Biology subjects

Johnson, M.

Publications and source records attributed to Johnson, M..

At least 19 recordsLinked to original sources

bioRxiv2026

A step beyond: age and sex-dependent changes in multidimensional locomotor and gait patterns of voluntary motion of C57BL/6 mice

Age-related decreases in physical activity drive frailty and reduced intrinsic capacity, promoting adverse health outcomes such as falls. Experimental tools for quantifying these metrics in preclinical models have lagged behind those used clinically; single-endpoint activity assessments (e.g., rotarod performance, treadmill activity) of rodents often require invasive handling and/or forced performance cues that may increase stress and variability, especially in aged animals. Here, we evaluated whether automated analysis of spontaneous movement using machine-learning-based software (Palmreader) could identify age-related changes in locomotor activity, gait, paw loading, or posture in male and female C57BL/6 mice across young, adult, and aged timepoints (4.5-, 7-, and 22 months of age). Parameters were quantified using the BlackBox One system during a 7-minute open-field recording. Principal component analysis demonstrated age-related separation of movement phenotypes, with individual parameters demonstrating increased hindlimb duty factor and longer stride lengths with age. Age-related changes in forelimb-to-hindlimb duty-factor imbalance, paw contact, and postural organization were also observed, and several gait alterations with age showed different patterns between males and females. Conventional measures of physical activity (e.g., distance traveled, walking time), in contrast, showed few age-related differences, particularly in females. These findings suggest that aging more consistently alters movement patterns rather than movement magnitude, and that gait and postural features may represent important age-related functional changes not captured by activity measurements alone. Automated assessment of spontaneous movement may therefore represent a useful complement to traditional physical activity testing, with the added benefit of minimal animal handling and short recording times.

animal behavior and cognition↗

bioRxiv2026

Nucleus remodeling activity is conserved amongst diverse SIV Vpr isolates

Human immunodeficiency virus (HIV) encodes four accessory proteins that are essential for virus replication in vivo, primarily through the counteraction of host innate immune defense mechanisms. One of these proteins, Vpr, induces constitutive DNA damage repair (DDR) signaling to drive global epigenetic remodeling and activation of transcription programs that enhance HIV-1 promoter activity during acute infection and virus reactivation from latency. Vpr is conserved amongst diverse simian immunodeficiency virus (SIV) strains; however, the evolutionary breadth of Vpr's nucleus remodeling activity has yet to be thoroughly characterized. Here, we investigate a diverse panel of 16 SIV Vpr isolates and demonstrate that 13 out of 16 are capable of significantly activating DDR signaling compared to control infected cells. Moreover, cells infected with these isolates also exhibit increased abundance of two histone marks associated with transcription and euchromatin formation, as well as increased activation of two transcription factors known to be critical for HIV-1 promoter activity. Furthermore, site-directed mutagenesis of a highly homologous SIV Vpr isolate that failed to engage the DDR response revealed previously uncharacterized amino acid residues required for HIV-1 Vpr DDR engagement. Finally, structural modeling and functional analyses revealed that phylogenetically diverse Vpr isolates from SIV African green monkey strains induce nucleus remodeling through an evolutionarily distinct set of amino acid residues. Together, these findings demonstrate that hijacking of DDR responses to promote remodeling of the nuclear environment is a broadly conserved Vpr function.

microbiology↗

bioRxiv2026

Dietary sugar type determines the response to protein restriction in females, but not males

Low protein (LP) diets improve metabolic health in rodents and humans. In rodents, LP diets are typically implemented by replacing protein with carbohydrates like sucrose or cornstarch, keeping diets isocaloric. However, humans can choose from many different types of carbohydrate, and how dietary carbohydrate quality - the precise composition of the dietary sugars - impacts the response to dietary protein remains largely unexplored. Here, mice were fed control (21% protein) or LP (7% protein) diets with four different carbohydrate sources: sucrose, a 1:1 glucose/fructose mixture, glucose, or fructose. While LP diets improved metabolic health across all groups in male mice, carbohydrate quality also significantly altered specific health outcomes, with fructose-fed mice having the lowest body weight and adiposity of all control diets. In female mice, responses to LP diets were influenced by carbohydrate quality, with certain sugars inducing a stronger metabolic response to LP diets than previously seen. Finally, in female APP/PS1 mice, a model of Alzheimer's disease, we find that although LP diets reduce A-beta; plaque burden irrespective of carbohydrate type, dietary sugar type does influence spatial memory. Together, these results demonstrate that while dietary protein is a critical determinant of metabolic and neurological health, carbohydrate quality influences these outcomes in a sex-specific manner.

