Biology subjects

Xu, S.

Publications and source records attributed to Xu, S..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Ptf1a robustly drives the gliogenic switch in the rodent embryonic cortex in a dosage-dependent manner by activating pro-glial gene expression programs

It is widely believed that the gliogenic switch during rodent embryonic development is governed by the orchestrated crosstalk between a cohort of genes and extracellular cues. Here we report that ectopic expression of the single bHLH factor, pancreas transcription factor 1 (PTF1A), is sufficient to drive radial glial progenitors (RGPs)-regardless of progenitor heterogeneity and developmental stage-to differentiate into glial cells. Ptf1a- expressing RGPs exhibit progenitor behaviors indicative of a neurogenic-to-gliogenic fate transition, resembling endogenous progenitors at the late stages of embryonic development, and preferentially produce OLIG2+ oligodendrocytes in vivo, some of which are positive for PDGFR or CC1. This robust gliogenic competency depends on the dosage of stable Ptf1a expression in RGPs. RNAseq reveals the glial transcriptome, including upregulated expression of several notch signaling components and the endothelin receptors in Ptf1a RGPs. We further identify Ednrb as one of the downstream targets of Ptf1a that directs RGPs toward gliogenesis via the ERK1/2 signaling pathway. In summary, our study uncovers a novel and robust role for Ptf1a in glial fate specification, offering a potential strategy for generating human oligodendrocytes in vitro.

neuroscience↗

bioRxiv2025

Cytosolic mannosyltransferases involved in the ER N-glycosylation pathway exhibit bacterial ancestry

N-glycosylation in eukaryotes begins with the assembly of a lipid-linked oligosaccharide on the endoplasmic reticulum membrane. As a pivotal post-translational protein modification, it is conserved across all three domains of life. However, the evolutionary origins of the N-glycosylation pathway remain a subject of ongoing debate in evolutionary biology, largely due to the limited availability of robust data regarding the evolutionary trajectories of the glycosyltransferases involved in this process. Here, we present phylogenetic analyses of the eukaryotic ALG1 and ALG2 mannosyltransferases (MTases), which are crucial for constructing the core trimannosyl Man3GlcNAc2 structure conserved in eukaryotic N-glycans. Our comprehensive phylogenetic study, combined with functional and structural analyses, suggests that the ALG2 MTase likely originated from a bacterial ancestor. This inference is further supported by the identification of sequential and functional ALG1 homologs exclusively within bacterial lineages, rather than in Asgard archaea or other archaeal groups. Our findings challenge the prevailing hypothesis that the eukaryotic N-glycosylation pathway primarily evolved from archaeal ancestors, instead suggesting a chimeric origin involving contributions from both bacterial and archaeal lineages. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/682550v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ac81a8org.highwire.dtl.DTLVardef@746054org.highwire.dtl.DTLVardef@604bdborg.highwire.dtl.DTLVardef@14235d_HPS_FORMAT_FIGEXP M_FIG C_FIG The ALG glycosyltransferase (GTases) involved in the initial stage of eukaryotic N-glycosylation on the cytosolic side of the ER membrane exhibit chimeric evolutionary origins. LUCA: Last Universal Common Ancestor; LECA: Last Eukaryotic Common Ancestor. HighlightsO_LIThe evolutionary trajectories of cytosolic ALG mannosyltransferases (MTases) were studied C_LIO_LIThe ancestor of ALG2 exhibited in the LUCA but evolved its MTase activity in bacterial lineages C_LIO_LIThe ALG1 MTase originated from bacterial lineages C_LI

molecular biology↗

bioRxiv2025

Genetically Engineered Cell Membrane-Coated Nanodrug for Targeted Treatment of Thyroid Cancer

