Biology subjects

Tian, X.

Publications and source records attributed to Tian, X..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Splenic red pulp macrophages eliminate the liver-resistant Streptococcus pneumoniae during bloodstream infection

The spleen is well-known for defense against invasive infections of encapsulated bacteria, particularly Streptococcus pneumoniae (pneumococcus). However, the precise mechanism of the splenic anti-bacterial immunity remains elusive. Here we report that red pulp (RP) macrophages execute the splenic defense against S. pneumoniae in mice, with the help of natural antibodies (nAbs) and complement system. The spleen slowly but substantially cleared encapsulated pneumococci from the bloodstream in the early phase of blood infection, especially the serotypes that resist to liver filtration. Among splenic macrophage subpopulations, only the lack of RP macrophages led to the complete loss of the splenic immunity. Intravital microscopy detected direct capture and phagocytic killing of circulating pneumococci by RP macrophages in the absence of neutrophils and inflammatory monocytes. Likewise, plasma nAbs targeting pneumococcal cell wall phosphocholine and complement protein C3 were essential for RP macrophage-mediated immune clearance. Our findings have thus uncovered the long-sought immune pathway against invasive pneumococcal infections in the spleen. Short summaryThe spleen plays a crucial role in defending against invasive infections caused by Streptococcus pneumoniae (pneumococcus) and other encapsulated bacteria in humans, but the mechanisms underlying this organ-specific immunity remain largely elusive. This study has revealed that the spleen eliminates pneumococci through a coordinated interplay among red pulp macrophages, serum nAbs targeting phosphocholine and the complement system.

microbiology↗

bioRxiv2024

Natural antibodies to polysaccharide capsules enable Kupffer cells to capture invading bacteria in the liver sinusoids

The interception of blood-borne bacteria in the liver defines the outcomes of invasive bacterial infections, but the mechanisms of this anti-bacterial immunity are largely speculative. This study shows that natural antibodies (nAbs) to capsules enable liver macrophage Kupffer cells (KCs) to rapidly capture and kill blood-borne encapsulated bacteria in mice. Affinity pulldown with serotype-10A capsular polysaccharides (CPS10A) of S. pneumoniae (Spn10A) led to the identification of CPS10A-binding nAbs in serum. The CPS10A-antibody interaction enabled KCs to capture Spn10A bacteria from the bloodstream, in part through complement receptors on KCs. The nAbs were found to recognize the {beta}1-6-linked galactose branch of CPS10A, and similar moieties of serotype-39 S. pneumoniae and serotype-K50 Klebsiella pneumoniae capsules. More importantly, the nAbs empowered KCs to capture serotype-39 S. pneumoniae and serotype-K50 K. pneumoniae in the liver. Collectively, our data have revealed a highly effective immune function of nAb against encapsulated bacteria, and provided a proof of concept for treating septic bacterial diseases with monoclonal antibodies. Short summaryRapid capture of potentially harmful bacteria in blood by liver macrophages are vital for the blood sterility and health. This work reports how naturally occurring antibodies in the plasma enable macrophages to capture and kill blood-borne bacteria in the liver.

microbiology↗

bioRxiv2024

Spatiotemporal transcriptomic profiling reveals the dynamic immunological landscape of alveolar echinococcosis

Alveolar echinococcosis (AE) is caused by the invasive growth of the metacestodes of Echinococcus multilocularis (E. multilocularis). The early diagnosis, management, and treatment of AE remains challenging. Herein, we integrated bulk RNA-seq, scRNA-seq, and ST technologies to reveal the immune characteristics both spatially and chronologically in E. multilocularis infected mouse liver. An unprecedented high-resolution spatial atlas of the E. multilocularis infection foci was obtained, revealing the pivotal role of neutrophils, Spp1+ monocyte-derived macrophages (MoMFs), and fibroblasts in AE progression. We observed continuous recruitment of neutrophils and macrophages into the infected tissues, indicating prolonged immune stimulation by the growing metacestodes. However, their spatial distribution patterns and functions changed during the infection cause. At the early infection stage, Il1bhi neutrophils aggregated in the lesion displayed the highest pattern-recognition receptor activity and may kill the metacestodes through NETosis, but their death in the infection foci might also trigger immune suppression. Meanwhile, Spp1+ MoMFs attached to the protoscoleces to clear the infection. At the late infection stage, the neutrophils failed to infiltrate into the lesion although their continuous recruitment into the infected liver. Even though the Spp1+ MoMFs enclosing the microcysts of E. multilocularis may retain their pathogen-killing ability, they may also promote fibrosis and angiogenesis through interaction with fibroblasts. The pro- and anti-inflammatory phenotype balance of these immune cells may be associated with the transition of parasite control strategy from "active killing" to "negative segregation" by the host. Our findings identify key molecular traits involved in AE development, which may benefit the treatment of echinococcosis.

