Biology subjects

Xie, X.

Publications and source records attributed to Xie, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Single-cell Characterization of DNA Hydroxymethylation of the Mouse Brain During Aging

DNA methylation dynamics, including 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC), critically regulate brain function, yet conventional methods cannot distinguish these modifications, obscuring their distinct roles in gene regulation and aging. We present Joint- Cabernet, a bisulfite-free single-cell platform enabling simultaneous profiling of 5hmC, 5mC, and transcriptomes. Applying Joint-Cabernet to 84,071 nuclei from adult and aged mouse brains, we resolved cell-type-specific DNA hydroxymethylation landscapes, revealing elevated 5hmCG and 5hmCH levels in transcriptionally active genes across neuronal subtypes and spatial gradients in cortical layers. During aging, 5hmCG accumulates globally but is selectively enriched at open chromatin loci, aligning with the upregulation of cell-type-specific genes in distinct brain cell types. This single-cell DNA methylation brain cell atlas provides a framework for studying methylation- driven mechanisms in brain aging and neurodegenerative diseases.

genetics↗

bioRxiv2025

Prenatal Alcohol Exposure Impairs Striatal Cholinergic Function and Cognitive Flexibility in Adult Offspring

Fetal Alcohol Spectrum Disorder (FASD), caused by prenatal alcohol exposure (PAE), is characterized by significant cognitive impairments, including reduced cognitive flexibility. Despite the critical role of cholinergic interneurons (CINs) in the dorsomedial striatum (DMS) for cognitive and behavioral flexibility, their contribution to neurobehavioral deficits in FASD remains unclear. To address this gap, this research explored the impact of PAE on CIN populations and activity, cognitive flexibility, and compulsive drinking behaviors in adult offspring. Using ChAT-Cre;Ai14-tdTomato mice combined with ChAT staining, we found substantial reductions in CIN number within the striatum of adult PAE offspring. Functional assessments revealed that PAE markedly decreased CIN firing activity and reduced acetylcholine (ACh) release in the DMS, as measured by electrophysiology recordings and live-tissue confocal imaging using a genetically encoded ACh sensor. Behaviorally, PAE-exposed offspring exhibited a significant deficit in adapting to reversed action-outcome contingencies despite intact initial learning capabilities. Moreover, PAE-exposed mice exhibited compulsive alcohol drinking behavior, characterized by elevated consumption and preference for quinine-adulterated alcohol. These findings collectively highlight the critical role of impaired cholinergic signaling in the cognitive and behavioral deficits observed following PAE. Understanding this cholinergic dysfunction provides valuable insights necessary for developing targeted interventions aimed at mitigating cognitive and behavioral consequences associated with FASD. HighlightsPAE reduces the number of CINs, their firing rate and ACh release in the DMS. PAE impaired instrumental reversal learning, known to be mediated by CINs. PAE increased compulsive-like drinking of quinine adulterated alcohol.

neuroscience↗

bioRxiv2025

Drosophila Metaxin-2 controls beta-barrel biogenesis and muscle growth in a stage-dependent fashion

Metaxin-2 (Mtx2) is an evolutionarily conserved mitochondrial outer membrane protein. Mutations in human Mtx2 cause mandibuloacral dysplasia (MADaM), a progeroid disorder. However, the pathologic mechanisms of Mtx2 loss-of-function remain largely unknown. Using Drosophila, we show Mtx2 null mutants exhibit pupal lethality, rescued by Drosophila or human Mtx2, underscoring functional conservation across species. Tissue-specific conditional knockout and rescue experiments reveal muscle as a critical site of dMtx2 action, with alternations of myofibril assembly and myogenic proteins being observed in dMtx2 mutants. Structural and functional mitochondrial abnormalities are also detected, verifying dMtx2s function in mitochondrial homeostasis. Notably, Mtx2 deficiency affects beta-barrel protein biogenesis and muscle development in pupa but not in larva, demonstrating Mtx2s dynamic regulation in mitochondrial proteostasis and muscle development. Our results elucidate mitochondrial mechanisms driving potential muscle-autonomous defects in MADaM patients and highlights stage-specific Mtx2 function as a prospective therapeutic target for this progeroid syndrome.

