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bioRxiv2021

Global fitting for high-accuracy multi-channel single-molecule localization

Multi-channel detection in single-molecule localization microscopy (SMLM) greatly increases information content for various biological applications. Here, we present globLoc, a graphics processing unit (GPU) based global fitting algorithm with flexible PSF modeling and parameter sharing, to extract maximum information from multi-channel single molecule data. We show, both in simulations and experiments, that global fitting can substantially improve the 3D localization precision for biplane and 4Pi SMLM and color assignment for ratiometric multicolor imaging.

molecular biology↗

bioRxiv2024

Human lymphoid-neutrophil/monocyte restriction co-ordinately activates increased proliferation despite parallel heterogeneity in transcriptional changes

Recent studies indicate the human lympho-myeloid restriction process to be a different and more heterogeneous one than historically inferred. Here we describe the development of bulk and clonal culture systems that efficiently support early B-lymphoid differentiation and their use to identify biological and molecular changes that accompany their initial restriction from subsets of CD34+ human cord blood cells with lympho-myeloid-limited potential. Analyses of the changes observed revealed the acquisition of B-lymphoid- and neutrophil/monocyte (NM)-restricted properties are accompanied by a concomitantly accelerated and lineage-shared cell cycling activity and loss of self-renewal properties. Parallel, single-cell transcriptome analysis identified reduced expression of multiple self-renewal-associated genes and an accompanying heterogeneous activation of lineage-regulatory modules during the production of B, NM and dendritic cell precursors. These results uncover a connected regulation of lineage-shared proliferation control with persistent heterogeneity in the biological and transcriptional changes in the same cells undergoing B and NM lineage restriction.

immunology↗

bioRxiv2024

Differential biological effect of low doses of ionizing radiation depending on the radiosensitivity in a cell line model

PurposeExposure to low doses (LD) of ionizing radiation (IR), such as the ones employed in computed tomography (CT) examination, can be associated with cancer risk. However, not all individuals respond the same to IR, and cancer development could depend on the individual radiosensitivity. Notably, inter-individual differences in the response to IR have been very well studied for high and medium doses, but not for LD. In the present study, we wanted to evaluate the differences in the response to a CT-scan radiation dose of 20 mGy in two lymphoblastoid cell lines with different radiosensitivity. Materials and MethodsSeveral parameters were studied: gene expression, DNA damage, and its repair (by analyzing gamma-H2AX foci, chromosome breaks, and sister chromatid exchange), as well as cell viability, proliferation, and death. ResultsAfter 20 mGy of IR, the radiosensitive (RS) cell line showed an increase in DNA damage, and higher cell proliferation and apoptosis, whereas the radioresistant (RR) cell line was insensitive to this LD. Interestingly, gene expression analysis showed a higher expression of an antioxidant gene in the RR cell line, which could be used by the cells as a protective mechanism. After a dose of 500 mGy, both cell lines were affected by IR but with significant differences. The RS cells presented an increase in DNA damage and apoptosis, but a decrease in cell proliferation and cell viability, as well as less antioxidant response. ConclusionsA differential biological effect was observed between two cell lines with different radiosensitivity, and these differences are especially interesting after a CT scan dose. If this is confirmed by further studies, one could think that individuals with radiosensitivity-related genetic variants may be more vulnerable to long-term effects of IR, potentially increasing cancer risk after LD exposure.

molecular biology↗

bioRxiv2018

Toxoplasma gondii ROP18 Inhibits Human Glioblastoma Cell Apoptosis through Mitochondrial Pathway by Targeting Host Cell P2X1