Thyroid cancer is the most common endocrine malignancy, particularly in patients with radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC), who have limited treatment options and poor clinical prognosis. In this study, doxorubicin (DOX) and sorafenib (SOR) were loaded into the photothermal conversion agent mesoporous polydopamine (mPDA), forming mPDS. Tumor cell membrane coating engineering resulted in the formation of mPDS@CAR-M, significantly improving tumor targeting. After internalization by tumor cells, the loaded drugs are rapidly released under near-infrared (NIR) laser irradiation. mPDS@CAR-M effectively inhibits tumor cell proliferation by enhancing oxidative stress, suppressing the PI3K-AKT-mTOR signaling pathway, and inducing cytotoxic autophagy. By activating harmful autophagy, mPDS@CAR-M further inhibits the epithelial-mesenchymal transition (EMT) process, reducing tumor cell migration capacity. In vivo experiments showed that mPDS@CAR-M significantly reduced tumor volume, with its therapeutic efficacy closely related to the expression level of the targeted surface antigen. Therefore, mPDS@CAR-M demonstrates significant potential in the treatment of RAIR-DTC, providing a novel direction for further clinical exploration.

cancer biology↗

bioRxiv2025

Diel rhythms shape viral community structure and activity across the host domains of life

Circadian rhythms, driven by endogenous clocks and synchronized with environmental cues, are fundamental to life on Earth. While extensively studied in diverse organisms, their influence on viral ecology remains largely unexplored. This study takes the temporal dynamics of viruses in Daya Bay as an example to uncover the rhythmic control of viral replication and activity. Using a high-resolution time-series dataset collected every 2 hours over a 3-day period, we identified a total of 22,151 viral operational taxonomic units (vOTUs) and 414 Nucleocytoplasmic Large DNA Virus (NCLDV) genomes. Our analysis revealed significant diel fluctuations, with 14.48% of vOTUs exhibiting diel patterns in metagenomic abundance and 1.97% showing diel transcriptional activity. We found that these abundant diel viruses infect hosts across the domains of life, including cyanobacteria, pelagibacteria, Marine Group II (MGII) archaea and protists, all known for their diel metabolism. The expanded spectrum of host diversity with diel viral interactions significantly broadens our understanding of virus-host rhythmic dynamics in natural environmental settings. A strong positive correlation was detected between the transcriptional activities of these diel viruses and their respective hosts. Contrary to bacteriophages, which mostly peaked during the day, we demonstrated that NCLDVs showed nocturnal diel abundance with a co-fluctuating diel transcriptomic activity pattern tightly hitched to their hosts, peaking at night and declining during the day. Furthermore, we identified a rich compendium of viral genes with significant diel expression patterns, including those related to structural protein production, DNA replication, and stress response. Notably, several essential viral genes involved in stress response and repair were found to be diel transcribed for the first time, including UV-endonuclease (UvdE), peroxidase, chaperones, and early light-induced protein (ELIP). Our findings suggest that viruses across host domains actively synchronize with environmental cues to optimize their replication and transmission, despite their dependence on host metabolism. This study provides novel insights into the rhythmic control of viral communities and their intricate interactions with hosts, with profound implications for microbial community succession and biogeochemical cycles.

ecology↗

bioRxiv2025

Species-specific basal fluidization shapes early forebrain development

The human forebrain exhibits an expanded surface area compared with other great apes. Despite progress in dissecting species-specific gene expression programs and cell lineages, the tissue-level physical mechanisms underlying human-specific neurodevelopment remain unclear. Here, by quantifying tissue dynamics and mechanics using oil microdroplets in cerebral organoids from humans, gorillas, chimpanzees, and mice, we uncover a transient tissue fluidization occurring exclusively in the basal region of human neuroepithelia. Enhanced droplet motility and frequent cell rearrangements reveal this basal fluidization, which gradually diminishes at the onset of neurogenesis in humans. By contrast, such basal fluidization is not detected in non-human neuroepithelia examined here. Mechanistically, basal fluidization is driven by basal nuclear fluctuations and facilitated by expanded intercellular spaces. Increasing N-cadherin expression in human neuroepithelia to gorilla levels suppresses both nuclear fluctuations and basal fluidization. Integrating simulations of neuroepithelial dynamics with experimental data, we further find that nuclear fluctuations underlying basal fluidization promote the insertion of newly divided nuclei into the basal side to support tangential surface expansion, a hallmark of human forebrain development.

developmental biology↗

bioRxiv2025

Trait motivation is associated with Fusiform face area Morphometry Evidence from a Chinese Youth Sample