developmental biology↗

bioRxiv2025

FASN Inactivation-induced Progranulin (GRN) Expression Promotes Lysosome-dependent Cell Death to Suppress Leukemogenesis

Cancer cells rely on lipogenesis in addition to exogenous lipid uptake, and fatty acid synthase (FASN) is aberrantly overexpressed in myeloid leukemia. However, the precise role of FASN in leukemogenesis in vivo remains elusive. Here, we demonstrated that FASN is essential for leukemogenesis. Ablation of FASN impeded leukemic cell growth, survival, clonogenicity in vitro, and the leukemic burden in vivo. Conditional knockout of FASN barely affected hematopoiesis but significantly attenuated the leukemic progression of MLL-AF9 transplants. By screening a library of platensimycin derivatives, we identified compound MS-C19 as a potent FASN inhibitor. Pharmacological targeting of FASN by MS-C19 suppressed leukemic cell growth and clonogenicity in clinical AML blasts. Mechanistically, both MS-C19 treatment and FASN deficiency triggered the activation of lysosomal and inflammatory gene expression. Loss of FASN led to lysosomal membrane permeabilization and subsequent lysosome-associated cell death, but not lysosome biogenesis. We further identified that GRN, a lysosomal and neuroinflammatory gene, was potently transcribed by TFEB upon FASN loss. Depletion of GRN significantly reversed the inhibitory effects caused by FASN knockdown. Our work demonstrates that FASN is a therapeutic target for myeloid leukemia, and inactivation of FASN by the lead compound MS-C19 provides an alternative approach for leukemic intervention. Key PointsO_LIGenetic ablation of FASN induces apoptosis, impedes leukemia cell growth, and improves the survival of leukemic xenografts. C_LIO_LITargeting FASN by the compound MS-C19, a derivative of the natural microbe antibiotic platensimycin, reduces the cell survival of leukemic cell lines, the clinical leukemic blasts, and MLL-AF9 transformed murine leukemic cells. C_LIO_LIFASN inhibition induces lysosome-dependent cell death that relies on Progranulin (GRN) expression. C_LI

cancer biology↗

bioRxiv2025

Persistent endotoxin exposure in macrophages elicits an immunometabolic profile susceptible to NF-kB and p53 perturbation

The prolonged exposure of macrophages to endotoxin occurs in various chronic inflammatory conditions, including long-term inflammation triggered by particle-releasing implanted biomaterials, progressive pulmonary obstructive conditions, chronic bronchitis, colitis and hepatitis characterized by sustained exposure to gut-derived lipopolysaccharide (LPS). However, immune cellular metabolism in models of persistent inflammation remains undercharacterized, especially when compared to the well-studied immunometabolic changes following short-term LPS exposure. Here, we demonstrate that persistent LPS stimulation induces increased oxygen consumption alongside reduced levels of ATP and elevated glycolysis, with a mixed inflammatory profile indicative of endotoxin tolerance in primary bone marrow-derived macrophages. Pharmacological blockade of complex I of the mitochondrial electron transport chain lowered oxygen consumption and mitochondrial ROS by reducing NF-{kappa}B signaling. Surprisingly, NF-{kappa}B inhibition at TAK1 or I{kappa}B phosphorylation, and p53 activation, reduced both oxygen consumption and glycolysis with minimal mitochondrial ROS impact. The absence of elevated mitochondrial membrane potential in prolonged LPS exposure suggests mitochondrial ROS production is likely reverse electron transport-independent, supported by the lack of effect of a mitochondrial depolarizer on oxygen consumption. These findings reveal a distinct immunometabolic phenotype in macrophage endotoxin tolerance, extending our understanding of immunobiology in chronic inflammatory diseases.

immunology↗

bioRxiv2025

Actin Depolymerization Factors (ADFs) Moonlighting: Nuclear Immune Regulation by Interacting with WRKY Transcription Factors and Shaping the Transcriptome