It is known that Toxoplasma gondii infection both initiates and inhibits host cell apoptosis through different proapoptotic signaling cascades, but the parasitic factors involved in these processes remain unclear. T. gondii virulence factor ROP18 has been reported to regulate host cell apoptosis, but the results of this regulation are few reported and contradictory. In this study, we found that immune or neuro cells infected by any one of the T. gondii strains (RH-type I, ME49-type II, and VEG-type III) showed a significantly lower apoptosis index than their uninfected controls when apoptosis was induced by staurosporine (STS). We further found that ROP18 of RH strain inhibited ATP induced apoptosis in human glioblastoma cells (SF268) with endogenous expression of human proapoptotic protein purinergic receptor 1 (P2X1), but had no effects on the immune cells of RAW264.7 and THP-1 without detectable P2X1 expression, which may indicate that ROP18s inhibition of host cell apoptosis is related to P2X1. Interestingly, we further identified that ROP18 (RH strain) interacted with P2X1, and over-expression of ROP18 in COS-7 cells inhibited the cell apoptosis mediated by P2X1. We also found that ROP18 of RH strain inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from mitochondria to cytoplasm, and 1 ATP-triggered caspases activation. Collectively, these findings supported that ROP18 inhibited the host cell apoptosis through the intrinsic mitochondria pathway by targeting host cell P2X1, thereby suggesting a sensor role of the host proapoptotic protein P2X1 in this process\n\nAuthor summaryThe obligate intracellular protozoan Toxoplasma gondii has been shown to modulate cell apoptosis through different apoptotic pathways. However, the consequences are various and even contradictory, and the parasite effectors and the precise biological mechanisms remain unclear. Herein we showed that T. gondii of type I, II, and III strains could inhibit the apoptosis of neuro cells and immune cells. Toxoplasma gondii ROP18 (RH strain) inhibited apoptosis of human glioblastoma cell SF268 by targeting C terminal of host cell P2X1 protein, but not through proteasome-dependent degradation of P2X1.

molecular biology↗

bioRxiv2026

Autolab HBH: A Rapid Hyperbaric Heating Device for Streamlined, PCR-Ready Sample Preparation Across Diverse Biological Matrices and Organisms

Traditional nucleic acid extraction methods are costly, lengthy, and highly variable depending on the complexity of the sample matrix or the organism of interest. Workflows may exceed twenty steps, require separate kits for RNA and DNA, and demand expensive instrumentation, creating barriers to both speed and scalability. The AutolabTM HBH system addresses these limitations by using hyperbaric heating (HBH) to achieve temperatures above 100 {degrees}C in a sealed, pressurized environment through induction heating, enabling rapid lysis of diverse organisms and neutralization of macromolecular PCR inhibitors within minutes. The combination of extreme heat and HBH-optimized lyophilized reagents rapidly inactivates nucleases while preserving free nucleic acids. The workflow is streamlined to two steps: heating up to 1 mL of sample in the proprietary HBH bullet, followed by a brief centrifugation to pellet additives. The resulting supernatant is immediately compatible with real-time reverse transcription polymerase chain reaction (RT-PCR) and other downstream molecular assays. Here, we evaluate the systems broad compatibility with diverse sample buffers, matrices, and organisms. Comparative testing was conducted alongside Qiagen extraction methods to benchmark performance.

molecular biology↗

bioRxiv2020

Amyloid beta acts synergistically as a pro-inflammatory cytokine

The amyloid beta (A{beta}) peptide is believed to play a central role in Alzheimers disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily-selected functions of A{beta} are incompletely understood. Here, we report that nanomolar concentrations of A{beta} act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that A{beta} can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by A{beta} have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of A{beta} at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of A{beta} often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for A{beta} and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

molecular biology↗

bioRxiv2023

Abundant capped RNAs are derived from mRNA cleavage at 3'UTR G-Quadruplexes

The 3 untranslated region (3UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis gene expression (CAGE), a method for the detection of capped 5 ends of mRNAs, additionally reveals a large number of apparently 5 capped RNAs derived from 3UTRs. Here we provide the first direct evidence that these 3UTR-derived RNAs are indeed capped and often more abundant than the corresponding full-length mRNAs. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA knockdowns, we find that these 3UTR-derived RNAs likely originate from AGO2-mediated cleavage, and most often occur at locations with potential to form RNA-G-quadruplexes and are enriched by RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validates several 3UTR-derived RNAs, demonstrates their abundance and shows that they tend not to co-localise with the parental mRNAs. We also find that production of 3UTR-derived RNA could explain the previously reported role of a 3UTR G-quadruplex in regulating the production of APP protein. Taken together, we provide new insights into the origin and abundance of 3UTR-derived RNAs, show the utility of CAGE-seq for their quantitative detection, and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/538568v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3f2ca7org.highwire.dtl.DTLVardef@18ca1b6org.highwire.dtl.DTLVardef@1ccdd8dorg.highwire.dtl.DTLVardef@e58938_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2018

