Biology subjects

Song, R.

Publications and source records attributed to Song, R..

At least 19 recordsLinked to original sources

bioRxiv2026

miR-34/449 miRNAs regulate choroid plexus ciliogenesis to control cerebrospinal fluid production

A developmental increase in cerebrospinal fluid (CSF) production during development is essential for neuronal growth and ventricular expansion. A key regulator of CSF production is the specialized sensory multicilia of the choroid plexus (ChP), which mediate non-canonical Sonic hedgehog (Shh) signaling to suppress water channel and ion transporter expression, thereby limiting CSF production. ChP multicilia progressively shortens during development, attenuating Shh signaling and promoting CSF production. Here, we identify miR-34/449 miRNAs as essential regulators of ChP multiciliogenesis. Whereas mutations in canonical ciliogenesis genes elevate CSF production and contribute to hydrocephaly, deletion of miR-34/449 reduces CSF volume and causes microcephaly. Loss of miR-34/449 miRNAs causes excessive basal body amplification, defective basal body docking, and failure of developmental multiciliary shortening. Consequently, miR-34/449-deficient ChP cilia remain abnormally long and fail to attenuate Shh signaling, resulting in sustained repression of water channel and ion transporter expression and reduced CSF production. Mechanistically, miR-34/449 miRNAs directly target Gmnc, a master transcriptional regulator of multiciliogenesis, to restrain basal body amplification and promote basal body docking. Together, our findings identify miR-34/449 miRNAs as critical regulators of ChP multiciliogenesis and establish the developmental remodeling of ChP multicilia as a mechanism to couple Shh signaling dynamics to developmental control of CSF production.

developmental biology↗

bioRxiv2026

Towards autonomous biology: Compiler-Verified Protocols as a Foundation for Real World AI Execution

Artificial intelligence has advanced from analyzing experimental data to autonomously generating hypotheses, designing experiments, and coordinating closed loop discovery. Yet the translation from computational reasoning to physical execution remains bottlenecked by the experimental protocol, which in biology still relies on ambiguous natural-language descriptions: a medium other engineering disciplines abandoned decades ago in favor of compiler verified specification languages. This deficit fragments reproducibility along three axes: protocol accuracy, pre execution verification, and cross platform portability. Existing formalisms address only subsets of these challenges, trading expressiveness for rigor, portability for standardization, or usability for provenance. Here we introduce the Biology Protocol Language (BPL), a domain specific language with a biology-native type system in which every quantity carries physical units, every reagent declares its physical form, and every container maintains compiler-tracked state, so that implicit assumptions must be stated explicitly and physically impossible operations are rejected at compile time. We further develop BPL-COGEN, a pipeline that couples a fine tuned 30 billion parameter language model with the deterministic compiler in a closed generate validate repair loop, iteratively correcting the translation from natural language SOPs to BPL through compiler diagnostics until all physical, dimensional, and state constraints are satisfied. On a benchmark of 300 published Nature Protocols papers, BPL COGEN achieved an overall fidelity score of 95.1 against the source protocols as ground truth. Wet-lab experiment and cross-platform validation in GFP expression library construction and HPLC to UHPLC method translation confirmed that a single BPL source yielded reproducible execution across manual and liquid handler assisted contexts. The results established a novel pipeline that generates compiler-verified protocols, which is an essential prerequisite for physically embodied AI in biology.

synthetic biology↗

bioRxiv2026

Radiosynthesis and Evaluation of Novel Cholesterol 24-Hydroxylase Positron Emission Tomography Tracers

Cholesterol 24-hydroxylase (CH24H or CYP46A1) is a pivotal enzyme in brain cholesterol metabolism and has emerged as a therapeutic and imaging target in neurodegenerative disorders. Although [18F]Cholestify ([18F]CHL-2205) has shown promise as a positron emission tomography (PET) tracer for imaging of CYP46A1, the impact of cyclopropyl moiety conformation on binding and imaging performance remains unexplored. Here, we report the rational design and preliminary evaluation of novel CYP46A1 PET tracers, in which the left-side cyclopropyl group was modified into bridged, spirocyclic, and fused bicyclic architectures to probe steric and conformational effects. All compounds 9-11 exhibited high CYP46A1 affinity (IC50 = 0.19-0.28 nM). Radiosynthesis of [18F]9-11 was achieved via copper-mediated [18F]fluorination, providing practical non-decay-corrected radiochemical yields of 10-34% with excellent radiochemical purity (>98%). In vitro autoradiography in rat brain sections demonstrated specific and regionally selective binding, comparable to that observed for [18F]CHL-2205. These cyclopropyl-derived scaffolds establish a scaffold-driven strategy for PET tracer development, providing a robust framework for further structure-activity relationship studies and the rational optimization of CYP46A1 PET tracers.