immunology↗

bioRxiv2024

MtrAB two-component system is crucial for the intrinsic resistance and virulence of Mycobacterium abscessus

Mycobacterium abscessus (Mab) poses serious therapeutic challenges, principally due to its intrinsic resistance to a wide array of antibiotics. The pressing issue of drug resistance has spurred an urgent need to explore novel targets and develop new therapeutic agents against Mab. The MtrAB two-component system, conserved among Actinobacteria, is pivotal for regulating various metabolic processes. Nevertheless, the role of MtrAB in Mab remains elusive. In this study, we uncovered that Mab strains with disrupted mtrA, mtrB or both exhibited heightened susceptibility to a variety of antibiotics with diverse mechanisms of action, in contrast to the wild-type strain. In a murine model, rifabutin, bedaquiline, and amikacin, which were inactive against the wild-type Mab strain, demonstrated efficacy against all the mtrA, mtrB and mtrAB knockout strains, significantly reducing pulmonary bacterial burdens compared to vehicle controls after ten days of treatment. Notably, the virulence of all the mtrA, mtrB, and mtrAB knockout strains was highly diminished in the murine model, as evidenced by a substantial decrease in bacterial load in the lungs of mice after 16 days. We observed that all three knockout strains exhibited a significantly reduced growth rate compared to the wild-type strain. We discovered that cells lacking mtrA, mtrB or both exhibited an elongated cell length and had multiple septa, suggesting that both MtrA and MtrB regulate cell division of Mab. Subsequently, an ethidium bromide accumulation assay disclosed that the absence of either mtrA or mtrB or both significantly increased cell envelope permeability. In summary, this study suggests that mtrA and mtrB play an important role in the intrinsic resistance and virulence of Mab by affecting cell division and altering cell permeability. Consequently, MtrA and MtrB represent promising targets for the discovery of anti-Mab drugs. HIGHLIGHTSO_LIKnockout of mtrA, mtrB or mtrAB leads to increased sensitivity of M. abscessus in vitro and in vivo. C_LIO_LIThe mtrA, mtrB or mtrAB knockout M. abscessus strains exhibit highly reduced virulence. C_LIO_LIMtrA and MtrB are potential targets for anti-M. abscessus drug discovery. C_LIO_LIKnockout of mtrA, mtrB or mtrAB results in defective cell division in M. abscessus. C_LI

microbiology↗

bioRxiv2024

Extracellular electron transfer drives efficient H2-independent methylotrophic methanogenesis by Methanomassiliicoccus, a seventh order methanogen

Methylotrophic methanogenesis is achieved via methyl group dismutation or H2 reduction. This study reports extracellular electron droving efficient methylotrophic methanogenesis. The 7th order methanogen Methanomassiliicoccus luminyensis exclusively implements H2-dependent methylotrophic methanogenesis, but strain CZDD1 isolated from paddy soil possessed a higher methane-producing rate in coculture with Clostridium malenominatum CZB5 or the electrogenic Geobacter metallireducens. Chronoamperometry detected current production from CZB5, and current consumption accompanied CH4 production in a methanol-containing electrochemical culture of CZDD1. This demonstrated that M. luminyensis was capable of both direct species electron transfer (DIET) and extracellular electron transfer (EET) in methylotrophic methanogenesis. EET and DIET also enabled CZDD1 to produce methane from dimethyl arsenate. Differential transcriptomic analysis on H2-versus EET- and DIET-cocultures suggested that a membrane-bound Fpo-like complex and archaella of M. luminyensis CZDD1 could accept extracellular electrons. Given the ubiquitous environmental distribution of Methanomassiliicoccus strains, EET driven methylotrophic methanogenesis may contribute significantly to methane emission.

microbiology↗

bioRxiv2024

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/581057v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ffeb68org.highwire.dtl.DTLVardef@b83871org.highwire.dtl.DTLVardef@81b34corg.highwire.dtl.DTLVardef@1224b0b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

High-throughput Screening of Compounds against Autoluminescent Nonreplicating Mycobacterium tuberculosis under Diverse Conditions