physiology↗

bioRxiv2026

LRRC57/RABIN is a presynaptic inhibitor of Rab GTPases in glutamatergic neurons

Synaptic vesicle cycling, if not properly constrained, can result in excessive neurotransmitter release and subsequent neural pathology. Rab GTPases orchestrate synaptic vesicle trafficking through GTP-dependent interactions with effector proteins, but the restraining mechanism of these interactions is unknown. Here we identify LRRC57 (or RABIN for RAB INhibitor), a conserved brain-enriched protein in glutamatergic synapses that binds multiple GTP-loaded synaptic Rabs and competitively blocks access to their effectors. Loss of Rabin increased glutamate release, expanded vesicle pools, accelerated vesicle turnover, and produced circuit hyperexcitability with epileptiform activity, which was mitigated by an antiepileptic agent that targets presynaptic function. Conversely, overexpression of the Rabin gene suppressed neurotransmitter release and protected against induced seizures and persistent epileptiform discharges. Together, these findings define a noncanonical decoy-effector mechanism that constrains presynaptic Rab signaling to preserve excitatory circuit stability.

neuroscience↗

bioRxiv2026

Reward predictability shapes decision making states and exploitative control in marmosets

Animals navigating dynamic environments must transition between behavioral states dominated by exploitation of known rewards or exploration of alternatives. Understanding this flexibility requires characterizing not only choices and outcomes but also how reward information updates strategy. Common marmosets offer a unique combination of genetic tractability and frontal cortical complexity, yet scalable behavioral platforms for studying flexible decision making in this species remain limited. Here, we developed an automated home-cage touchscreen platform that enables voluntary behavioral testing across extended periods without water restriction. We trained marmosets on a series of reversal learning tasks of increasing complexity, culminating in a fully uncued probabilistic two-armed bandit task, and applied reinforcement learning models to examine how reward predictability shapes adaptive choice in marmosets. A Q-learning model with choice perseveration captured trial-by-trial dynamics, with choice variability consistent with stochastic sampling from the fitted policy. A trial-level behavioral state classifier identified distinct and persistent modes of exploitation and exploration that differed in their sensitivity to reward history and their transition structure. Comparing behavioral patterns within probabilistic and deterministic reward contingencies revealed that reward predictability reorganized state occupancy and sharpened error correction within predominately exploitative states, with animals responding to single negative outcomes more rapidly under deterministic feedback. These findings establish both a behavioral and computational framework to dissect the computations and circuits underlying adaptive decision making in marmosets.

animal behavior and cognition↗

bioRxiv2026

Competition against the intestinal microbiota selects for pathoadaptive traits in ESBL E. coli

The intestinal tract is a reservoir for Extended-Spectrum {beta}-Lactamase (ESBL)-producing Escherichia coli. Asymptomatic gut colonization by these pathobionts represents a major risk for extraintestinal infections. Despite clinical relevance, the genetic basis of gut colonization by ESBL E. coli remains poorly understood. Here, we determined how the microbiota shapes the fitness landscape of diverse ESBL E. coli strains, defining the functional requirements for intestinal colonization. In microbiota-depleted hosts, colonization relies mostly on metabolic functions. In contrast, in mice harbouring a microbiota, pathoadaptive functions associated with adhesion and biofilm formation are dominant determinants of E. coli fitness, together with accessory virulence functions. Consistent with these observations, experimental evolution in mice reveals convergent adaptation of ESBL E. coli to the presence of a complex microbiota through enhanced adhesion. These findings establish the microbiota as a major ecological driver of pathoadaptation in antibiotic-resistant pathobionts.

microbiology↗

bioRxiv2026

Scaling up polarization-sensitive optical coherence tomography to image the whole macaque brain