An automated workflow for the discovery and docking simulation of the protein-protein complexes using in vivo chemical cross-linking

Introduction Introduction Results Discussion Materials and Methods Competing interests Contributions Materials and Correspondence References Chemical cross(X)-link mapping assisted by mass spectrometry (XL-MS, also CXMS and CLMS) is a low-resolution hybrid method of structural biology, yielding a set of pairwise distance restraints between reactive solvent-accessible amino acids1-9. Most commonly used X-linkers at present belong to the class of amino-reactive homo-bifunctional NHS-esters, which act as protein proximity sensors, connecting predominantly Lys residues (as well as far less common and informative N-terminal amines)10-14. Main structural application of XL-MS to date i ...

molecular biology↗

bioRxiv2025

SwitchClass: dissecting attenuated and escalated molecular features via a label-switch classification framework

Biological systems exhibit complex molecular trajectories in response to perturbations, ranging from changes that revert or attenuate towards homeostasis to alterations that persist or escalate. Capturing these complex patterns is essential for understanding molecular resilience and maladaptive persistence. Here, we introduce SwitchClass, a label-switch classification framework that distinguishes attenuated and escalated molecular features across biological states. By training dual classifiers with inverted outcome labels, SwitchClass computes a differential feature importance score ({delta}) that quantifies directional change in high-dimensional data. Applied to colorectal cancer proteomics spanning healthy, pre-treatment, and post-treatment samples, Switch-Class reveals proteins that normalise after therapy and those remaining dysregulated, uncovering partial molecular recovery. In phosphoproteomics of dietary perturbation and reversal, it uncovers phosphorylation sites linked to incomplete restoration of insulin signalling. In single-cell transcriptomes from COVID-19 patients with varying severities, it identifies cell-type-specific transcripts that mark either the resolution or persistence of inflammatory activity. Together, these analyses establish SwitchClass as a generalisable and interpretable framework for mapping directional molecular changes underlying adaptation, divergence, and disease severity across biological systems. SwitchClass is freely available from https://github.com/PYangLab/SwitchClass.

bioinformatics↗

bioRxiv2021

CZON-cutter: a CRISPR-Cas9 system with multiplexed organelle imaging in a simple unicellular alga

The simple cellular structure of the unicellular alga Cyanidioschyzon merolae consists of one nucleus, one mitochondrion, one chloroplast, and one peroxisome per cell and offers unique advantages to investigate mechanisms of organellar proliferation and the cell cycle. Here, we describe an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, CZON-cutter, for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease to target editing at a locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants by fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows validation of phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. SummaryAn engineered CRISPR-Cas9 system, named CZON-cutter, for simultaneous genome editing and fluorescent protein labeling of organelles in Cyanidioschyzon merolae can be used to validate intracellular function of a particular gene, even if it is essential.

molecular biology↗

bioRxiv2023

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

Proximity between proteins plays an essential and ubiquitous role in many biological processes. Molecular tools enabling to control and observe the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced REcognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible, and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allowed furthermore the design of innovative biosensors for the study of various processes, such as signal transduction and apoptosis.

cell biology↗

bioRxiv2023

The selfish yeast plasmid exploits a SWI/SNF-type chromatin remodeling complex for hitchhiking on chromosomes and ensuring high-fidelity propagation

Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.