Remodeling of the actin cytoskeleton is a critical process for plant immunity, essential for the transport, activation, and stabilization of immune-regulatory molecules and organelles. In this process, actin depolymerization factors (ADFs) function as key players through severing and depolymerizing actin microfilaments. However, recent evidence suggests that ADFs may possess non-canonical immune functions inside the nucleus, in addition to the canonic cytosolic role, a phenomenon not adequately explained by the traditional mechanistic model of ADF-actin dynamics. In this study, we demonstrate that Arabidopsis ADFs exhibit a moonlighting function in the nucleus, where they interact with transcriptional machinery to regulate the transcriptome during both the resting state and the immune responses. We show that ADF2/3/4 have redundant functions in defense against virulent and avirulent Pseudomonas syringae. Notably, it is nuclear - rather than cytosolic - ADFs that contribute to defense against P. syringae and mediate pro-immune transcription. Mechanistically, we demonstrate that nuclear ADFs interact with transcription factors, histone complexes, and other components of the transcriptional machinery. Specifically, ADF2/3/4 can form a complex with WRKY transcription factors, such as WRKY22/29/48, thereby directly regulating WRKY activity to shape the pro-immune transcriptome. In summary, our study reveals that ADFs moonlight as direct regulators of transcription factors, mediating a broad range of nuclear-cytoplasmic regulation in plant immunity and potentially other biological processes.

plant biology↗

bioRxiv2025

Alcohol Attenuates CRF-Induced Excitatory Effects from the Extended Amygdala to Dorsostriatal Cholinergic Interneurons

Alcohol relapse is associated with corticotropin-releasing factor (CRF) signaling and altered reward pathway function, though the precise mechanisms remain unclear. Here, we investigated how CRF modulates cholinergic interneurons (CINs) in the dorsal striatum, a region critical in mediating cognitive flexibility and action selection. Using monosynaptic and retrograde circuit tracing, we identified direct inputs from CRF-expressing (CRF) neurons in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) to dorsal striatal CINs. We showed that CINs express CRF receptor 1 (CRFR1) and established their functional connectivity with CeA/BNST CRF projections. Functional recordings revealed that CRF enhanced CIN excitability and promoted acetylcholine release in the dorsal striatum. However, acute alcohol exposure and withdrawal attenuated the excitatory effect of CRF on CIN firing, suggesting a mechanism by which alcohol disrupts CRF-dependent neuromodulation. These findings reveal a previously unrecognized CRF-CIN pathway linking the extended amygdala to the dorsal striatum and provide new insight into how CRF and alcohol interact to impair striatal function. This work highlights CRF signaling as a potential target for understanding stress-induced changes to the reward pathway. HighlightsO_LIDorsal striatal CINs receive monosynaptic CRF+ inputs from CeA and BNST neurons. C_LIO_LICRFR1 is expressed in striatal CINs, and CRF+ fibers are present in the dorsal striatum. C_LIO_LICRF enhances dorsal striatal CIN activity via CRFR1 signaling. C_LIO_LIAcute alcohol exposure impairs CRF-induced cholinergic activity. C_LI Significance StatementThe dorsal striatum regulates goal-directed behavior and is implicated in alcohol use disorder (AUD). Within this region, cholinergic interneurons (CINs) support cognitive flexibility and receive input from limbic areas, including the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). In this study, we identified direct projections from CRF-producing neurons in the CeA and BNST to dorsal striatal CINs, a subset of which express CRF receptor 1 (CRFR1). Electrophysiological recordings confirmed these projections provide functional input that is disrupted by acute alcohol exposure. These findings lay the groundwork for future studies on how CRF and alcohol interact to impair striatal function.

neuroscience↗

bioRxiv2025

Pharmacological interrogation of crosstalk between TLR signaling and stress granules reveals a compound with antiviral effect.