pharmacology and toxicology↗

bioRxiv2025

Distributed fMRI patterns coupled to low-frequency cardiorespiratory dynamics provide markers of aging

How aging affects brain-body connections can be investigated through changes in the coupling between functional magnetic resonance imaging (fMRI) signals and bodily autonomic processes across the adult lifespan. Recent studies using univariate approaches have identified age-related changes in the association between fMRI signals from multiple individual brain regions and low-frequency respiratory and cardiac activity. Here, we investigate if whole-brain spatial fMRI patterns associated with low-frequency physiological processes (heart rate and respiratory volume fluctuations) present generalizable changes with age. Data from human participants of both sexes are included in the analysis. We find that chronological age can be predicted statistically beyond chance from patterns of low-frequency fMRI-physiology coupling, even after accounting for individual differences in physiological signal characteristics and brain anatomy. Notably, brain areas implicated in central autonomic regulation, including nodes within salience and ventral attention networks (e.g., insula and middle cingulate cortex), are amongst the strongest contributors to age prediction. Further, we observe that after removing physiological effects from fMRI data, the residual blood oxygen level-dependent (BOLD) signal variability is still a reliable indicator of age. Together, these findings underscore the close integration between brain and body physiology, and highlight this interaction as a potential biomarker of the aging process. Significance StatementThe association between brain activity and respiratory or cardiac activity is often dismissed as "noise" in functional magnetic resonance imaging (fMRI) studies. However, emerging evidence suggests that coupling between fMRI and peripheral physiological signals can provide valuable insight into the brain-body connection. In this study, we show that whole brain patterns of coupling between fMRI signals and low-frequency respiratory and cardiac processes can reliably predict age across the adult lifespan. Brain regions involved in autonomic regulation, such as insula and cingulate cortex, were among the most informative predictors of age. These findings suggest that fMRI-physiology coupling may capture aging-related changes in brain vascular health and autonomic function and may have broader relevance for tracking disease-related disruptions in brain-body interaction.

neuroscience↗

bioRxiv2025

Ethylene signal-driven plant-multitrophic synergy boosts crop performance

Efficient nutrient use in agriculture depends on the dynamic interplay between plant roots, soil, and microbial communities. The root-rhizosphere interface is central to nutrient uptake and serves as a key hub for interactions with beneficial microbes. Arbuscular mycorrhizal (AM) fungi, positioned at the nexus between plant roots and soil microbiota, play a critical role in enhancing crop performance under nutrient-limited conditions. In this study, we dissected the genetic and molecular basis of AM fungi-induced lateral root development in maize (Zea mays), focusing on the role of ethylene-responsive transcription factors (ERFs). We identified ERF genes as essential regulators of pericycle cell division, acting downstream of ethylene biosynthesis genes (ACS6 and ACS7) and AM fungal signaling. Our findings reveal that AM fungi promote lateral root initiation by activating ERF expression and reprogramming flavonoid metabolism, particularly reducing the accumulation of flavonols such as kaempferol and quercetin, which otherwise inhibit root development when over-accumulated. Furthermore, we demonstrated that Massilia, a beneficial rhizobacterium, synergizes with AM fungi to enhance lateral root formation by colonizing fungal hyphae, degrading flavonoids, and contributing to auxin production. Together, our results uncover a tripartite signaling network linking ethylene signaling, flavonoid-mediated microbial recruitment, and AM symbiosis. This study highlights ERFs as central integrators of plant-microbe interactions and provides a molecular framework for engineering root architecture and microbiome assembly to improve nutrient acquisition and support sustainable crop productivity.

