Biology subjects

Wu, A.

Publications and source records attributed to Wu, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A single-plane electrical impedance tomography framework for integrated liver and kidney assessment

Chronic liver and kidney diseases frequently coexist, but their assessment typically requires separate clinical workflows involving serum-based estimated glomerular filtration rate (eGFR) testing and liver elastography-based controlled attenuation parameter (CAP). Electrical impedance tomography (EIT) has recently shown promise for providing portable, non-invasive proxies for both reference measurements. However, existing multi-organ EIT assessment still requires different acquisition protocols for each organ. To simplify this procedure, we assessed the feasibility of a single-plane EIT framework for reconstructing organ-specific images and estimating liver and kidney health from one abdominal belt placement. Across diverse abdominal belt locations, opposite-plane deviation was significantly greater for eGFREIT estimates (2.92%; IQR, 0.46-12.53%) than for CAPEIT estimates (0.94%; IQR, 0.66-1.70%) (p < 0.01). For both organs, compared to images acquired at the conventional organ-specific planes, image correlation significantly decreased (both, p < 0.05) and normalized root mean squared error significantly increased (both, p < 0.05) at the furthest imaging plane, with these differences being numerically larger for the kidney. Together, these results supported the kidney plane as the preferred location for multi-organ assessment. Test-retest evaluation after belt removal and reapplication demonstrated that the correlation between two repeated CAPEIT estimates was just as strong at the kidney plane (R2 = 0.98, p < 0.001) as it was at the conventional liver plane (R2 = 0.97, p < 0.001). Finally, in an independent clinical cohort, kidney-plane CAPEIT estimates were significantly higher in participants with a high metabolic liver-risk profile (i.e. diabetes and high blood pressure) (p < 0.005). CAPEIT estimates also distinguished high and low metabolic risk groups with an AUC of 0.756. Together, these findings support the feasibility of a portable multi-organ EIT imaging framework capable of extracting complementary liver and kidney information from a single belt placement.

bioengineering↗

bioRxiv2026

Topotecan Syndergizes with SP141 in the Management of Uveal Melanoma

Purpose: Uveal melanoma (UM) is a rare but aggressive intraocular malignancy originating in the uvea (choroid, iris, and ciliary body). Despite primary treatment, 50% of UM patients develop metastases to the liver, lungs, bones, and skin. Topotecan, a topoisomerase I inhibitor, and SP141, an MDM2 inhibitor, have been studied in cancers, including retinoblastoma. This study explores a combined approach with these drugs in the 92-1 UM cell line. Methods: Primary choroidal melanoma-derived 92-1 cells were treated with topotecan and SP141 individually to determine IC50 values. Cell viability and proliferation were assessed using the MTT assay and the xCELLigence real-time monitoring system. The synergistic effects were analyzed quantitatively using the Loewe Additivity and Highest Single-Agent (HSA) models. Flow cytometry was employed to analyze cell cycle distribution, while cell death and apoptosis were monitored via high-throughput, real-time quantitative analysis using the IncuCyte live-cell kinetic imaging system. Results: Both Topotecan and SP141 induced significant cell death in 92-1 cells. xCELLigence showed dose- and time-dependent inhibition of proliferation. Combining sub-IC50 concentrations of topotecan and SP141 exhibited significant synergistic effects, markedly reducing growth. Cell cycle analysis revealed that topotecan causes a dose-dependent biphasic arrest in G2 and S phases, while SP141 causes G2/M arrest, leading to cell death. Cytotoxicity assays corroborated increased cell death following combination therapy, and the Caspase 3/7 assay indicated that apoptosis is the mechanism underlying cell death. Conclusions: The multi-targeted approach combining topotecan and SP141 drugs demonstrated synergistic therapeutic effects in 92-1 cells, suggesting a potential treatment strategy for managing UM.

cancer biology↗

bioRxiv2026

A diffusion model of viral evolution predicts mutation fitness and evolutionary trajectories

