherapy of schistosomiasis japonica, which has broad spectrum of action against various developmental stage, potentially addressing the deficiency of PZQ. It might be promoted as a drug candidate for use either alone or in combination with PZQ for the treatment of schistosomiasis. Author SummarySchistosomiasis is one of the neglected tropical diseases caused by infection of Schistosoma spp. Currently, in the absence of effective vaccines for schistosomiasis, PZQ is the first line drug chosen for the treatment and control of schistosomiasis in most developing countries. However, after long-term and large-scale administration of PZQ, drug-resistance has been a great concern. Therefore, there is a need for new therapies. In this study, with the aim of preventing the formation of less effective metabolite 4-trans-cyclohexanol, a novel PZQ derivative, P96, is synthesized with the cyclohexyl group substituted by cyclopentyl group. It is this small modification that gives us a big surprise. In vitro, all the biological assessments, including viability reduction rate and morphological properties by scanning electron microscopy, demonstrate that P96 has superior anti-schistosomula activity compared to PZQ, and retains similar or even higher anti-adult S. japonicum activity to PZQ. The antischistosomal effect of P96 is dose-dependent. In vivo, P96 displays high efficacy against all developmental stages of S. japonicum, with significantly improved efficacy against young stage worms compared to PZQ. Furthermore, the quantitative detection results of specific circulatory SjR2 DNA prove that P96 has similar activity to PZQ against adult schistosome at molecular level in rabbit sera with infection of schistosomiasis. In conclusion, the novel PZQ derivative, P96 is a promising drug candidate for chemotherapy of schistosomiasis, potentially addressing the deficiency of PZQ, and might be promoted for use either alone or in combination with PZQ for treatment and control of schistosomiasis.

pharmacology and toxicology↗

bioRxiv2023

Efficient transgene-free genome editing in plants in the T0 generation based on a co-editing strategy

Transgene-free genome editing of plants in the T0 generation is highly desirable but challenging, especially in perennials and vegetatively propagated plants. Here, we investigated the co-editing strategy for generating transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta. Specifically, CBE/gRNA was used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants were efficiently generated for various genes (either individual or multiplex) in tomato, tobacco, potato, and citrus in the T0 generation. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 8% to 50%. Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. The co-editing strategy is efficient for generating transgene-free, genome-edited plants in the T0 generation, thus being a potent tool for plant genetic improvement.

plant biology↗