Stress granules (SG) are cytoplasmic membraneless compartments that regulate cellular stress responses and have been implicated in antiviral defense against Influenza A Virus (IAV). SG stabilization has been reported to be a viable strategy to develop antiviral drugs. To expand the repertoire of SG-modulating compounds, we performed a targeted pharmacological screen focusing on inhibitors of TLR signaling pathway, based on our previous work demonstrating an antagonistic relationship between SGs and TLR signaling. Using the synthetic dsRNA analog polyinosinic:polycytidylic acid (poly(I:C)) to induce SGs in A549 human lung cancer cell line, we screened a panel of TLR signaling pathway inhibitors for their effect on SG. We developed a robust image analysis pipeline utilizing ilastik for pixel classification and CellProfiler for object quantification, enabling high-throughput analysis of SG alterations. This screen identified multiple small molecules that destabilize SGs, including inhibitors of RIPK3, TBK1, PERK, IRAK1/4, and JNK1/2/3 and ERK1/2 kinases. Several inhibitors of NF-{kappa}B signaling also disrupted SG integrity. Most strikingly, PPM18, an NF-{kappa}B inhibitor, emerged as a dual-action compound, triggering spontaneous SG assembly via PERK-mediated eIF2 phosphorylation, while simultaneously suppressing poly(I:C)-induced SGs. Intriguingly, PPM18 exhibited potent IAV inhibition, but crucially, this antiviral activity was decoupled from SG formation as it robustly inhibited replication in G3BP1-/- cells which did not assemble SGs upon PPM18 treatment. Our work not only identifies PPM18 as a unique SG modulator and an antiviral agent but also challenges the prevailing belief linking SG assembly directly to antiviral activity as a general phenomenon. Our findings demonstrate that SG formation and antiviral activity can be functionally uncoupled, providing new insights into host-virus interactions. In summary, our results demonstrate feasibility of using SG screening for finding novel antiviral compounds while raising important questions about the role of SGs themselves in modulating host-virus interactions.

immunology↗

bioRxiv2025

Crosstalk Among Disrupted Blood-Brain Barrier, Neuroinflammation, and Coagulopathy in AC70 hACE2 Tg Mice with Prolonged Neurological Manifestations Induced by the Omicron BA.1 Variant of SARS-CoV-2

Long COVID, or post-acute sequela of COVID (PASC), has emerged as a major post-pandemic challenge, with lasting neurological effects in a substantial number of patients. The underlying pathophysiological mechanisms of PASC remains elusive, largely due to the lack of suitable animal models that replicate key clinical and pathological features, especially in survivors of acute infection. Here, we employed the Omicron BA.1-infected AC70 hACE2 Tg mice to explore the pathogenesis of COVID-19. Our findings in the AC70/BA.1 model demonstrated that surviving mice developed lasting neurological sequelae of COVID-19, making it a promising platform for studying the pathogenesis of neuro- or long-COVID. Histopathological examinations revealed a transient pulmonary inflammatory response that correlated with the levels of live virus, whereas neuroinflammation within the brains persisted and progressed beyond the acute phase, without a clear linkage to direct virus effects. We showed that the severity of neurological sequelae directly correlated with the degree of blood-brain-barrier (BBB) disruption, alterations in tight junctions (TJs) and fibrinogen extravasation into the brain parenchyma. Additionally, BA.1-infected mice elicited a sustained systemic prothrombotic state, with dysregulated coagulation and fibrinolytic pathways in an organ-specific manner, as evidenced by distinct pattern of intracellular fibrin(ogen) depositions, elevated d-dimers, and tissue-plasminogen activator (t-PA) expression in the lungs and brains. We also found a positive correlation between progressive neuroinflammation and t-PA expression, which was closely co-localized with Iba-1-positive cells, indicating that activated microglia may serve as an additional source of t-PA in the CNS. Lastly, we showed that BA.1 variant triggered prolonged anti-Annexin A2 (ANXA2) autoantibody production. ANXA2 is essential for the neurovascular system, coordinating multiply dynamic processes within the endothelium, including cell surface fibrinolysis and TJ assembly. Our analysis revealed that impaired TJ structures coexisted with diminished ANXA2 levels around the brain vessels, suggesting its involvement in BBB dysfunction and neuroinflammation. Author SummaryLong COVID symptoms, especially neurological complications, remains a key public health concern worldwide. Alterations in the cerebrovascular system, along with ongoing coagulation disorders, may play a crucial role in the pathogenesis of this condition. Here, we established murine model for study post-acute sequela of COVID (PASC). We demonstrated that AC70 Tg mice infected with a high dose of BA.1 Omicron variant developed prolonged neurological symptoms, accompanied by progressive neuroinflammation in survivors. Our findings clearly showed the involvement of blood-brain-barrier (BBB) disfunction in the severity of neuropathology induced by SARS-CoV-2 infection. We also showed that the bidirectional interaction between dysregulated coagulation/ fibrinolysis and inflammatory systems may exhibit a distinct pattern in the CNC, significantly contributing to persistent neurological symptoms. Finally, we highlighted a potential role of malfunctioning Annexin A2 (ANXA2) in this phenomenon that may serve as a promising drug target for neuro-COVID interventions.