Trait motivation is fundamental in shaping human behaviors. Previous studies have primarily focused on their impact on affective and motivational processing, with their role in perceptual processes less investigated. The present study takes face perception, a crucial and well-studied perceptual process, as a representative specimen to examine the perceptual effect of trait motivation. We investigated whether the behavioral activation system (BAS) and the behavioral inhibition system (BIS) were associated with structural characteristics of the inferior temporal face-selective regions as well as face recognition performance. With a sample of Chinese young adults (N = 264), voxel-based morphometry revealed that BIS scores correlated with greater gray matter volume in the fusiform face area. Further, a higher BIS score was associated with slightly better performance in face recognition. These findings provide novel evidence that trait motivation, particularly behavioral inhibition, is linked to both the structure and function of the face processing system. This underlines the intrinsic coupling between motivational and perceptual systems, blurring the presumed divide between affective and perceptual processes.

neuroscience↗

bioRxiv2025

Probe-Based Identification of Metal-Binding Sites Using Deep Learning Representations

Metalloproteins play indispensable roles in a multitude of cellular processes. They incorporate metal ions as vital cofactors to catalyze biochemical reactions, stabilize protein structures, and mediate electron transfer. Given their prevalence and importance, identifying metal-binding sites within proteins remains a challenging task, due to the intricate complexity of the protein environments and the promiscuous binding behavior of metal ions. Although computational approaches for predicting metal-binding sites have been developed for decades, their performance often suffers from limited accuracy due to constrained methodologies and data scarcity. Here, we introduce PRIME, a hybrid deep learning framework that harnesses both evolutionary and structural signals to predict metal-binding sites with high accuracy and efficiency. PRIME employs protein language models (PLMs) and pre-trained structure models (PSMs), following the paradigm of deep representation learning, to extract essential information of protein sequences and structures. PRIME integrates a novel probe generation algorithm that bridges sequence- and structure-based predictions by efficiently scanning candidate binding sites. The resulting framework achieves superior accuracy across a wide range of metal ions, including both abundant ions such as Zn2+ and Ca2+ and less abundant ions such as K+ and Na+, surpassing the performance of existing methods. In addition, ablation analysis shows that PSMs significantly enhance the accuracy of metal-binding site prediction. Case studies on AlphaFold2 models, along with the high prediction speed of PRIME, further demonstrate its potential for high-throughput applications in metalloproteomics.

bioinformatics↗

bioRxiv2025

Maladaptive aphid-induced transgenerational plasticity is overcome in nature

Inherited stress responses are widely assumed to be adaptive for plants by preparing offspring for renewed attack. To date, it remains unclear whether and under which conditions these responses may benefit the attacker rather than the host. Studying the interaction between the aphid Rhopalosiphum nymphaeae and its native host, the aquatic duckweed Spirodela polyrhiza, we show that aphids can either benefit or suffer from inherited stress responses, depending on metabolite identity and ecological context. In multigenerational clonal plant lineages maintained indoors, ancestral aphid herbivory increased aphid reproduction and reduced plant fitness under renewed attack, indicating that the net effect of inherited stress responses can favor the herbivore. However, not all inherited responses benefited the aphid: using genetic and chemical manipulations, we found that transgenerational priming of jasmonate enhanced aphid performance, whereas transgenerational priming of tyramine reduced it. Notably, tyramine--but not jasmonates--accumulated to higher levels across generations also in large mesocosms outdoors, albeit only within and not across growing seasons. Together, our results reveal that inherited responses of hosts can favor the attackers and highlight ecological context and life-cycle transitions as key determinants of the persistence, duration, and ecological consequences of inherited stress responses.

ecology↗

bioRxiv2025

Dual-patterned pluripotent stem cells self-organize into a human embryo model with extended anterior-posterior patterning