Viral evolution arises from random mutations and natural selection, yet computational approaches rarely model these two forces in a unified way. We present Viral Evolution Simulator (VES), a diffusion model-based framework that mirrors this duality by design: forward noise injection simulates stochastic mutation, and reverse denoising recapitulates selective filtering. Trained solely on viral protein sequences, VES predicts mutational fitness without functional data, measuring fitness as the reconstruction difficulty of a mutated sequence relative to its wild-type counterpart. Across immune escape, receptor binding, and deep mutational scanning datasets, VES outperforms state-of-the-art generative models, achieving a 31.78% error reduction over the best baseline in immune escape mutation fitting evaluation. When trained on sequences collected before June 2024 and evaluated against H1N1 strains that later emerged, VES assigned high scores to 16 of 20 mutations that subsequently showed the sharpest frequency shifts. Extending to avian influenza H5, the framework reveals a dynamic interplay between antigenic escape and human-type receptor binding. Both functions dropped sharply in 2021, followed by a sustained rise in receptor affinity that could connect to recent epidemiological trends. VES offers a generalizable, sequence-only foundation for tracing evolutionary trajectories and prioritizing mutations for surveillance and experimental validation, pointing toward where functional efforts might matter most.

bioinformatics↗

bioRxiv2026

Seasonal influenza vaccine strain selection by quantifying viral fitness

The long-standing challenge of seasonal influenza vaccines providing poor protection is attributed to the rapid and continuous evolution of the virus. In theory, the recommendation of vaccine strain is to predict the dominant strain with best fitness in the upcoming season. Here, we develop FutureFlu, a biologically-grounded framework that quantifies the fitness score of influenza variants in upcoming seasons, by integrating metrics on three levels: molecular genetic divergence, individual immune escape, and population-scale transmission dynamics. The fitness scores of influenza variants present strong positive correlation with their actual observed frequencies in the next seasons. Validation across 24 seasons of three influenza subtypes shows FutureFlu recommends antigenically matched vaccine strains more frequently than annual recommendations, particularly for challenging subtypes: 83.3% versus 45.8% seasons for A/H3N2, and 75.0% versus 33.3% seasons for B/Victoria. Furthermore, FutureFlu significantly outperforms the currently used methods in vaccine strain selection whether or not there is available antigenic data from hemagglutination inhibition (HI) assay, which provides a valuable supplement for WHO vaccine recommendation. To support global public health implementation, an open online platform (futureflu.com.cn) has been established to offer real-time viral fitness predictions and vaccine recommendations.

evolutionary biology↗

bioRxiv2026

Accurate and scalable decontamination of imaging-based spatial transcriptomics via optimal transport

Imaging-based spatial transcriptomics enables molecule-resolved profiling of gene expression and tissue organization in situ. However, segmentation errors, transcript spillover and three-dimensional cell overlap can introduce misassigned transcripts into cell-level expression profiles, compromising biological interpretation and obscuring genuine signals. Existing methods either remove suspect expression at the cost of signal loss or lack a biologically grounded criterion for transcript assignment. Here we present CellDot, an optimal-transport framework that determines the fate of each transcript by retaining it in its host cell, reassigning it to a plausible neighboring cell or removing it as background. By integrating reference-guided expression compatibility with spatial information and data-adaptive constraints, CellDot enables accurate and traceable molecule-level correction while preserving biologically meaningful variation. In evaluations across multiple human tumor datasets, CellDot exhibited superior performance compared to existing decontamination methods, successfully restoring spatial expression patterns that matched independent cross-platform measurements. Moreover, it significantly enhanced the recovery of cellular states, intercellular communication, and spatial niche programs. Our experiments using real data demonstrated CellDot's scalability and established it as the only method applicable to a whole-transcriptome Atera dataset, underscoring its distinct advantages in the field of spatial transcriptomics.