molecular biology↗

bioRxiv2018

PLZF limits enhancer activity during hematopoietic progenitor aging

PLZF (promyelocytic leukemia zinc finger) is a transcription factor acting as a global regulator of hematopoietic commitment. PLZF displays an epigenetic specificity by recruiting chromatin-modifying factors but little is known about its role in remodeling chromatin of cells committed towards a given specific hematopoietic lineage. In murine myeloid progenitors, we decipher a new role for PLZF in restraining active genes and enhancers by targeting acetylated lysine 27 of Histone H3 (H3K27ac). Functional analyses reveal that active enhancers bound by PLZF are involved in biological processes related to metabolism and associated with hematopoietic aging. Comparing the epigenome of young and old myeloid progenitors, we reveal that H3K27ac variation at active enhancers is a hallmark of hematopoietic aging. Taken together, these data suggest that PLZF, associated with active enhancers, appears to restrain their activity as an epigenetic gatekeeper of hematopoietic aging.

molecular biology↗

bioRxiv2024

Laminin 111 triggers cell quiescence and long-term survival by inducing IQGAP1-mediated cytosolic scaffolding of ERK and BAD inactivation

In an adult human body, only a minority ([~]1%) of cells are dividing; all others are either quiescent, senescent or terminally differentiated. Cellular quiescence, also called G0, is a reversible non-proliferative state in which cells, such as adult stem cells, exist until stimuli trigger their re-entry into the cell cycle. Quiescent cells are known to reside within microenvironment niches of specific extracellular matrix (ECM) composition, but the molecular mechanisms that control their entry and maintenance into G0 and their long-term survival are poorly understood. Here, using a reproducible and homogenous in vitro model of quiescence, ex vivo tissue histology, phosphoproteomics, and molecular cell biological assays, we revealed that Laminin 111 was sufficient to trigger i) reversible cell cycle exit into G0; ii) sustained and elevated MAPK/ERK signaling; and iii) long-term survival. We found that ERK was activated through the Rap1-BRAF-MEK arm underneath Laminin-binding Integrin 3{beta}1. Activated pERK was scaffolded into the cytoplasm by IQGAP1, thereby blocking its translocation into the nucleus and the activation of proliferative transcription factors. Instead, cytoplasmic pERK inhibited pro-apoptotic protein BAD, which mediated the survival of quiescent cells even in absence of mitogen stimuli. Importantly, we confirmed that pERK was elevated and retained in the cytoplasm of Lgr5+ stem cells when they were located within Laminin 1-positive niches in porcine intestine. These findings uncovered a molecular mechanism that may explain how quiescent cell pools, such as dormant adult stem cells, can survive many years despite low mitogen stimuli and be resistant to apoptotic challenges, including chemotherapy. HIGHLIGHTSO_LILaminin 111 is sufficient to induce cellular quiescence (G0) and long-term survival. C_LIO_LILaminin 111 triggers the sustained and elevated activation of ERK during G0. C_LIO_LIERK is activated not by growth factor receptors but through the Rap1-BRAF-MEK arm underneath Laminin-binding Integrin 3{beta}1. C_LIO_LIActive, phosphorylated ERK (pERK) is scaffolded by IQGAP1, which prevents it from translocating into the nucleus and activating proliferative transcription factors. C_LIO_LIInstead, cytoplasmic pERK mediates the phosphorylation, and thus inhibition, of BAD, thereby raising the threshold at which G0 cells enter apoptosis. C_LI

cell biology↗

bioRxiv2020

Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy

DNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA imagers to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.

molecular biology↗

bioRxiv2021

Transcriptome analysis of the NR1H3 mouse model of multiple sclerosis reveals a pro-inflammatory phenotype with dysregulation of lipid metabolism and immune response genes