Human gastruloids are a powerful class of stem cell-derived models that recapitulate key features of early embryonic development, including symmetry breaking and the emergence of three germ layers1-3. However, they lack anterior embryonic structures and coordinated axial organization4-6. To address this limitation, we pre-patterned human pluripotent stem cells (hPSCs) by exposing them to either anterior (FGF2) or posterior (CHIR99021 [CHIR] & retinoic acid [RA]) cues. Upon mixing, these dual-patterned hPSCs interacted and self-organized into elongated structures with both anterior and posterior features--which we term anterior-posterior (AP) human gastruloids. Anteriorly pre-treated cells robustly intercalated into posteriorly pre-treated cells, collectively giving rise to a continuum of neural tissues--including a brain-like domain, a neural tube-like structure, and neuro-mesodermal progenitors (NMPs)--with segmented somites arrayed bilaterally. Single cell RNA sequencing (scRNA-seq) revealed that human AP gastruloids contain cell types resembling the midbrain-hindbrain boundary (MHB), regionalized hindbrain structures (i.e. rhombomeres 1-8), regionalized neural crest (i.e. cranial, vagal, trunk)7,8 and head mesoderm. Transcriptomic comparisons to primate embryos revealed that human AP gastruloids most closely resemble Carnegie stage 11 (CS11) embryos. While they lack a notochord and full dorsal-ventral polarity, human AP gastruloids recapitulate key spatial and temporal features of early neurulation and somitogenesis. Perturbation of folic acid metabolism or rho-associated kinase (ROCK) signaling induced spinal cord defects, phenocopying aspects of spina bifida and other neural tube defects, highlighting this models potential for studying congenital disorders9. AP gastruloids may serve as a simple, robust, scalable platform for modeling coordinated human AP body axis development. More broadly, our results suggest that controlled interactions between differentially prepatterned progenitors can initiate self-organization of complex body axis features. The "pattern-and-mix" strategy may serve as a generalizable framework for assembling spatially organized stem cell models of mammalian development.

developmental biology↗

bioRxiv2025

Re-evaluating the Impact of Biological Sex on Atherosclerosis in Apoe-/- and Ldlr-/- mice

BACKGROUNDThe progression of cardiovascular disease shows significant sexual dimorphism: although females generally develop the disease later in life, they exhibit a higher age-related incidence than males. Despite this clinical pattern, preclinical studies often overlook both sexes in their design, and existing research on sex-specific atherosclerosis in mice remains inconsistent. This study was designed to assess the influence of sex on atherosclerosis using two widely used atherosusceptible mouse models--Apoe-/- and Ldlr-/- mice. METHODSTo investigate the influence of sex on atherogenesis, we conducted a 20-week study using both male and female Apoe-/- and Ldlr-/- mice. The mice were fed an atherogenic ALMN diet (40% trans-fat, 2% cholesterol, 22% fructose) to promote plaque development. We performed comprehensive analyses of: (1) systemic metabolic parameters (lipid profile, glucose metabolism); (2) atherosclerotic burden (en face and aortic sinus plaque area); and (3) plaque composition (necrotic core size, collagen content, macrophage infiltration). RESULTSIn both models, male mice showed higher lipid levels, worse glucose tolerance, and reduced insulin sensitivity compared to females. Apoe-/- mice showed minimal sex differences in atherosclerosis with a trend toward increased plaque size in females. Plaque stability markers-- including collagen content, necrotic core size, and macrophage infiltration--did not differ significantly between sexes. In contrast, Ldlr-/- males exhibited greater en face plaque burden than females, yet plaque stability remained similar across sexes. CONCLUSIONComparative analysis of two widely used murine atherosclerosis models revealed genotype-dependent sexual dimorphism. Female Apoe-/- mice showed a non-significant trend toward larger plaque areas than males, whereas male Ldlr-/- mice developed significantly larger en face atherosclerotic plaques than females. By evaluating plaque area and composition across these models, our findings underscore the importance of including both sexes in atherosclerosis studies, in accordance with guidelines from the National Institutes of Health and the American Heart Association.

cell biology↗

bioRxiv2025

P-cadherin and desmoglein-2 interact as strand-swap dimers and facilitate desmosome assembly.