The screening of new anti-mycobacterial chemicals is primarily focused on inhibiting the active growing bacteria. However, a major challenge in tuberculosis control is the ability of Mycobacterium tuberculosis to enter a nonreplicating state for extended periods, rendering it resistant to many clinical drugs and complicating eradication efforts. Existing low-oxygen-recovery assays designed for screening compounds targeting nonreplicating M. tuberculosis have limitations, including the colony-forming unit counting for non-luminous M. tuberculosis and the instability of the free plasmid carrying luxAB genes in luminescent M. tuberculosis, along with exogenous substrate requirements for light producing. Moreover, these assays fail to accurately replicate the growth conditions of nonreplicating M. tuberculosis in vitro, thus resulting in less convincing results. To address these challenges, we have developed an autoluminescence-based, cholesterol-enriched culture evaluation model to assess 17 anti-tuberculosis drugs of different classes against nonreplicating M. tuberculosis. Our findings indicate that the relative light unit, measured in real-time, serves as a reliable surrogate marker for colony-forming unit, which typically becomes available one month later. This suggests the utility of our model for the rapid determination of drug susceptibility dynamically. The autoluminescent M. tuberculosis, harbouring luxCDABE gene cluster within its genome, can emit blue-green light stably and autonomously without requiring an external substrate supplement. The minimal inhibitory concentrations of all the drugs tested under anaerobic conditions are significantly different from that detected in aerobic environment. Our model allows for rapid, precise, and efficient assessment of drug activity under anaerobic conditions, thereby enabling a more comprehensive evaluation of anti-mycobacterial efficacy. Overall, our model represents a significant advancement in anti-tuberculosis drug discovery and pharmaceutical development.

microbiology↗

bioRxiv2024

Differentiating specific and non-specific protein-metabolite interactions using gradient open port probe electrospray ionization mass spectrometry

Native electrospray ionization mass spectrometry (ESI-MS) using nano ESI or desorption electrospray ionization (DESI) has been widely used to study interactions between macromolecules and ligands, usually protein-metabolite interactions (PMIs). In MS spectra the charge state distributions (CSD) of proteins differ between native and non-native conditions and based on this, we report a method that can differentiate specific protein-metabolite interactions from non-specific binding. Our approach is based on a 3D-printed open port probe electrospray system (gOPP-ESI) using mobile phase gradients (aqueous to methanol) for the ionization of protein/protein-metabolite complex. Notably, we found that a true protein-metabolite complex is more resistant to the denaturing effect of methanol compared to the free protein. This is corroborated by the observation that forming high charge states of protein-metabolite complexes requires higher proportions of methanol than free protein while, for non-specific complexes, there is no obvious difference in the CSD. Therefore, by comparing the changes in the CSD of free protein and protein-metabolite complex versus the increase of methanol, we can distinguish metabolites that specifically interact with the target protein. The approach is evaluated with well-characterized protein-ligand pairs, and we confirmed that cytidine phosphates, N, N', N''-triacetylchitotriose, and fluvastatin are specific ligands for ribonuclease A, lysozyme, and beta-lactoglobulin respectively. However, cytidine-5-triphosphate (CTP) interacts non-specifically with lysozyme and beta-lactoglobulin. We believe that after first-round native-MS assays to identify which metabolites cause mass shifts to the free protein, the gOPP-ESI-MS could be used as a quick second-round check to exclude non-specific binding and discover metabolites truly interacting with the protein of interest, reducing the number of candidates for subsequent validation experiments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/583904v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@e2aad7org.highwire.dtl.DTLVardef@13e287borg.highwire.dtl.DTLVardef@1d3c6c0org.highwire.dtl.DTLVardef@9da2a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Single-cell multiomics reveals a gene regulatory circuit promoting leukemia cell differentiation

Cancer differentiation therapy aims to induce maturation of neoplastic cells, yet how cell fate is determined in oncogenic cellular contexts is poorly understood. Here we integrate chromatin accessibility and transcriptome analysis at single-cell levels to investigate the mechanism driving the differentiation trajectory of the PML/RAR+ acute promyeloid leukemia (APL) cell line model, NB4, under the induction of all-trans-retinoid acid (ATRA). We show that differentiating NB4 cells navigate through an intermediate cell fate decision point that leads to either terminal granulopoiesis or a transient immature-lymphocyte-like state. Functional perturbation studies indicate that ATRA signaling activates discrete PML/RAR-target enhancers to induce a positive feedforward gene regulatory circuit involving two myeloid-lineage transcription factors, SPI1 and CEBPE, which is both necessary and sufficient to activate a myelocytic gene expression program in NB4. Further, ectopic expression of SPI1 and CEBPE also promotes myelocytic differentiation in non-APL leukemia cell lines HL60 and K562. Together, these results shed light on the differentiation trajectory of therapy-induced cell decisions of APL and suggest a gene regulatory circuit that may be broadly exploited to promote terminal differentiation of leukemia.