bioinformatics↗

bioRxiv2026

Sexually Dimorphic Visual Processing in the Yellow-Fever Mosquito, Aedes aegypti

Flying insects process motion across the visual scene to regulate their ground speed, correct course deviations, and behaviorally relevant stimuli such as avoiding predators or finding hosts. The yellow-fever mosquito Aedes aegypti provides a useful model for studying these visually guided behaviors, as both sexes use vision to track moving features. Approaching a moving target presents mosquitoes with a demanding visual problem: they must extract target motion from the changing visual scene while simultaneously controlling their own flight and responding to potentially dangerous movements. To understand this, we first studied free-flight responses to static and moving vertical targets in a wind tunnel system capable of projecting dynamic visual stimuli, and found that males and females are sexually dimorphic with respect to their responses to target motion. When the feature was static, both males and females advanced toward it during flight; however, when it oscillated laterally at 2 Hz, only females continued to pursue it. In a subsequent yaw-unrestrained magnetic tethering paradigm, we showed that this divergence cannot be simply explained by sex-specific differences in visuomotor tuning dynamics, while micro-CT reconstructions revealed only modest sexual differences in eye morphology and broadly similar patterns of estimated spatial acuity. Together, these results indicate that males retain the biomechanical capacity to track oscillating targets but actively suppress approach behavior during free flight, whereas females pursue salient features independently of target motion. This pattern aligns with sex-divergent visual ecologies because the risk of approaching a large moving feature too closely likely outweighs the benefits for males seeking mates, whereas for host-seeking females the reproductive necessity of blood feeding overrides this avoidance.

neuroscience↗

bioRxiv2026

Visualizing zinc-starved slow-replicating Mycobacterium tuberculosis in neutrophil-rich lung lesions

The months-long treatment for tuberculosis is partially attributed to a minor Mycobacterium tuberculosis (Mtb) subpopulation exhibiting phenotypic drug resistance, although origins of these bacilli in hosts remain unknown. Previously, we observed that mycobacteria respond to growth-restrictive limitations of zinc by accumulating Mrf, and inducing Mpy recruitment to the decoding center of the ribosome, thereby hibernating the ribosome in an inactive state. Mrf accumulation - regulated at both transcriptional and post-translational levels by Zur and Clp, respectively, in a zinc-dependent manner - facilitates the formation of stable Mpy-70S ribosome complex in zinc-starved cells. Here, using a translational fusion of Mrf with a fluorescent-reporter protein we visualized zinc-starved mycobacteria with a physiology conducive for Mpy-dependent ribosome hibernation. About half of zinc-starved mycobacterial population expressed microscopically detectable levels of the reporter and exhibited Mpy-dependent slowdown of growth and isoniazid tolerance in vitro. In chronic, high-burden mouse lung infections, ~3% of Mtb bacilli expressed the reporter, and the frequency increased to ~35% upon isoniazid treatment. Transcriptomics of mouse lungs suggests an important role of S100A8/A9 and ZIP2 in neutrophils in zinc mobilization during Mtb infections. Moreover, depletion of neutrophils, or loss of S100A9 caused significant attenuation of zinc starvation response in Mtb. Altogether, the study offers a new approach for visualizing and manipulating slow-replicating Mtb in host tissues.

microbiology↗

bioRxiv2026

Diverse Intestinal Injuries Drive Heterogeneous Transcriptional Responses and Limited Reactivation of Developmental Gene Programs in Human Enteroids

The murine intestine reactivates developmental gene programs following various forms of damage in vivo and in vitro; however, injury response mechanisms used by the human intestine remain unclear. Using adult human small intestinal epithelium-only organoids ("enteroids"), we characterized the early response to eight injury conditions and injury-associated signaling pathways (P53, PGE2, YAP, TGFB) to interrogate whether human developmental genes were activated. P53 activation and decreased proliferation were common features across treatments. Most (7/8) injuries did not activate human development genes. Butyrate is a notable exception given it inhibited P53 and promoted a human developmental transcriptional signature. We observe that P53 induces a human adult gene signature while TGFB and YAP promote a developmental signature. Together our data characterizes various transcriptional responses to injury, supports injury-associated signaling pathways as regulators of human adult and developmental genes, and highlights how our data can be mined to predict injury-specific interventions for epithelial protection.

cell biology↗

bioRxiv2026

A Conserved Flexible N-Terminal Domain Tunes the Calcium Sensitivity of Sorcin by Stabilizing Its Active Conformation