Desmosomes are essential adhesive junctions that mediate the integrity of tension prone tissue like the heart and skin. The classical cadherin, P-cadherin (Pcad), is known to play an important role in forming desmosomes. However, the molecular mechanisms by which Pcads enable desmosome assembly are unknown. Here, we combine single molecule Atomic Force Microscopy, super-resolution and confocal imaging, mutagenesis assays, and atomistic simulations to resolve the structural mechanisms by which Pcads facilitate the formation of desmosomes. We show that Pcads interact with the desmosomal cadherin Desmoglien-2 (Dsg2) on opposing cells, by mutually swapping {beta}-strands terminated by conserved Tryptophan residues. A key determinant of this trans heterophilic strand-swap dimerization is the flexibility of a hinge on the swapped {beta} -strands: stiffening this hinge reduces the likelihood of heterophilic strand-swap dimer formation. We demonstrate that while desmosome formation is impaired in cells that either lack classical cadherins or that express strand-swap deficient Pcad, introduction of strand-swap competent Pcad into these cells rescues desmosome assembly. We show that the heterophilic dimers formed by Pcad and Dsg2 interact in a robust and persistent manner, which retains Pcad within the desmosome throughout the maturation process. SignificanceDesmosomes are essential intercellular junctions that mediate the integrity of tissue like the heart and skin. Desmosomal defects are associated with numerous skin disorders, heart diseases, and cancer. The adhesive protein P-cadherin is known to play an important role in forming desmosomes. However, the molecular mechanisms by which P-cadherins enable desmosome assembly are unknown. Here, we show that desmosome formation is facilitated by the direct interaction of P-cadherin with the transmembrane desmosomal adhesion protein Desmoglein-2. We demonstrate that P-cadherin and Desmoglien-2 interact robustly by swapping flexible portions of their protein backbone and that this molecular complex nucleates desmosome formation. Our results resolve the biophysical basis by which different adhesive proteins interact and mediate stable desmosome assembly.

biophysics↗

bioRxiv2025

A hardwired neural circuit for temporal difference learning

The neurotransmitter dopamine plays a major role in learning by acting as a teaching signal to update the brains predictions about rewards. A leading theory proposes that this process is analogous to a reinforcement learning algorithm called temporal difference (TD) learning, and that dopamine acts as the error term within the TD algorithm (TD error). Although many studies have demonstrated similarities between dopamine activity and TD errors1-5, the mechanistic basis for dopaminergic TD learning remains unknown. Here, we combined large-scale neural recordings with patterned optogenetic stimulation to examine whether and how the key steps in TD learning are accomplished by the circuitry connecting dopamine neurons and their targets. Replacing natural rewards with optogenetic stimulation of dopamine axons in the nucleus accumbens (NAc) in a classical conditioning task gradually generated TD error-like activity patterns in dopamine neurons by specifically modifying the task-related activity of NAc neurons expressing the D1 dopamine receptor (D1 neurons). In turn, patterned optogenetic stimulation of NAc D1 neurons in naive animals drove dopamine neuron spiking according to the TD error of the stimulation pattern, indicating that TD computations are hardwired into this circuit. The transformation from D1 neurons to dopamine neurons could be described by a biphasic linear filter, with a rapid positive and delayed negative phase, that effectively computes a temporal difference. This finding suggests that the time horizon over which the TD algorithm operates--the temporal discount factor--is set by the balance of the positive and negative components of the linear filter, pointing to a circuit-level mechanism for temporal discounting. These results provide a new conceptual framework for understanding how the computations and parameters governing animal learning arise from neurobiological components.

neuroscience↗

bioRxiv2025

Emergence of Genetic Sex Determination in an Environmentally Sex-Determined Animal

The evolution of genetic sex determination (GSD) from environmental sex determination (ESD) remains a fundamental issue in evolutionary biology. However, the mechanisms driving such a transition, particularly in its earliest stages, are largely unknown. Here, we report on a genetic variant in the ESD species, Daphnia pulex, in which structural variations and selection on chromosome have suppressed recombination. This suppression has facilitated the accumulation of antagonistic features of the two chromosome- haplotypes, resulting in heterozygosity and differentiation of the sex-determining gene Dfh on two haplotypes. Consequently, this process has driven the emergence of a mixed-mating system, in which sex determination is jointly regulated by genetic and environmental factors. Additionally, we detected ongoing divergence in chromosomal structure between the two haplotypes at the population level. These findings suggest that processes analogous to early sex-chromosome evolution are occurring in this ESD species, offering novel insights into the molecular mechanisms underlying the emergence of GSD.

evolutionary biology↗

bioRxiv2025

ATF4 alleviates doxorubicin-induced cardiomyopathy through H2S-mediated antioxidation