microbiology↗

bioRxiv2025

MKFGO: Integrating Multi-Source Knowledge Fusion with Pre-Trained Language Model for High-Accuracy Protein Function Prediction

Accurately identifying protein functions is essential to understand life mechanisms and thus advance drug discovery. Although biochemical experiments are the gold standard for determining protein functions, they are often time-consuming and labor-intensive. Here, we proposed a novel composite deep-learning method, MKFGO, to infer Gene Ontology (GO) attributes through integrating five complementary pipelines built on multi-source biological data. MKFGO was rigorously benchmarked on 1522 non-redundant proteins, demonstrating superior performance over 11 state-of-the-art function prediction methods. Comprehensive data analyses revealed that the major advantage of MKFGO lies in its two deep-learning components, HFRGO and PLMGO, which derive handcraft features and protein large language model (PLM)-based features, respectively, from protein sequences in different biological views, with effective knowledge fusion at the decision-level. HFRGO leverages an LSTM-attention network embedded with handcraft features, in which the triplet loss-based guilt-by-association strategy is designed to enhance the correlation between feature similarity and function similarity. PLMGO employs the PLM to capture feature embeddings with discriminative functional patterns from sequences. Meanwhile, another three components provide complementary insights for further improving prediction accuracy, driven by protein-protein interaction, GO term probability, and protein-coding gene sequence, respectively. The source codes and models of MKFGO are freely available at https://github.com/yiheng-zhu/MKFGO.

bioinformatics↗

bioRxiv2025

Effect of Shufeng Xingbi Therapy on Th1/Th2 immune balance and intestinal flora in rats with allergic rhinitis

ObjectiveTo study the effect of Shufeng Xingbi Therapy (SFXBT) on Th1/Th2 immune balance and intestinal flora in allergic rhinitis (AR) rats. Methods32 SD rats were randomly divided into control group, OVA group, antibiotic + SFXBT group and acetic acid + SFXBT group. Evaluate the AR behavioral score, observe the pathological changes of nasal mucosa with H&E, detect the composition and content of colonic contents with 16S rDNA, the levels of serum IgE, IL-4 and short-chain fatty acid (SCFAs) with ELISA, the mRNA expressions of STAT5, STAT6 and GATA3 in nasal mucosa with RT-qPCR, and the protein expressions of IL-4, STAT5, STAT6 and GATA3 in nasal mucosa with Western Blot. ResultsCompared with the OVA group, the AR behavioral score in the antibiotic + SFXBT group and acetic acid + SFXBT group decreased (P < 0.01), and the pathological changes of nasal mucosa were alleviated. At the door level, the relative abundance of Firmicutes in feces increased significantly, while the relative abundance of Bacteroidetes decreased significantly. At the genus level, The relative abundance of fecal Lactobacillus, Romboutsia, Allobaculum and Dubosiella increased significantly, the levels of serum IgE and IL-4 decreased (P < 0.05), the content of SCFAs increased significantly (P < 0.05), and the expression levels of STAT5, STAT6 and GATA3 mRNA and protein in nasal mucosa decreased significantly (P < 0.05). ConclusionShufeng Xingbi Therapy can significantly improve the inflammatory symptoms of nasal mucosa in AR rats, and its mechanism may be closely related to regulating Th1/Th2 immune balance and intestinal flora.

immunology↗

bioRxiv2025

Inherited resilience to clonal hematopoiesis by modifying stem cell RNA regulation