plant biology↗

bioRxiv2025

Open source software for tube vocal tract modeling, resonance prediction, illustration, and 3D printing

We present accessible code-free tube vocal tract modeling software. The software implements a transfer function. Applications involve exploratory speech acoustics-based basic research and education in phonetic sciences. The program has been made publicly available and presents researchers and students in speech-centric sciences with easily accessible vocal tract modelling and vowel synthesis.

scientific communication and education↗

bioRxiv2025

Neural network-assisted analysis of tube vocal tract models

We present a pipeline for deep neural network assisted modeling and analysis of tube vocal tract models. Such models are composed of a series of cylindrical tube segments, each characterized by length and cross-sectional area. A large synthetic dataset of such tube configurations is generated, and a circuit theory-based algorithm predicts corresponding formant frequencies. To explore the mapping between tube sequence shapes and predicted resonance (formant) values, the pipeline integrates both linear regression and nonlinear machine learning models --including multi-layer perceptrons. Model interpretability is assessed using Shapley Additive Explanations (SHAP), which quantifies the contribution of each segment to predicted formant frequencies. The proposed framework enables detailed exploration of the articulatory-acoustic relationships inherent to an acoustic tube and vocal tract simulacrum. We present and describe the pipeline in the context of modeling effects of perturbations on the first three predicted resonances for a 16- cm tube, divided into 1 cm segments. Our pipeline can be applied to any method that models predictions of behavior of an acoustic tube, where the tube is conceived as a series of segmented units.

bioengineering↗

bioRxiv2025

A handheld microfluidic manifold for massively multiplexed CRISPR-based nucleic acid detection

Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. Here, we introduce Scalable On-site Nucleic Acid Testing Architecture (SONATA), enabling >100-chamber reaction partitioning for multiplexed nucleic acid amplification and detection with a portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized reagents and a small stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. We demonstrate the integration of the platform with Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based detection technology. We show that deployed with SONATA, SHINE retains its sensitivity, enabling highly multiplexed pathogen detection in [≤] 1 hour. In addition, we demonstrate the detection of single-nucleotide variants, including mutations associated with drug susceptibility.

bioengineering↗

bioRxiv2025

The Rab32-LRMDA-Retriever Complex is a Key Regulator of Intestinal Immune Homeostasis

Maintaining intestinal homeostasis relies on the intricate interplay among the mucosal epithelium, immune system, and host microbiome. A key question is how innate immune cells sense and process microbes in the gut lumen, eliciting appropriate protective responses without causing tissue injury. Clearance of invading microbes and initiation of downstream inflammatory responses are central to this process and require proper function of the endolysosomal system. Dysfunction of this system can predispose the host to chronic inflammatory disorders and acute infections. Here, through forward genetic screening of N-ethyl-N-nitrosourea (ENU)-mutagenized mice and CRISPR/Cas9 validation, we identify Lrmda, encoding leucine-rich melanocyte differentiation-associated protein (LRMDA), as a key regulator of intestinal homeostasis. Using hematopoietic chimera and conditional knockouts, we show that LRMDA functions primarily in CD11c+ cells, including mucosal dendritic cells (DCs) and macrophages, but not in non-hematopoietic cells. Proteomic, cellular, and biochemical analyses reveal that LRMDA directly and cooperatively interacts with the endolysosome-specific small GTPase Rab32 and the endosomal recycling complex Retriever. Loss of LRMDA or Retriever function increases susceptibility to dextran sodium sulfate (DSS)-induced colitis and impairs clearance of Listeria monocytogenes. Together, our findings establish the Rab32-LRMDA-Retriever complex as a critical regulator of endolysosomal trafficking in innate immune cells, essential for maintaining intestinal immune homeostasis.

immunology↗

bioRxiv2025

Mitochondrial Genome-Based Phylogeny of Turbellarians and Evidence for Accelerated Mitochondrial Evolution in Symbiotic Species