BackgroundDoxorubicin (DOX) remains a cornerstone in the treatment of various cancers. However, its clinical utilization is significantly hampered by dose-dependent cardiotoxicity. The generation of reactive oxygen species (ROS) constitutes the central component of the pathogenesis of DOX-induced cardiotoxicity. Activating transcription factor 4 (ATF4) has been demonstrated to exert a cardioprotective effect and augment cardiac antioxidative capacity in settings of heart failure. However, the role of ATF4 in DOX-induced cardiomyopathy (DIC) remains unknown. MethodsTo explore the role of ATF4 in DOX-induced cardiomyopathy, cardiac-specific ATF4 conditional heterozygous mice and AAV9 mediated ATF4 overexpression mouse models were utilized. Cardiac function was assessed by echocardiography. The upstream regulator and downstream mediator of ATF4 were evaluated using RNA-seq analysis and further verified using ChIP assay and luciferase reporter assay. ResultsWe found a substantial decrease in ATF4 expression levels in the heart of DIC mice. ATF4+/- mice exhibited a higher degree of susceptibility to DOX-induced cardiotoxicity in comparison with ATF4flox/flox mice, as evidenced by the manifestation of more severe cardiac dysfunction and a significantly earlier mortality rate. In contrast, cardiacc-specific overexpression ATF4 by AAV9 confers robust cardioprotection against DOX-induced cardiomyopathy. Mechanistically, we identified the upstream regulator of ATF4 as KLF16, which was significantly suppressed during DOX treatment. Further, the decrease of ATF4 led to a reduction in cystathionine {gamma}-lyase (CSE) transcription and hydrogen sulfide (H2S) production in the context of DOX-induced cardiotoxicity. ChIP and luciferase reporter assays revealed that ATF4 functioned as the transcription factor of the CSE gene, which is a key enzyme in the synthesis of H2S to counteract oxidative stress. Consistently, ROS scavengers or H2S donors was shown to mitigate the consequences of ATF4 deficiency. In contrast, the ectopic expression of ATF4 mitigated oxidative stress and apoptosis in DOX-induced cardiotoxicity, both in vivo and in vitro. ConclusionsOur study revealed a novel function of ATF4 in counteracting oxidative stress in DOX cardiotoxicity by promoting the transcription of CSE. ATF4 may represent a promising therapeutic target for the treatment of DOX-induced cardiomyopathy.

pathology↗

bioRxiv2025

Low nucleus to cytoplasmic ratio compromises cytokinesis, genome stability and development in haploid embryonic cells

Mammalian species exist as diploid organisms, with only gametes existing as stable, nonproliferating haploids. Haploid mammalian pluripotent stem cells, in all derived species to date - mouse, human and rat - undergo spontaneous diploidization. Here, we investigated the mechanisms affecting the stability of the haploid state using the mouse and human embryo, as well as a human embryonic stem cells. We demonstrate that diploidization occurs early in preimplantation development and is often unproductive, with haploid embryos exhibiting decreased developmental potential. Haploid embryos show increased chromosome segregation errors at the first mitosis, failure to align chromosomes on the metaphase plate in the second mitosis. Delayed mitotic progression and failure to form a central spindle and a midbody are followed by cytokinesis failure, diploidization, increased DNA damage marked by {gamma}H2AX and RPA32 foci, and low developmental potential. By increasing the nucleo-cytoplasmic ratio in diploids, or reducing this ratio in haploids, these phenotypes can be induced in diploids or reduced in haploids. Changing ploidy alone from haploid to diploid without also adjusting nucleo-cytoplasmic ratio does not improve mitotic phenotypes and developmental outcomes. Thus, the most upstream driver responsible for the instability of the haploid state is a stage- specific ratio of nucleus to cell size.

cell biology↗

bioRxiv2025

The Gene Encoding Ornithine Decarboxylase for Putrescine Biosynthesis Is Essential for the Viability of Fusobacterium nucleatum