Somatic mutations that increase hematopoietic stem cell (HSC) fitness drive their expansion in clonal hematopoiesis (CH) and predispose to blood cancers. Although CH frequently occurs with aging, it rarely progresses to overt malignancy. Population variation in the growth rate and potential of mutant clones suggests the presence of genetic factors protecting against CH, but these remain largely undefined. Here, we identify a non-coding regulatory variant, rs17834140-T, that significantly protects against CH and myeloid malignancies by downregulating HSC-selective expression and function of the RNA-binding protein MSI2. By modeling variant effects and mapping MSI2 binding targets, we uncover an RNA network that maintains human HSCs and influences CH risk. Importantly, rs17834140-T is associated with slower CH expansion rates in humans, and stem cell MSI2 levels modify ASXL1-mutant HSC clonal dominance in experimental models. These findings leverage natural resilience to highlight a key role for post-transcriptional regulation in human HSCs, and offer genetic evidence supporting inhibition of MSI2 or its downstream targets as rational strategies for blood cancer prevention.

genetics↗

bioRxiv2025

Respiratory Airway Secretory Cells act as Immune Sentinels in Human Distal Airways

Pulmonary immunity in the human distal respiratory airways is essential for lung function but remains poorly explored, mainly due to limited physiologically relevant models. Here, we develop distal lung organoids containing respiratory airway secretory (RAS) cells, a recently identified epithelial population unique to the distal airways of humans and large mammals, using human pluripotent stem cells (hPSCs). Lineage tracing identified RAS cells as descendants of SOX9brightNKX2-1bright progenitors. Single-cell transcriptomics elucidated that RAS cells exhibited a distinct immune-competent phenotype with enriched genes associated with viral host entry and pattern recognition receptor signaling. Functionally, RAS cells infected by respiratory syncytial virus (RSV) exhibited enhanced antiviral activity marked by upregulation of interferon-stimulated genes (ISGs) and complement component 3 (C3). In contrast, bacterial flagellin or Pseudomonas aeruginosa (PAO1) triggered a TLR5-driven immune response in RAS cells, inducing complement system activation, a response suppressed by selective TLR5 inhibition. Notably, in chronic obstructive pulmonary disease (COPD), RAS cells displayed dysregulated immune activation, characterized by elevated expression of adaptive immune mediators. Together, our findings identify RAS cells as previously unrecognized sensors and effectors of mucosal immunity in the human distal airways and reveal their roles in viral infection, bacterial stimulation, and chronic inflammatory lung disease, providing potential therapeutic targets for respiratory diseases.

cell biology↗

bioRxiv2025

Isl1 Controls Axon Pathfinding of Retinal Ganglion Cells in the Binocular Visual Pathway

Axons of retinal ganglion cells (RGCs) face a choice to project ipsilaterally or contralaterally at the optic chiasm. This decision is crucial for the establishment of binocular vision in mammals. The transcription factor ZIC2 is well known as a key determinant of ipsilateral RGC identity, however the transcriptional programs controlling contralateral RGC identity have only begun to be elucidated more recently. Here we show that inactivation of the LIM-HD transcription factor ISL1 results in an expansion of the ZIC2+ ventrotemporal (VT) domain of the retina and an increase in ipsilateral projections from outside the VT retina. RNA-Seq, gene expression and CUT&Tag analyses show that ISL1 regulates RGC laterality through a set of unique downstream effectors as well as a few common ones, as other transcription factors. Our data also show that RGC axons misrouted at the midline in Isl1-null mutants could innervate the appropriate eye-specific regions of the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC), in agreement with earlier studies that have shown that eye specific targeting of visual nuclei in the brain is independent of laterality decisions at the optic chiasm.

neuroscience↗

bioRxiv2025

CyclicBoltz1, fast and accurately predicting structures of cyclic peptides and complexes containing non-canonical amino acids using AlphaFold 3 Framework

Cyclic peptides exhibit favourable properties making them promising candidates as therapeutics. While the design and modeling of peptides in general has seen rapid advances since the advent of modern machine learning methods, existing deep learning models cannot effectively predict cyclic peptide structures containing non-canonical amino acids (ncAAs), which are often crucial for peptide therapeutics. To address this limitation, we here extend the recent AF3-style model Boltz to cyclic peptides with ncAAs. In addition to the positional encoding offset, we used a simple yet effective extension of the cyclic offset encoding based on AlphaFold3s tokenization scheme that allows the modeling of cyclic peptides with modified residues. On a test set of peptides with ncAAs, our approach outperforms HighFold2 on 13/17 cases, with an average C RMSD of 1.877[A] and an average all-atom RMSD of 3.361[A]. Our results show that the cyclic offset encoding shown for AlphaFold2 generalizes to AlphaFold3-based models and can be extended to incorporate ncAAs, showing potential in the design of novel cyclic peptides with ncAAs for therapeutic applications.