BackgroundFlatworms are a highly diverse phylum with over 26,500 predominantly parasitic species. A minor portion of this diversity comprise predominantly free-living "turbellarians" Phylogenetic relationships within turbellarian orders remain debated, with recent mitochondrial genome studies also questioning the monophyly of the "Neoophora clade". Some unique mitochondrial gene features have also been observed in this group. Within Turbellaria, the order Rhabdocoela includes significant symbiotic lineages, such as the endosymbiotic Umagillidae, Pterastericolidae, and Graffillidae, and the ectosymbiotic Temnocephalidae, which notably exhibits characteristics akin to a parasitic lifestyle. Given evidence linking parasitic lifestyles to accelerated mitogenomic evolution, we hypothesize that similar patterns: symbiotic turbellarians have mitochondrial genomes that accelerate evolution compared to free-living turbellarians. This study presents the first complete mitochondrial genome of the ectosymbiont Craspedella pedum, provides mitogenomic insights into turbellarian phylogeny, and addressing our hypothesis. ResultsThe mitochondrial genome of Craspedella pedum is a circular DNA molecule of 18,456 base pairs, containing the standard 36 flatworm mitochondrial genes, a duplicated trnT, two cox1 pseudogene fragments, a putative atp8 gene, and several distinctive NCRs. Phylogenetic analyses based on 47 mitochondrial genomes, including the newly sequenced C. pedum and two assembled species from SRA database, using CAT-GTR model and BI and ML algorithms further confirmed that the paraphyly of the Neoophora clade and the basal position of Catenulida and Macrostomida. Different from previous study, Rhabdocoela forms a distinct clade within the turbellarians diverged immediately after Macrostomida before Polycladida and Tricladida. Within Rhabdocoela, C. pedum formed a direct sister clade with Typhloplanidae, suggesting a close phylogenetic relationship between the two. We also identified the paraphyletic of the Planoceridae of Polycladida and the unstable position of Planariidae within Tricladidain. Furthermore, we found a trend that the nad4L-nad4 gene box in turbellarians may have evolved from an overlapping state, to the insertion of a non-coding region (NCR), and subsequently to a separated configuration, correlating with species divergence. The selection pressure analysis showcased selective relaxation from free-living species of Rhabdocoela to symbiotic species of Rhabdocoela. Furthermore, we also detected a relaxation from certain tubellarian lineages to Rhabdocoela. Along with higher GORR and longer Brl, Rhabdocoela and its symbiotic group possesses a more rapidly evolving mitochondrial genome. ConclusionsThe mitochondrial genome of Craspedella pedum displays uncommon characteristics, combined extended branch lengths and elevated GORR, suggesting rapid evolution and extensive rearrangements. Our phylogenetic analysis, integrating additional mitogenome data, further corroborates the paraphyly of the Neoophora clade and offers novel insights into turbellarian phylogeny. For the first time, we confirmed the accelerated evolution of mitochondrial genomes in symbiotic tubellarians compared to free-living ones, as evidenced by a longer average Brl, a higher average GORR, and relaxed selection pressure. Additionally, with the same evidences, we found that Rhabdocoela also exhibited accelerated mitochondrial genome evolution in planarians.

genomics↗

bioRxiv2025

High-throughput Monoclonal Antibody Screening from Immunized Rabbits via Droplet Microfluidics

The discovery of monoclonal antibodies (mAbs) is critical to advancing therapeutics, diagnostics, and biomedical research. While mouse-derived mAbs dominate current applications, their limitations--short serum half-life, human immunogenicity, and restricted recognition of human-specific antigens--underscore the need for alternative sources. Rabbit-derived mAbs have been gaining significant traction with their superior antigen-binding affinity, broader epitope diversity, and higher yield potential. However, the absence of well-defined surface markers on rabbit B cells has hindered efficient enrichment strategies, limiting the exploration of this valuable antibody repertoire. In this study, we present an integrated workflow that combines magnetic negative selection with high-throughput droplet microfluidics to overcome these barriers. By optimizing a pan B cell enrichment protocol using a tailored antibody cocktail, we achieved a notable boost in IgG secretion and B cell enrichment. Through two complementary droplet-encapsulated assays using particle aggregation for soluble antigens and reporter cells for membrane-bound antigens, we identified target cells capable of secreting high-affinity IgGs. Subsequent sequencing, in vitro antibody production and characterization confirmed the high affinity rate of the discovered antibodies, outperforming rates previously reported. The use of droplet microfluidics streamlines the analysis of rabbit IgG repertoires, providing a robust platform for rabbit single B cell antibody discovery with promising applications in precision medicines and diagnostics.