Fusobacterium nucleatum is a Gram-negative anaerobe associated with periodontitis and colorectal cancer. It secretes putrescine, a polyamine that promotes biofilm formation by oral co-colonizers and enhances the proliferation of cancer cells. However, the physiological importance of putrescine for F. nucleatum itself remains unexplored. Here, we show that putrescine biosynthesis, mediated by the ornithine decarboxylase gene oda, is essential for F. nucleatum viability. Deletion of oda was only possible when a functional copy was provided in trans, and CRISPR interference of oda expression resulted in complete growth arrest. The essentiality of oda was conserved across multiple subspecies. Supplementation with exogenous putrescine enabled the isolation of a conditional oda mutant whose growth was strictly putrescine-dependent. Putrescine depletion caused filamentation, membrane disruption, detergent hypersensitivity, and lysis in hypoosmotic conditions, indicating a critical role in maintaining cell envelope integrity. RNA sequencing revealed broad transcriptional remodeling under putrescine-limited conditions, including upregulation of genes involved in lipid metabolism, osmoprotection, and cell wall remodeling. Notably, oda transcript levels increased when putrescine was depleted, suggesting a negative feedback mechanism. These findings demonstrate that putrescine is not only an extracellular communal metabolite but is also vital for the cellular integrity and survival of F. nucleatum under anaerobic conditions. IMPORTANCEFusobacterium nucleatum is a prominent member of the oral microbiota and has been linked to various human diseases, including periodontitis, preterm birth, and colorectal cancer. Despite its clinical significance, the metabolic requirements that support its growth and viability remain poorly understood. In this study, we identify the oda gene, which encodes ornithine decarboxylase, as essential for F. nucleatum survival due to its role in putrescine biosynthesis. We demonstrate that depletion of putrescine leads to severe growth and morphological defects, accompanied by widespread transcriptional changes. These findings reveal an underappreciated metabolic vulnerability and highlight the critical role of polyamine homeostasis in maintaining cellular integrity in this notorious anaerobe.

microbiology↗

bioRxiv2025

Strain, procedures, and tools for reproducible genetic transformation and genome editing of Spirodela polyrhiza (L.) Schleid.

Duckweeds (Lemnaceae) have excellent potential for fundamental and applied research due to ease of cultivation, small size, and continuous fast clonal growth. However, their usage as model organisms and platforms for biotechnological applications is often limited by the lack of universal genetic manipulation methods necessary for transgene expression, gene editing, and other methods to modify gene expression. To identify suitable strains for genetic manipulation of the giant duckweed, Spirodela polyrhiza, we screened several genotypes for callus induction and regeneration and established genetic transformation. We have identified SP162 to be amenable to Agrobacterium-mediated transformation via tissue culture. The procedure is robust and reproducible across laboratories, allowing stable expression of different reporter genes and selectable markers, enabling CRISPR/Cas9-mediated genome editing. In addition, due to a weak small RNA-based silencing response, S. polyrhiza sustains prolonged periods of transgene activity in transient expression assays. To promote duckweed research and encourage the adoption of S. polyrhiza, we have made SP162 (ID#: 5676) and its genome publicly available and provide here detailed procedures for its cultivation and transformation. Furthermore, we created a web server to explore its genome, retrieve gene sequences, and implemented orthologous gene search and a gRNA design function for diverse CRISPR/Cas-based applications (https://agxu.uni-mainz.de/SP162/).

plant biology↗

bioRxiv2025

C9orf72/SMCR8 complex maintains microglial homeostasis via RAB8A-ESCRT-mediated lysosomal repair

Microglia are critical regulators of neuroinflammation and neurodegeneration. Haploinsufficiency of C9orf72, the most common genetic cause of ALS and FTD, has been mainly linked to autophagy-lysosomal pathway defects, but its precise role in microglial lysosomal function remains unclear. Here we identify the C9orf72/SMCR8 complex as a key regulator of microglial homeostasis through lysosomal repair. Loss of C9orf72 and SMCR8 in mice causes age-dependent neuroinflammation and microgliosis, with microglia adopting a disease-associated state. In aged CNS tissue, microglia display lysosomal damage, marked by galectin-3 upregulation and its accumulation on lysosomes. To model this process, we applied the lysosomotropic agent LLOMe to microglia, which recapitulated lysosomal damage and revealed defective recruitment of phosphorylated RAB8A and the ESCRT machinery in C9orf72/SMCR8-deficient cells. Notably, mutant microglia accumulate GTP-bound RAB8A, which becomes aberrantly hyperphosphorylated and mislocalized to non-lysosomal vesicles. We further show that the GAP activity of the C9orf72/SMCR8 complex is essential for lysosomal repair. These findings uncover a previously unrecognized role for the C9orf72/SMCR8 complex in coordinating RAB8A-ESCRT-mediated lysosomal repair, thereby safeguarding microglial homeostasis and limiting neuroinflammation.

cell biology↗