Sorcin is a penta-EF-hand Ca2+-binding protein that acts as a Ca2+ sensor and regulator of Ca2+ homeostasis. Although the structure and Ca2+-dependent activation of Sorcin is well characterized, the function of its flexible N-terminal domain (NTD) remains unclear. We combined sequence analysis, Ca2+-induced aggregation assays, multidimensional NMR spectroscopy, and long-timescale molecular dynamics (MD) simulations to define the NTDs role in Sorcin activation. Sequence comparisons showed that the NTD is conserved across vertebrates despite its intrinsic disorder, indicating functional importance. NTD deletion markedly reduced Ca2+ responsiveness. Relative to full-length Sorcin, the construct with NTD truncation required more than twofold higher Ca2+ concentrations and over tenfold higher protein concentrations to initiate aggregation, while aggregation kinetics slowed by nearly three orders of magnitude. Temperature-dependent measurements yielded an apparent activation energy of 36.7 kJ{middle dot}mol-1, consistent with aggregation driven by Ca2+-induced conformational activation rather than denaturation. NMR chemical shift perturbations localized the effects of NTD removal to the EF-hand Ca2+-binding loops and adjacent D-helix. Apo-state MD simulations revealed transient intra- and intermolecular NTD-SCBD contacts that explain some perturbations and support direct and allosteric regulation. Ca2+-bound simulations further showed that SCBD departs more readily from the crystallographic active conformation than full-length Sorcin, indicating that the NTD stabilizes the active state through dynamic contacts. Together, these findings establish the NTD as a critical regulator that enhances Ca2+ responsiveness by shifting Sorcins conformational equilibrium toward the active state and suggest that flexible N-terminal extensions regulate signaling within the penta-EF-hand protein family.

biophysics↗

bioRxiv2026

MSstatsBioNet: Integrating Statistical Analyses with Prior Knowledge Biomolecular Networks for Quantitative Proteomics and Phosphoproteomics

A common outcome of quantitative mass spectrometry-based proteomic and phosphoproteomic experiments is a list of proteins that are differentially abundant between conditions. However, biological interpretation requires evaluation in the context of prior knowledge of biological mechanisms and protein function. One approach to facilitate mechanistic biological interpretation is to integrate such lists with biological network databases, built from manually curated resources and text mining systems. This manuscript automates this process with MSstatsBioNet, a Bioconductor package that integrates MSstats, a family of open-source packages for detecting differentially abundant proteins, and INDRA, a system that extracts biomolecular networks from biomedical literature using text mining and merges those networks with the content of curated knowledge bases. Taking as input a list of differentially abundant proteins from MSstats, MSstatsBioNet retrieves a protein subnetwork from INDRA and overlays experimental fold changes onto the underlying subnetwork. Users can then interact with the network and overlaid data, interrogating primary literature evidence to construct granular mechanistic narratives for iterative hypothesis generation. We demonstrate the utility of this approach with three case studies, two measuring changes in protein abundance and one measuring changes in phosphorylation.

bioinformatics↗

bioRxiv2026

Suspension-Based Human Esophagoids Recapitulate WNT2B-Dependent Regulation of Esophageal Basal Progenitors