molecular biology↗

bioRxiv2025

Acquired resistance to sotorasib in mutant NSCLC is vulnerable to PI3K-mTOR pathway inhibition mediated by 4E-BP1 regulator of cap-dependent translation

Sotorasib and adagrasib have shown significant efficacy in KRASG12C mutant NSCLC; however, acquired resistance (AR) occurs within 6-12 months. While some resistance arises from new mutations, over half of the resistant cases lack identifiable genomic alterations. We hypothesize that resistance is driven by signaling network rewiring, creating new therapeutic vulnerabilities. To investigate AR mechanisms, multiple AR models, including cell lines, PDXs, CDXs, and PDXOs, were developed. H23AR and H358AR cells displayed >600-fold and 200-fold; and PDXO303AR and PDXO314AR organoids exhibited >300-fold and >100-fold resistance to sotorasib, respectively; however, no additional mutations in KRAS or other potential genetic alterations were identified. The AR cells and PDXOs also showed comparable resistance to adagrasib. Distinct protein signatures associated with KRAS reactivation, mTORC1 signaling upregulation, and PI3K/AKT/mTOR pathway activation were identified in TC303AR & TC314AR PDXs. PI3K protein levels were significantly elevated in AR PDXs, H23AR, and H358AR cells. Pharmacological inhibition of PI3K with copanlisib or genetic knockout via CRISPR-Cas9 restored sotorasib sensitivity, suppressed colony formation, and inhibited downstream effectors, including p-AKT, p-mTOR, p-S6, p70S6K, p-GSK3{beta}, and p-PRAS40 in AR cells. Copanlisib also sensitized both acquired and primary resistant PDXOs and synergized with sotorasib in restoring drug sensitivity. p4E-BP1 was significantly upregulated in H23AR and H358AR cells, which is suppressed by copanlisib. The level of p4E-BP1 expression was correlated with Sotorasib sensitivity in PI3K knockout clones, where the most sensitive clone displayed reduced or no p4E-BP1 expression. CRISPR-Cas9-mediated knockout of 4E-BP1, either alone or in combination with PI3K knockout, dramatically restored sotorasib sensitivity to levels comparable to parental cells. Suppression of 4E-BP1 hyperphosphorylation required dual inhibition of mTORC1 and mTORC2, and treatment with AZD8055 or sapanisertib (mTORC1/2 dual inhibitors) significantly dephosphorylated 4E-BP1 and restored sotorasib sensitivity in resistant cells and PDXOs. In PDX, CDX, and xenograft models in vivo, the combination of sotorasib with either copanlisib or sapanisertib resulted in robust, synergistic, and durable tumor regression at well-tolerated doses. These findings showed the critical role of PI3K/mTOR signaling as a bypass mechanism of resistance to KRASG12C inhibitors. We conclude that mTORC1/2 mediated inhibition of p4E-BP1 and combination strategies targeting this pathway effectively overcome acquired resistance to KRASG12C inhibitors in NSCLC.

cancer biology↗

bioRxiv2025

Multicilia dynamically transduce Shh signaling to regulate choroid plexus functions

Choroid plexus is a major site for cerebrospinal fluid (CSF) production, characterized by a multiciliated epithelial monolayer that regulates CSF production. We demonstrate that defective choroid plexus ciliogenesis or Intraflagellar transport yields neonatal hydrocephalus, at least in part, due to increased water channel Aqp1 and ion transporter Atp1a2 expression. We demonstrate choroid plexus multicilia as sensory cilia, transducing both canonical and non-canonical Shh signaling. Interestingly, it is the non-canonical Shh signaling that represses Aqp1 and Atp1a2 expression by Smo/Gi/cAMP pathway. Choroid plexus multicilia exhibit unique ciliary ultrastructure, carrying features of both primary and motile cilia. Unlike most cilia that elongate during maturation, choroid plexus ciliary length decreases during development, causing a decline of Shh signaling intensity in developing choroid plexus, a derepression of Aqp1 and Atp1a2, and ultimately, an increased CSF production. Hence, developmental dynamics of choroid plexus multicilia dampens the Shh signaling intensity to promote CSF production.