Polarization-sensitive optical coherence tomography (PS-OCT) is a label-free imaging technique that exploits birefringence to visualize myelinated axons at micrometer resolution. However, serial PS-OCT imaging has been limited to small volumes, including tissue blocks from larger species, owing to constraints in acquisition speed, system stability, and data processing. These limitations have prevented its application to whole-brain mapping in large mammals. Here we present a scalable PS-OCT acquisition system and computational pipeline for whole-brain imaging in the rhesus macaque. The framework integrates high-throughput serial imaging with automated reconstruction and processing, enabling volumetric imaging at micrometer-scale resolution across decimeter-scale brain volumes. Using this approach, we acquired two complete macaque brains at a voxel size of 5.5 x 5.5 x 3.4 m and an effective resolution of approximately 10 x 10 x 5.5 m, generating multi-terabyte datasets consisting of multiple contrasts including fiber orientation information. The datasets and associated processing tools are made publicly available. This platform establishes a method for large-scale, high-resolution mapping of white matter architecture in primate brains. The resulting datasets provide a reference for validating MRI models and support the development of neurotechnological applications, including deep brain stimulation, where accurate characterization of axonal organization is required.

neuroscience↗

bioRxiv2026

Structure of the proton-powered secretion motor at the heart of the bacterial flagellum

Bacterial type III secretion systems export proteins across the inner membrane using the proton-motive force (PMF), but how proton flow is coupled to opening of the export channel is unknownXX,XX. Here we present single-particle cryo-EM structures at 2.5-4.2 [A] resolution of the intact flagellar Export Apparatus from Salmonella enterica, obtained using optimized extraction conditions of the endogenous assembly that retain the complete transmembrane complex. The transmembrane domain of FlhA forms a nonameric funnel-shaped basket beneath the Export Gate, with each subunit harboring a buried pathway of conserved hydrophilic residues spanning the hydrophobic core of the membrane. FlhB is resolved for the first time within the intact gate, revealing helices that thread through the FlhA channel, completely sealing it at rest. A symmetry-free reconstruction captures one FlhA protomer hinged outward at the water-filled cavity, breaking the rotational symmetry. Together, these structures define the architecture of the proton-transducing element of the type III secretion system and suggest a rotary gating mechanism, with parallels to the F0 motor of ATP synthase, in which PMF-driven conformational cycling of FlhA drives regulated opening of the export channel.

microbiology↗

bioRxiv2026

Modeling CISH-KO TIL therapy: T-cell persistence and endogenous competition determine response in gastrointestinal cancer

BackgroundTumor-infiltrating lymphocyte (TIL) therapies have shown promise in murine models; however, early-phase clinical trials reveal substantial heterogeneity in treatment response across patients. Elucidating the biological drivers of this variability is critical for improving patient selection and therapeutic design. MethodsWe developed a mechanistic mathematical model of Cish-inactivated TIL therapy capturing interactions among tumor cells, endogenous TILs, and infused CISH-KO (Cish gene knockout) T-cells. The model was calibrated to longitudinal tumor size data from two murine studies and also fitted to clinical data from a first-in-human phase 1 trial enrolling 22 and treating 12 patients with metastatic gastrointestinal cancers (27 independently tracked tumor lesions). We used the calibrated model to identify patient-level determinants of response and to simulate combination and fractionation treatment strategies in silico. FindingsFour mechanistically distinct parameters emerged as dominant determinants of therapeutic outcome: CISH-KO T-cell persistence (r = 0.827, p < 10-4) was the single strongest predictor of therapeutic failure; other major determinants included endogenous T-cell proliferation rate (r = 0.648, p < 10-4) , CISH-KO T-cell killing rate, and intrinsic tumor growth rate. In silico simulations predicted that the addition of anti-PD-1 checkpoint blockade to standard-dose CISH-KO TIL therapy achieved comparable or superior tumor control relative to dose doubling, while maintaining lower peak T-cell level. Additionally, fractionating the same total dose across multiple delayed infusions further improved predicted tumor control by prolonging CISH-KO T-cell persistence, again without raising peak effector burden. InterpretationThe efficacy of current CISH-KO TIL protocols is influenced not only by the cytotoxic potency of infused T-cells, but also by their persistence within the tumor microenvironment and competitive pressure from the reconstituting host immune compartment. These findings support strategies that enhance the functional efficiency and prolong the availability of infused T-cells--such as concurrent PD-1 blockade or fractionated dosing of the same total cell product--rather than dose escalation alone and identify CISH-KO T-cell persistence and endogenous immune competition as actionable targets for improving TIL-based immunotherapies.