Background & AimsThe human esophagus undergoes a tightly regulated developmental program, transitioning from a simple columnar epithelium in early development to a mature stratified squamous tissue essential for adult barrier function. Here, we constructed a developmental cell atlas spanning early development to adulthood and leveraged it to generate physiologically relevant in vitro models. MethodsWe utilized single-cell RNA sequencing and spatial multiplex proteomics of human esophageal tissue from early development through adulthood. We established a feeder-supported 2D culture system and a Matrigel-free, suspension-based 3D esophagoid model in a 96-well format. To interrogate WNT2B function, we analyzed patient tissue harboring WNT2B loss-of-function mutations and performed WNT inhibition in esophagoids. ResultsSequencing profiling identified stage-specific epithelial populations: multiciliated and GPC3 basal cells were unique to early development; KRT14 basal and CRNN luminal cells were adult-specific; and COL17A1, LY6D, and KRT4 populations were shared across stages. Spatially organized WNT2B, KIT, and VWC2 mesenchymal subtypes were identified. The 2D system preserved both epithelial and mesenchymal compartments with transcriptional fidelity. Esophagoids exhibited basal-to-luminal stratification, mesenchymal compartmentalization, and required stromal interactions for formation. WNT2B repressed self-renewal of TP63 basal progenitors and inhibited proliferation, confirmed by pharmacologic inhibition of WNT in the in vitro esophagoids. ConclusionsWe present a stage-resolved atlas of human esophageal development and a scalable esophagoid platform recapitulating esophageal architecture. WNT2B regulates progenitor dynamics by restraining basal cell self-renewal. Esophagoids provide a physiologically relevant system for modeling esophageal development and disease. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/733451v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1af5743org.highwire.dtl.DTLVardef@8a061dorg.highwire.dtl.DTLVardef@1975c3forg.highwire.dtl.DTLVardef@292ce9_HPS_FORMAT_FIGEXP M_FIG C_FIG Key Findings and ImplicationsO_LIDevelopmental Atlas: The study presents a comprehensive transcriptional and structural atlas of the human esophageal epithelium, identifying conserved and stage-specific epithelial populations from early development to adulthood. Notably, stage-specific gene expression of multiciliated and GPC3 basal cells were unique to early development, while KRT14 basal and CRNN luminal cells were adult specific, with COL17A1+ (basal), LY6D+ (epibasal), and KRT4+ (middle), shared at all stages. C_LIO_LIMesenchymal Diversity: Spatial and transcriptional profiling revealed distinct mesenchymal subtypes, including WNT2B, KIT, and VWC2 populations, which are spatially organized and contribute to epithelial-mesenchymal signaling. These findings reinforce the role of stromal-epithelial interactions in esophageal development. C_LIO_LI2D Esophagus Cell Culture System: A feeder-supported 2D cell culture system was developed that retains both epithelial and mesenchymal populations, preserving transcriptional fidelity and enabling long-term expansion for mechanistic studies. C_LIO_LI3D Esophagoid Model: A suspension-based 3D organoid system was optimized using a 96-well format, enabling high-throughput generation of esophagoids with robust epithelial stratification and mesenchymal compartmentalization. These organoids recapitulate key features of the human esophagus, including basal-to-luminal organization, and require stromal interactions for formation. C_LIO_LIFunctional Role of WNT2B in esophagus development: Both in vivo and in vitro analyses demonstrated that WNT2B regulates epithelial progenitor dynamics and tissue architecture by repressing self-renewal of basally localized TP63+ cells and inhibiting proliferation. Loss-of-function models and WNT pathway modulation confirmed its role in epithelial-mesenchymal crosstalk and organoid integrity. C_LI

developmental biology↗

bioRxiv2026

Discrete Cognitive Resolution in Alzheimer's Disease: Cross-Cohort Reanalysis of ADNI and NACC Longitudinal Data

INTRODUCTIONDo continuous cognitive totals capture patient-relevant transitions, or does decline have discrete structure? METHODSWe formalize a discrete cognitive resolution (DCR) model in which decline is loss of binary discriminative coordinates, with deterministic emissions tying items to a shared low-dimensional mask. Using natively discrete item-level data (ADAS-Cog, 688 ADNI participants; MoCA sub-items, 13,323 NACC participants), we tested pre-specified signatures by out-of-sample log-loss against continuous-drift, per-item, and mixed-effects IRT competitors, with a coordinate-label permutation null (S2). RESULTSDCR beat both pre-specified baselines (ADNI 0.436 vs 0.965; NACC 0.566 vs 0.602). S2 was decisive in ADNI (AUC 0.782; null 0.529, P < .001); in NACC the signal concentrated in orientation (AUC 0.718). Mixed-effects IRT achieved lower log-loss than DCR. DISCUSSIONCognitive decline shows discrete coordinate structure when items are single-coordinate probes. The claim is structural, not predictive; encoding is decisive.

neuroscience↗

bioRxiv2026

RubiSpec-MS: Determination of Rubisco CO 2 /O 2 Specificity Factor using Liquid Chromatography-Mass Spectrometry