BackgroundThe development of effective treatments for multiple sclerosis (MS), and in particular its progressive forms, is hampered by the lack of etiologically relevant cellular and animal models of human disease. Models that recapitulate the biological and pathological processes leading to the onset and progression of MS in patients are likely to afford better translational efficacy. Following the discovery of the NR1H3 p.Arg415Gln pathogenic mutation for progressive MS in two Canadian families, we developed a knock-in mouse model harboring a homologous mutation in the endogenous gene to provide a more physiologically relevant model of human MS. MethodsGene expression was evaluated in constitutive heterozygote (which recapitulates the human disease genotype) and homozygote Nr1h3 p.Arg413Gln knock-in mice on a C57BL/6 background, and compared to wild-type littermates. AmpliSeq Transcriptome Mouse Gene Expression kits analyzed on an Ion Proton sequencer were used to generate the gene expression profiles of spleen, liver, brain and spinal cord tissue from three-month-old male and female mice. Differential expression between genotypes was assessed with DESeq2, and Gene Ontologies pathways enrichment analysis performed with DAVID v6.8. Benjamini-Hochberg false discovery rate (FDR) correction for multiple testing was applied. ResultsTranscriptome analysis of spleen tissue from Nr1h3 p.Arg413Gln mice revealed 23 significantly dysregulated genes (FDR<0.05) with greater than a two-fold change in expression. These include CD5 antigen-like (Cd5l), complement component 6 (C6), procollagen C-endopeptidase enhancer 2 (Pcolce2), interleukin 22 receptor, alpha 2 (Il22ra2), and T cell immunoglobulin and mucin domain containing 4 (Timd4). Gene Ontology enrichment analysis support upregulation of cell cycle pathways and downregulation of immune system response in splenic cells. The liver transcriptome identified 27 significantly dysregulated genes with greater than a two-fold change in expression compared to wild-type littermates. Cd5l, Timd4, C-C motif chemokine receptor 3 (Ccr3), ADAM metallopeptidase domain 11 (Adam11) and macrophage expressed 1 (Mpeg1) were amongst those most significantly dysregulated. Enrichment analysis supported altered immune function with upregulation of sterol and steroid metabolic processes and downregulation of fatty acid biosynthesis and inflammatory and immune system responses. Although brain and spinal cord transcriptome profiles identified several genes significantly dysregulated in Nr1h3 mice compared to wild-type littermates (FDR<0.05), none presented greater than two-fold changes in gene expression. DiscussionThe analysis of the Nr1h3 p.Arg413Gln mouse model of MS suggests that the predominance of a pro-inflammatory over a healing or reparative phenotype, combined with deficiencies in myelination and remyelination, are the biological mechanisms implicated in the onset of MS and the development of a more severe progressive disease course observed in patients with NR1H3 mutations. Association of NR1H3 common variants with MS risk indicates that the disruption of these biological and immunological processes is not only informative for familial forms of disease but MS patients at large. Differences in transcriptome profiles underline the value of this model for the development and validation of novel therapeutic strategies and ultimately treatments with the potential to delay or even halt the onset of progressive MS and to ameliorate the severity of clinical symptoms.

molecular biology↗

bioRxiv2021

Inhibition of BET family proteins suppresses African swine fever virus infection

African swine fever (ASF), an acute, severe, highly contagious disease caused by African swine fever virus (ASFV) infection in domestic pigs and boars, has a mortality rate of up to 100%. Because effective vaccines and treatments for ASF are lacking, effective control of the spread of ASF remains a great challenge for the pig industry. Host epigenetic regulation is essential for the viral gene transcription. Bromodomain and extraterminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT, are epigenetic "readers" critical for gene transcription regulation. Among these proteins, BRD4 recognizes acetylated histones via its two bromodomains (BD1 and BD2) and recruits transcription factors, thereby playing a pivotal role in transcriptional regulation and chromatin remodeling during viral infection. However, how BET/BRD4 regulates ASFV replication and gene transcription is unknown. Here, we randomly selected 12 representative BET family inhibitors and compared their effects on ASFV infection in pigs primary alveolar macrophages (PAMs). They were found to inhibit viral infection by interfering with the different stages of viral life cycle (attachment, internalization, desencapsidation and formation of viral factories). The four most effective inhibitors (ARV-825, ZL0580, I-BET-762 and PLX51107) were selected for further antiviral activity analysis. These BET/BRD4 inhibitors dose-dependently decreased the ASFV titer, viral RNA transcription and protein production in PAMs. Collectively,our study reported novel activity of BET/BRD4 inhibitors in inducing suppression of ASFV infection, providing insights into role of BET/BRD4 in epigenetic regulation of ASFV and potential new strategies for ASF prevention and control. IMPORTANCESince the continuing spread of the ASFV in the world, and lack of commercial vaccines, the development of improved control strategies including antiviral drugs are urgently needed. BRD4 is an important epigenetic factor and has been commonly used for drug development for tumor treatment. Furthermore, the latest research showed that BET/BRD4 inhibition could suppress replication of virus. In this study, we first showed the inhibitory effect of agents targeting BET/BRD4 on ASFV infection with no significant host cytotoxicity. Then, we found 4 BET/BRD4 inhibitors which can inhibit ASFV replication, RNA transcription and protein synthesis. Finally, we analyzed 4 inhibitors biological effect on BRD4 according to the structure of BRD4, and docking analysis of BET-762, PLX51107, ARV-825 and ZL0580 binding to BD1 and BD2 domains of BRD4 was performed. Our findings support the hypothesis that BET/BRD4 can be considered as attractive host targets in antiviral drug discovery against ASFV.