cancer biology↗

bioRxiv2024

Pathway-enhanced Transformer-based model for robust enumeration of cell types from the cell-free transcriptome

Analyzing cell types of origin of cell-free RNA can enhance the resolution of liquid biopsies, thereby deepening the understanding of molecular and cellular changes in development and disease processes. Existing deconvolution methods typically rely on meticulously curated gene expression profiles or employ deep neural network with vast and complex solution spaces that are difficult to interpret. These approaches overlook the synergistic and co-expression effects among genes in biological signaling pathways, compromising their generalizability and robustness. we developed Deconformer, a Transformer-based deconvolution model that integrates biological signaling pathways at the embedding stage, to address these issues. Compared to popular methods on multiple datasets, Deconformer demonstrates superior performance and robustness, and is capable of tracking the developmental process of the fetal and placenta. Additionally, pathway-level interpretability of Deconformer offers new insights into crosstalk, dependencies, and other interactions within cell-free RNA pathways, supporting further biological discoveries. We posit that Deconformer represents a significant advancement in the precise analysis of the cell-free transcriptome. It holds the promise of describing disease progression and severity with a new level of accuracy, focusing on the contributions of originating cell types and their pathway dependencies. This model has the potential to catalyze the development of non-invasive diagnostic tools and enhance our understanding of the underlying biology of diseases.

bioinformatics↗

bioRxiv2024

Establishment of an Inhalation Administration Noninvasive Murine Model for Rapidly Testing Drug Activity against Mycobacterium tuberculosis

The efficacy of many compounds against Mycobacterium tuberculosis is often limited when administered via conventional oral or injection routes due to suboptimal pharmacokinetic characteristics. Inhalation delivery methods have been investigated to achieve high local therapeutic doses in the lungs. However, previous models, typically employing wild-type M. tuberculosis strains, were intricate, time-consuming, labor-intensive, and with poor repeatability. In this study, we developed an autoluminescence-based inhalation administration model to evaluate drug activity by quantifying relative light units (RLUs) emitted from live mice infected with autoluminescent M. tuberculosis. This novel approach has several improvements: it eliminates the need for anesthesia in mice during administration and simplifies the instrument manipulation; it is cost-effective by utilizing mice instead of larger animals; it shortens time from several months to 16 or 17 days for obtaining result; it is non-invasive by measuring the live RLUs of mice; up to six mice can be administrated daily and simultaneously, even 2-3 times/day; results are relatively objective and repeatable minimizing human factors. Proof-of-concept experiments demonstrated that inhalable rifampicin, isoniazid, and ethambutol showed anti-M. tuberculosis activity at concentrations as low as 0.5, 0.5, and 0.625 mg/mL, respectively, as evidenced by comparing the live RLUs of mice. Furthermore, consistency between RLUs and colony-forming units of the lungs reaffirms the reliability of RLUs as an indicator of drug efficacy, highlighting the potential of this approach for accurately assessing anti-M. tuberculosis activity in vivo. This autoluminescence-based and non-invasive inhalation model offers a substantial reduction in the time, effort, and cost required for evaluating the efficacy of screening new drugs and repurposing old drugs in vivo via inhalation administration.

microbiology↗

bioRxiv2024

Ilamycins are dual inhibitors of ClpX and ClpC1 involved in proteostasis in Mycobacteria

The mycobacterial caseinolytic protease (Clp) system has been recognized as a promising therapeutic target. In this study, we identified two novel ilamycin analogs, ilamycin E (ILE) and ilamycin F (ILF), both targeting the ClpC1 component of the ClpC1P1P2 proteasome. ILE potently disrupts ClpC1P1P2-mediated proteolysis, leading to delayed bactericidal activity, while ILF also binds ClpC1, albeit with lower affinity. Notably, we discovered a unique mutation in clpX and a novel insertion in clpC1, both conferring resistance to ILE and ILF in mycobacterium, validated by gene editing. Furthermore, ILE can also inhibit the proteolytic activity of ClpXP1P2 in a manner dependent on the substrates tag sequence and adaptor. This first demonstration of clpX- and clpC1-mediated ilamycin resistance underscores the potential of ilamycins to target multiple components of the Clp protease system, offering a novel dual-target strategy for combating mycobacterial infections.