O_LIThe specificity factor(SC/O) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a key kinetic parameter quantifying the ratio of carboxylation to oxygenation reactions. Current methods to measure SC/O are expensive, labor-intensive, require specialized expertise, and/or rely on radiolabeled compounds, hindering Rubisco research. C_LIO_LIWe present RubiSpec-MS (Rubisco Specificity by LC-MS/MS), a method to determine the SC/O of purified Rubiscos using liquid chromatography-coupled mass spectrometry (LC-MS/MS) for separation and sensitive detection of non-radioactive reaction products. C_LIO_LIWe benchmark RubiSpec-MS using multiple model Rubisco enzymes. We find that the assay accurately reproduces corresponding literature values for each enzyme and performs similarly in a side-by-side comparison using an established radiometric method. C_LIO_LIRubiSpec-MS provides a faster, more accessible, and highly parallelizable method to measure Rubisco SC/O using equipment common in academic core facilities and industrial laboratories. C_LI

Plant Biology↗

bioRxiv2026

Inorganic sulfate transport by the Mycobacterium tuberculosis PE22/PPE36 complex

Mycobacterium tuberculosis (Mtb) encodes two multigene families with 169 members that are exclusive to mycobacteria, the pe and ppe genes. These genes have unusual sequences including low-complexity repeat regions, but their functions--and whether they share a common function--have long been unclear. Recently, several members of the pe/ppe family were shown to transport nutrients across the outer Mtb membrane, a role for which no other proteins have yet been identified. Whether nutrient transport is a family-wide function and the range of nutrients transported by the PE/PPEs remains unclear. Sulfur is an essential nutrient for Mtb physiology and pathogenesis. To test whether PE/PPE transporters contribute to sulfur acquisition, we analyzed the transcriptional response of Mtb to sulfate by RNA sequencing. The pe22/ppe36 genes were induced in sulfate-limiting conditions. Deletion of pe22/ppe36 impaired growth in low-sulfate media and reduced intracellular sulfate levels, effects that were reversed by heterologous expression of the Mycobacterium smegmatis porin MspA. The response of sulfur-responsive genes to sulfur was muted in the pe22/ppe36 deletion strain, and mass spectrometry showed lower sulfolipid and sulfur metabolite levels in the deletion strain. These findings identify PE22/PPE36 as a specific sulfate uptake system and supports the emerging idea of PE/PPE proteins as nutrient uptake systems across the Mtb outer membrane. Significance statementThe mechanism of nutrient transport across the porin-less mycobacterial outer membrane and the function of the large pe/ppe gene family have been longstanding questions in mycobacterial biology. This study shows that the Mycobacterium tuberculosis PE22/PPE36 complex serves as a selective conduit for inorganic sulfate. Deletion of this complex disrupts intracellular sulfur homeostasis and triggers a metabolic seesaw that depletes cell-surface sulfolipids. Our findings expand the PE/PPE transport paradigm and suggest a direct link between nutrient acquisition and TB transmission.

microbiology↗

bioRxiv2026

Pharmacological inhibition of LIN28A promotes imatinib sensitivity in CML resistance

Resistance to tyrosine kinase inhibitors (TKIs) remains a critical challenge in chronic myeloid leukemia (CML), particularly when driven by mechanisms independent of BCR-ABL1 kinase-domain mutations. Building on the identification of the RNA-binding protein LIN28A as a driver of imatinib resistance, we evaluated emerging LIN28 inhibitors as potential sensitizing agents. Screening three small molecules in an imatinib-resistant (ImR) K562 model identified LIN28i-1632 as uniquely synergistic with imatinib (synergy score: 12.07), reducing cell proliferation by 71.15%. Quantitative DIA and TMT proteomics revealed that this synergy is characterized by significant proteomic remodelling, including the downregulation of the canonical LIN28 target HMGA1 and the activation of apoptotic and G2/M cell cycle checkpoint programs. Mechanistically, phosphoproteome and kinome profiling showed suppressed AKT/RPS6K and CDK signalling. We further demonstrate that LIN28i-1632 promotes sensitivity by reducing BCR-ABL protein abundance and attenuating the AKT survival axis through RICTOR downregulation and PTEN restoration. Collectively, our findings establish pharmacological LIN28 inhibition as a viable strategy to overcome TKI resistance by simultaneously engaging cell-cycle arrest and dismantling the AKT-mediated survival network.

cancer biology↗

bioRxiv2026

Generation of KARRIKIN INSENSITIVE2 loss-of-function mutants in Ceratopteris richardii using a CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology

Plant hormones regulate almost every aspect of plant growth and development. KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling, which is thought to transduce signals derived from an unidentified ligand known as the KAI2 ligand (KL), regulates numerous traits, including seed germination in angiosperms and vegetative reproduction in bryophytes. The origin of KAI2 is believed to be ancient, and the evolution of its signaling pathways remains of significant interest. Ferns represent critical lineages for elucidating the evolution of land plant traits and growth mechanisms that enabled adaptation to terrestrial environments. Therefore, functional studies of key components of this pathway in ferns are essential for understanding the evolutionary trajectory of KAI2-dependent signaling during vascular plant diversification. However, experimental platforms for the CRISPR/Cas9 system, a powerful tool for investigating gene function, remain undeveloped in ferns. Here, we report an efficient CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology in the model fern, Ceratopteris richardii (C. richardii). We generated loss-of-function mutants of the KAI2 ortholog in C. richardii (CrKAI2), as well as the signaling components CrMAX2 and CrSMXL. We demonstrate that exogenous application of an artificial KL agonist increases the expression of KAI2-dependent signaling responsive genes in wild type plants; this response is abolished in Crkai2 mutants. These findings indicate that KAI2-dependent signaling is conserved in C. richardii. Furthermore, this study proposes an efficient CRISPR/Cas9 method that will facilitate genetic studies in ferns.

plant biology↗

bioRxiv2026

Protein domain characterization reveals human MIC60 tolerates loss of helical bundle domain

The mitochondrial contact site and cristae organizing system (MICOS) is essential for cristae junction formation and inner mitochondrial membrane architecture. To define how MICOS integrity is established and maintained, we generated conditional deletion models of Immt (encoding MIC60), a core MICOS subunit, in tissue-specific settings and in cultured cells. Liver-specific deletion of Immt in mice induced profound defects in mitochondrial ultrastructure and function, establishing MIC60 as essential for mitochondrial integrity. Notably, despite the severity of the defects, we did not detect increased apoptosis in liver tissue or in cells. To directly link MIC60 structure to its function, we performed a systemic structure-function analysis of human MIC60 using domain-specific deletion mutants expressed in Immt-deleted cells. We identified that the transmembrane, coiled-coil, and mitofilin domains are required for MICOS assembly, mitochondrial morphology, and respiratory function. Unexpectedly, deletion of the predicted helical bundle (a region spanning 229 amino acids) substantially restored mitochondrial structure and function, nearly matching full-length MIC60. A mutation (K299E) associated with human disease within this domain similarly preserved most MIC60-dependent functions. Together, these results establish MIC60 as a non-redundant regulator of mitochondrial architecture while revealing that a large predicted structural domain is largely dispensable for MIC60s core functions, refining current models of MICOS organization and uncovering unexpected modularity within MIC60.

cell biology↗

bioRxiv2026

Microencapsulation of a novel Bacteroides thetaiotaomicron strain: a promising strategy to fortify intestinal barrier fortification in weaned pig model

Porcine Bacteroides thetaiotaomicron LYH5 demonstrated in vitro antimicrobial activity, suggesting probiotic potential. Due to poor gastric juice tolerance, LYH5 was encapsulated via extrusion using sodium alginate (SA) and gellan gum. Box-Behnken design optimization yielded optimal parameters: SA 1.5%, gellan gum 0.4%, CaCl2 0.9%, bacteria:glue ratio 1:4, achieving an encapsulation rate of 84.22{+/-}0.17%. Its effect on weaned piglet intestinal health was evaluated using 78 piglets (7.69{+/-}0.52 kg) randomly assigned to 4 groups for 40 days: CON (control), T (basal diet + LYH5 live bacteria, 1x10{superscript 1} CFU/mL), TJ (basal diet + LYH5 microcapsules, 1x10{superscript 1} CFU/mL, J (basal diet + empty capsules). The results of this experiment showed that compared with the control group, LYH5 microcapsule can improve the intestinal barrier function without affecting the growth performance of piglets, and provide ideas and references for the development of human next-generation probiotics (NGP). IMPORTANCEThis study addresses the key bottleneck of poor gastric acid tolerance of probiotics via microencapsulation and provides a practical reference for the development of human next-generation probiotics.

microbiology↗