bioinformatics↗

bioRxiv2024

Increased versatility and convenience: advances and strategy optimization of ReMOT-mediated genetic modification in insects

Genetic modification via gene editing has become a widely adopted and demonstrably effective method in functional gene research within entomology. However, the optimal efficiency and simplicity of delivering exogenous guide RNA-clustered regularly interspaced short palindromic repeats-associated protein 9 (gRNA-Cas9) complexes into target tissues are crucial for successful gene editing. The Receptor-Mediated Ovary Transduction of Cargo (ReMOT) strategy, which simplifies the delivery process, target site selection, technical requirements, and delivery cost compared with embryonic microinjection, enabling efficient editing at the germline level, is gaining increasing attention. Although the feasibility and advantages of this technique have been demonstrated in various insect species, further optimization of operational details and the overcoming of further bottlenecks are still required. This review focuses on advances in developing ReMOT as a valuable technology, exploring its applicability, rationale for selecting the ovary as a delivery target site, factors influencing its efficiency, and recommendations for improvement. The versatility and effectiveness of ReMOT make it a promising method for researchers looking to make precise genetic modifications with greater ease and efficiency.

genetics↗

bioRxiv2024

Development of a novel peripherally acting alpha2A-adrenergic receptor antagonist for anti-diabetic

Yohimbine, a potent alpha2A-adrenergic receptor (2AAR) antagonist, was found therapeutic potential for type 2 diabetes through improving insulin release. However, the adverse side effects mediated by its actions in the brain hampered its use. Here, based on molecular docking analysis and structural modification, we have developed a novel peripherally acting yohimbine derivative (CDS479-2). CryoEM data found that yohimbine and CDS479-2 have similar interactions with the structure of 2AAR. Importantly, CDS479-2 shows similar 2AAR antagonist activity as yohimbine, but with very limited access to the brain, and thus avoiding the unwanted central effects such as hypertension and anxiety. Acute administration of CDS479-2 by injection or gavage lowered blood glucose levels and improved glucose tolerance in the high-fat diet-induced obesity (DIO) mice, an animal model for human type 2 diabetes. Remarkably, DIO mice received 2 weeks of daily administration of CDS479-2, but not yohimbine, exhibited sustained normoglycaemia, and increased density of the insulin-producing beta cells, in which important proliferation genes were found upregulated. Moreover, the overall protein expression levels of their pancreas were more similar to that of the healthy chow-fed mice. Thus, CDS479-2 may indicate a new direction for type 2 diabetes treatment. Importantly, the strategy we employed in this study will inspire the optimization for drugs that with both peripheral and central targets. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/628818v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1d3d7corg.highwire.dtl.DTLVardef@1afbad3org.highwire.dtl.DTLVardef@12a1aborg.highwire.dtl.DTLVardef@1341a02_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

The structural basis for de novo DNA methylation in chromatin

De novo cytosine methylation is essential for mammalian development and is deposited by DNMT3A and DNMT3B. In cells, DNA methylation occurs in the context of chromatin, where nucleosomes are connected by DNA linkers. Here, we report Cryo-EM structures of DNMT3A2/3B3 bound to di-nucleosomes with different linker lengths. We show that DNMT3A2/3B3 preferentially binds di-nucleosomes separated by short DNA linkers by inducing large-scale changes to the di-nucleosome structure, enabling each DNMT3B3 subunit to bind each nucleosome. Linker length and the position of cytosines within the linker control DNA methylation, indicating that a significant fraction of linkers in chromatin are naturally resistant to DNMT3A2/3B3 activity. Finally, DNMT3A2/3B3 scans for H3K36me2-3 modifications, explaining how H3K36 methylation simulates DNMT3A2 activity. Our structure is the first example of a DNA methyltransferase interacting with higher-order nucleosome substrates and provides new insights on how DNA methylation takes place in chromatin.

biophysics↗