microbiology↗

bioRxiv2024

EmbB and EmbC Regulate the Sensitivity of Mycobacterium abscessus to Echinomycin

Treatment of Mycobacterium abscessus (Mab) infection is a major challenge due to its intrinsic resistance to most available drugs. It is thus imperative to find new anti-Mab drugs. In this study, we investigated the activity and intrinsic resistance mechanism of echinomycin (ECH) against Mab. ECH is active against Mab (MIC: 2 {micro}g/mL). The embC gene knockout strain (Mab{Delta}embC) showed hyper-sensitive to ECH (MIC: 0.0078-0.0156 {micro}g/mL). The MICs of ECH-resistant strains screened based on the Mab{Delta}embC strain were 0.25-1 {micro}g/mL. Mutations were found in the EmbB, including Asp306Ala, Asp306Asn, Arg350Gly, Val555Ile, and Gly581Ser, which led to increased resistance to ECH when overexpressed in Mab{Delta}embC individually (0.25-0.5 {micro}g/mL). The EmbB mutants edited by the CRISPR/Cpf1 system became more resistant to ECH (MIC: 0.25-0.5 {micro}g/mL). The permeability of gene-edited and overexpressed Mab strains was reduced, as shown by the ethidium bromide accumulation assay, but it was still significantly higher than that of the parent Mab. To summarize, our study demonstrates that ECH has a strong anti-Mab activity and confirms that EmbB and EmbC are related to the sensitivity of Mab to ECH. EmbB mutation may partially compensate for the function of EmbC. Impact StatementMycobacterium abscessus (Mab) is a rapidly growing, intrinsic multidrug-resistant Mycobacterium. This study demonstrated that echinomycin (ECH) has potent antibacterial activity against Mab, and the mechanism of ECH resistance to Mab is related to EmbB and EmbC. EmbB and EmbC can alter the sensitivity of Mab to ECH by altering the permeability of its cell wall. In addition, there is a functional complementary evolution between EmbB and EmbC to regulate sensitivity to ECH. Overall, our study provides a novel anti-Mab drug candidate ECH and a scientific foundation for developing effective strategies to prevent and control Mab.

microbiology↗

bioRxiv2024

Spatially Exploring RNA Biology in Archival Formalin-Fixed Paraffin-Embedded Tissues

Spatial transcriptomics has emerged as a powerful tool for dissecting spatial cellular heterogeneity but as of today is largely limited to gene expression analysis. Yet, the life of RNA molecules is multifaceted and dynamic, requiring spatial profiling of different RNA species throughout the life cycle to delve into the intricate RNA biology in complex tissues. Human disease-relevant tissues are commonly preserved as formalin-fixed and paraffin-embedded (FFPE) blocks, representing an important resource for human tissue specimens. The capability to spatially explore RNA biology in FFPE tissues holds transformative potential for human biology research and clinical histopathology. Here, we present Patho-DBiT combining in situ polyadenylation and deterministic barcoding for spatial full coverage transcriptome sequencing, tailored for probing the diverse landscape of RNA species even in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for five years. Furthermore, genome-wide single nucleotide RNA variants can be captured to distinguish different malignant clones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA-mRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis trajectory. High resolution Patho-DBiT at the cellular level reveals a spatial neighborhood and traces the spatiotemporal kinetics driving tumor progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to study human tissue biology and aid in clinical pathology evaluation.

systems biology↗

bioRxiv2024

Electrophysiological responses to syntactic and 'morphological' structures: evidence from Mandarin Chinese