molecular biology↗

bioRxiv2025

Origins of cin: Lateral Gene Transfer of Cytoplasmic Incompatibility Nuclease Operon to Orientia tsutsugamushi

CinB nucleases are Wolbachia proteins that induce cytoplasmic incompatibility (CI) through tandem nuclease domains nuc1 and nuc2 1. CI is a form of reproductive parasitism (RP) whereby males are conditionally sterilized 2. The system behaves as a toxin-antidote (TA) system 1-8 where operon gene cinA encodes an antidote and gene cinB encodes a toxin 1,5. Cin operons are purportedly the cause of gene-drive induced by wolbachiae infecting Drosophila simulans 1,9-12. An unanswered research question is whether lateral transfer of CI operons to bacteria outside wolbachiae would transfer RP phenotype and activate gene-drive. We demonstrate that a cin operon, capable of gene-drive, has jumped into the genome of Orientia tsutsugamushi, a human pathogen and causative agent of lethal scrub typhus. When expressed in transgenic Drosophila melanogaster, the wildtype cinBoTsu was capable of inducing CI independent of its partner antidote cinAoTsu. In addition, cinAoTsu rescued the phenotype in accordance with strict TA functionality. To understand the diverging roles of the tandem nuclease domains we mutated the domains and tested all permutations of active/inactive forms. Finally, we isolated IS5 transposon variants flanking the operon in O. tsutsugamushi and re-activated them to test their mobility. We demonstrate that these transposons can transfer genes and initiate lateral gene transfers into E. coli. These data demonstrate that active bacterial transposons can mobilize and transfer CI factors (cifs) to diverse bacteria. Overall, our data contribute mechanistic understanding in support of the TA model of CI and illuminate biochemical mechanisms that mobilize cifs across genomes from phylogenetically diverse taxa. Significance StatementCI operons are foundational genes that directly contribute to the success of Wolbachia-based bio-control strategies. Two applications of RP-inducing Wolbachia strains are insect population replacement and the incompatible insect technique. Both these techniques do not work if cifs do not function. Thus, a mechanistic understanding of cif function contributes to worldwide bio-control implementations. Furthermore, certain wolbachiae have long been studied as broad-spectrum inducers of diverse RP phenotypes, including CI, parthenogenesis, male-killing, and feminization. How these diverse phenotypes evolve and if they are all induced by cif genes is a long-standing field question. For example, Orientia tsutsugamushi, the causative agent of a deadly scrub typhus, induces parthenogenesis in Leptotrombidium mites, which putatively have reproductive advantages over uninfected mites. How Orientia induces RP is an important epidemiological question in vector biology. In our report, we show that an active cin operon is capable of CI and jumped into Orientia genomes via an IS5 transposon. We reconstructed this transposon and engineered it as a biotechnological tool. In toto, our study leads to the proposal of a new hypothesis whereby CI and parthenogenesis phenotypes might both be connected to the same cif expressed under divergent host genetic contexts.

molecular biology↗