developmental biology↗

bioRxiv2024

Spatiotemporal Regulatory Logics of Mouse Gastrulation

Spatiotemporal coordination of cellular and molecular events is crucial for cell fate commitment during mouse gastrulation. However, the high-precision mechanisms governing the timing and spatial dynamics remain poorly understood. Here, we present a time-series single-cell multi-omic dataset from the mouse gastrulating embryos and construct a hierarchical gene regulatory landscape. Integrating this with real three-dimensional transcriptomic coordinate, we created ST-MAGIC and ST-MAGIC (+) atlas, dissecting the spatiotemporal logics of regulatory networks and signaling responsiveness underpinning the lineage commitment at gastrulation. Specifically, we delineated the multi-omic basis for left-right symmetry breaking events in the gastrula and also revealed the spatiotemporal molecular relay for axial mesendoderm lineage, where early and intermediate transcription factors first open the chromatin regions and setup the responsiveness to signaling, followed by terminal factors to consolidate the transcriptomic architecture. In summary, our study presents a spatiotemporal regulatory logic framework of mouse gastrulation, that advances our understanding of mammalian embryogenesis.

developmental biology↗

bioRxiv2024

Chromosome-scale assembly and annotation of the wildwheat relative Aegilops comosa

Wild relatives of wheat are valuable sources for enhancing the genetic diversity of common wheat. Aegilops comosa, an annual diploid species with an MM genome constitution, possesses numerous agronomically valuable traits that can be exploited for wheat improvement. In this study, we report a chromosome-level genome assembly of Ae. comosa accession PI 551049, generated using PacBio high-fidelity (HiFi) reads and high-throughput chromosome conformation capture (Hi-C) data. The assembly spans 4.47 Gb, featuring a contig N50 of 23.59 Mb and a scaffold N50 of 619.05 Mb. A total of 39,063 gene models were annotated through a combination of homoeologous proteins, Iso-Seq, and RNA-Seq data. Comparative genome analysis revealed a terminal intrachromosomal translocation in chromosome 2M of Ae. comosa (and Ae. umbellulata) compared to its homoeologous chromosomes in other diploid wheat species. Phylogenetic analysis showed a close relationship between Ae. comosa and Ae. umbellulata. This newly constructed reference genome of Ae. comosa will serve as an important genomic resource for comparative genomic studies and the cloning of agriculturally important genes.

genomics↗

bioRxiv2024

Manipulation of the brown glume and internode 1 gene leads to alterations in lignified tissue coloration, lignification, and pathogen resistance in wheat

Lignin is a crucial component of the cell wall, providing mechanical support and protection against biotic and abiotic stresses. However, little is known about wheat lignin-related mutants and their roles in pathogen defense. In this study, we identified an ethyl methanesulfonate (EMS)-derived Aegilops tauschii mutant named brown glume and internode 1 (bgi1), which exhibits reddish-brown pigmentation in various tissues, including internodes, spikes, and glumes. Using map-based cloning and single nucleotide polymorphism (SNP) analysis, we identified AET6Gv20438400 (BGI1) as the leading candidate gene, encoding the TaCAD1 protein. The mutation occurred in the splice acceptor site of the first intron, resulting in a premature stop codon in BGI1. We validated the function of BGI1 using loss-of-function EMS and gene editing knockout mutants, both of which displayed reddish-brown pigmentation in lignified tissues. BGI1 knockout mutants exhibited reduced lignin content and shearing force relative to wild type, while BGI1 overexpression transgenic plants showed increased lignin content and enhanced disease resistance against common root rot and Fusarium crown rot. We confirmed that BGI1 exhibits CAD activity both in vitro and in vivo, playing an important role in lignin biosynthesis. BGI1 was highly expressed in the stem and spike and localized in the cytoplasm. Transcriptome analysis revealed the regulatory networks associated with BGI1. Finally, we demonstrated that BGI1 interacts with TaPYL-1D, potentially involved in the abscisic acid signaling pathway. The identification and functional characterization of BGI1 significantly advance our understanding of CAD proteins in lignin biosynthesis and plant defense against pathogen infection in wheat.