Although psycho-/neuro-linguistics has assumed a distinction between morphological and syntactic structure building as in traditional theoretical linguistics, this distinction has been increasingly challenged by theoretical linguists in recent years. Opposing a sharp, lexicalist distinction between morphology and syntax, non-lexicalist theories propose common morpho-syntactic structure building operations that cut across the realms of "morphology" and "syntax", which are considered distinct territories in lexicalist theories. Taking advantage of two pairs of contrasts in Mandarin Chinese with desirable linguistic properties, namely compound vs. simplex nouns (the "morphology" contrast, differing in morphological structure complexity per lexicalist theories) and separable vs. inseparable verbs (the "syntax" contrast, differing in syntactic structure complexity per lexicalist theories), we report one of the first pieces of evidence for shared neural responses for morphological and syntactic structure complexity in language comprehension, supporting a non-lexicalist view where shared neural computations are employed across morpho-syntactic structure building. Specifically, we observed that the two contrasts both modulated neural responses in left anterior and centro-parietal electrodes in an a priori 275:400 ms time window, corroborated by topographical similarity analyses. These results serve as preliminary yet prima facie evidence towards shared neural computations across morphological and syntactic structure building in language comprehension.

neuroscience↗

bioRxiv2024

Disrupting Homologous Recombination or Single-Strand Annealing Significantly Hinders CRISPR-Cas12a-Assisted Nonhomologous End-Joining Gene Editing Efficiency in Mycobacterium abscessus

Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, causes many types of serious infections in humans, posing a significant public health challenge. Currently, effective genetic manipulation tools for M. abscessus are still being developed, which hampers research and therapeutic development. However, the clustered regularly interspaced short palindromic repeats (CRISPR) - associated protein (Cas) systems have emerged as promising methods for generating highly specific double-strand breaks (DSBs) in its genome. These DSBs can be repaired by the error-prone nonhomologous end joining (NHEJ) mechanism, facilitating targeted gene editing. Here, our study marks a pioneering application of the CRISPR-NHEJ strategy in M. abscessus. Additionally, we discovered that NrgA from Mycobacterium marinum is crucial for the repair of DSBs caused by the CRISPR-Cas system in M. abscessus. Finally, contrary to previous findings, our study also indicates that inhibiting or overexpressing homologous recombination/single-strand annealing significantly decreases the efficiency of NHEJ repair in M. abscessus. This discovery challenges established perspectives and suggests that the NHEJ repair in M. abscessus may require the involvement of components from homologous recombination and single-strand annealing, demonstrating the complex interactions among the three DSB repair pathways in M. abscessus. Impact statementThere are still very few genetic manipulation tools available for Mycobacterium abscessus. Here we report the successful application of CRISPR-Cas12a-assisted nonhomologous end joining (NHEJ) in efficient gene editing in M. abscessus. Contrary to previous research suggesting that homologous recombination (HR) inhibition may enhance such editing efficiency in other Mycobacterium species, our results showed that disruption or overexpression of either HR or single-strand annealing not only failed to enhance but also significantly reduced the gene editing efficiency in M. abscessus. This suggests that NHEJ repair in M. abscessus may require components from both HR and single-strand annealing, highlighting a complex interaction among the DSB repair pathways in M. abscessus.

microbiology↗

bioRxiv2024

Targeting transcription factors through an IMiD independent zinc finger domain

Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/574032v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@b8e4e8org.highwire.dtl.DTLVardef@bc6f01org.highwire.dtl.DTLVardef@11bbe85org.highwire.dtl.DTLVardef@791cb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Lithocholic acid targets TULP3 to activate sirtuins and AMPK to retard ageing

Lithocholic acid (LCA), accumulated in the body during calorie restriction (CR), can confer administered metazoans with the ability to activate AMP-activated protein kinase (AMPK) and retard ageing. However, how LCA is signalled to activate AMPK and elicit the biological effects is unclear. Here, we show that LCA can enhance sirtuins (SIRTs) to deacetylate and subsequently inhibit vacuolar H+-ATPase (v-ATPase), thereby triggering AMPK activation via the lysosomal glucose-sensing pathway. Through proteomic analysis of SIRT1-coimmunoprecipitated proteins, we identify and validate that TUB like protein 3 (TULP3) is a constitutive component of SIRTs. Surprisingly, we found that TULP3 is an LCA receptor, and that the LCA-bound TULP3 activates SIRTs. The activated SIRTs in turn deacetylate the V1E1 subunit of v-ATPase on K52, K99 and K191 residues. Muscle-specific expression of the 3KR mutant of V1E1, mimicking the deacetylated state, dominantly activates AMPK and rejuvenates muscles in aged mice. Moreover, LCA once administered also activates AMPK and extends lifespan and healthspan in nematodes and flies, depending on the TULP3 homologues tub-1 and ktub, respectively. Our study thus elucidates that LCA triggers the TULP3-sirtuin-v-ATPase- AMPK route to manifest benefits of calorie restriction.

physiology↗