plant biology↗

bioRxiv2024

Boundary-Sensing Mechanism in Branched Microtubule Networks

The self-organization of cytoskeletal networks in confined geometries requires sensing and responding to mechanical cues at nanometer to micron scales that allow for dynamic adaptation. Here, we show that the branching of microtubules (MTs) via branching MT nucleation combined with dynamic instability constitutes a boundary-sensing mechanism within confined spaces. Using a nanotechnology platform, we observe the self-organization of a branched MT network in a channel featuring a narrow junction and a closed end. Our observations reveal that branching MT nucleation occurs in the post-narrowing region only if that region exceeds a certain length before it terminates at the channels closed end. The length-dependent occurrence of branching MT nucleation arises from the dynamic instability of existing MTs when they interact with the channels closed end, combined with the specific timescale required for new MTs to nucleate at a point distant from the closed end, creating a mechanical feedback. Increasing the concentration of the base branching factor TPX2 accelerates nucleation kinetics and thus tunes the minimum length scale needed for occurrence of branching MT nucleation. As such, this feedback not only allows for adaptation to the local geometry, but also allows for tunable formation of MT networks in narrow (micron and submicron scale) channels. However, while a high concentration of TPX2 increases the kinetic rate of branching MT nucleation, it also stabilizes MTs at the channels closed end leading to MT growth and nucleation in the reversed direction, and thus hinders boundary sensing. After experimental characterization of boundary-sensing feedback, we propose a minimal model and execute numerical simulations. We investigate how this feedback, wherein growing MTs dynamically sense their physical environment and provide nucleation sites for new MTs, sets a length/time scale that steers the architecture of MT networks in confined spaces. This "search- and-branch" mechanism has implications for the formation of MT networks during neuronal morphogenesis, including axonal growth and the formation of highly branched dendritic networks, as well as for plant development and MT-driven guidance in fungi, and engineering nanotechnologies.

bioengineering↗

bioRxiv2024

The Phylogeny and the Evolution of Parasitic Strategies in Trematoda

Trematodes are obligatory parasites that generally must transmit between hosts to complete their life cycle. They parasitize varying numbers of intermediate hosts (0, 1 or 2), but the evolutionary history of these strategies and the ancestral states remain unknown. We conducted the ancestral state reconstruction of the number of intermediate hosts using mitogenomic (Trematoda) and nuclear-genomic (Neodermata) topologies. Aspidogastrea was identified as the sister-group ("basal") to all other Trematoda using a range of approaches, so it is crucial for studying the evolutionary history of trematodes. However, there is only one transcriptome available for this lineage, and mitochondrial genomes (mitogenomes) remain unavailable. Herein, we sequenced mitogenomes of two aspidogastreans: Aspidogaster ijimai and Aspidogaster conchicola. As the ancestral state reconstruction analysis is topology-sensitive, we tested multiple phylogenetic strategies, comprising the outgroup selection, phylogenetic models, partitioning strategies, and topological constraints. These mitogenomic phylogenies exhibited pronounced topological instability, with Aspidogastrea resolved as the "basal" radiation in most, but not all, topologies. Based on our analyses, Cestoda was the optimal outgroup choice, and the "heterogeneous" CAT-GTR model in PhyloBayes was the optimal model choice. We inferred the time tree and conducted ancestral state reconstruction analyses using this "optimal" topology, as well as constrained mitogenomic and nuclear genomic topologies. Results were ambiguous for some lineages, but scenario that received the strongest support is the direct life cycle (no intermediate hosts) in the ancestors of Trematoda (proto-trematodes) and Aspidogastrea (proto-aspidogastreans), while the ancestor of Digenea (proto-digeneans) had two intermediate hosts. The inferred scenario indicates that host strategies are relatively plastic among trematodes, putatively comprising several independent host gains, and multiple host losses. We propose a timeline for these events and discuss the role that alternating sexual and asexual generations putatively played in the evolution of complex parasitic life histories in digeneans.

evolutionary biology↗