Cancer Biology↗

bioRxiv2026

Loss of PSAE redirects PGRL1 to photosystem I and enhances PGR5-dependent cyclic electron transfer in Arabidopsis

Electrons energised by light energy at photosystem I (PSI) primarily enter the linear photosynthetic electron transfer (LET) or cyclic electron transfer (CET) pathway. The balance between LET and CET activity is a crucial factor in the regulation of photosynthesis, since CET increases the stoichiometry of proton to electron transfer. The additional transmembrane proton gradient ({Delta}pH) triggers feedback control of light harvesting and electron transfer photoprotection via non-photochemical quenching (NPQ) and photosynthetic control (PCON), maintaining the balance between the output of the light reactions and the downstream metabolism. Previously, it was found that Arabidopsis mutants lacking the stromal-facing membrane-extrinsic PSAE subunit of PSI (psae1-3) show enhanced CET activity, decreased LET and lower PSI oxidation in excess light. Here we show that high CET activity in psae1-3 primarily depends on the Proton Gradient Regulation 5 (PGR5)-dependent CET pathway rather than the NDH-dependent pathway. High CET is abolished in the psae1-3 pgr5CAS double mutant. In the psae1-3 ndho double mutant the elevated proton flux and CET are largely maintained; however, CO2 fixation and growth are significantly worsened, indicating that NDH still makes a physiologically meaningful contribution in the psae1-3 background. Biochemical analysis revealed that PGRL1 is redistributed from its normal mixed membrane distribution to become predominantly PSI-associated in psae1-3, providing a physical basis for the enhanced PGR5-dependent CET. These results underscore the primary importance of the PGR5-dependent CET pathway for optimal photosynthesis and CO2 fixation in Arabidopsis and establish the organisation of the PSI acceptor side as a key regulatory determinant of the CET/LET balance. HighlightLoss of the PSI acceptor-side subunit PSAE enhances predominantly PGR5-dependent cyclic electron transfer and redirects PGRL1 to PSI in Arabidopsis, while NDH contributes to maintaining CO2 fixation when the PSI stromal side is disrupted.

plant biology↗

bioRxiv2026

Epithelial sensing of vitamin A shapes intestinal antimicrobial defense

Vitamin A is a central regulator of intestinal adaptive immunity, but its role in innate immunity is less defined. Antimicrobial proteins form a chemical barrier that protects the intestinal epithelium from microbial invasion. Among these, REG3 family lectins are induced by the microbiota, yet how nutritional cues intersect with microbial signals to control their expression remains unclear. Here, we show that dietary vitamin A promotes expression of REG3 antimicrobial lectins, including REG3G, in intestinal epithelial cells from both mice and humans. This induction is mediated by retinoic acid and requires retinoic acid receptor (RAR) signaling. Mechanistically, RARs bind directly to the Reg3g promoter adjacent to a STAT3 binding site. As STAT3 mediates microbiota-induced IL-22 signaling in epithelial cells, this arrangement provides a molecular framework for integrating nutritional and microbial inputs at the level of REG3G transcription. Extending these findings, we demonstrate that vitamin A-retinoic acid signaling similarly promotes expression of -defensin antimicrobial proteins. Together, these findings define a transcriptional mechanism by which vitamin A enhances epithelial antimicrobial defenses and strengthens mucosal innate immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/710399v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@18dd8aforg.highwire.dtl.DTLVardef@18cf2fdorg.highwire.dtl.DTLVardef@a4dc89org.highwire.dtl.DTLVardef@18aa9c7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIVitamin A promotes epithelial expression of REG3 antimicrobial proteins in the intestine C_LIO_LIRetinoic acid receptors (RARs) directly activate mouse Reg3g and human REG3G transcription C_LIO_LIRARs bind the Reg3g promoter adjacent to STAT3, integrating nutritional and microbial signals C_LIO_LIVitamin A-RAR signaling broadly regulates epithelial antimicrobial programs, including -defensins C_LI

immunology↗

bioRxiv2026

A Novel Pilus System in Candidate Phyla Radiation Bacteria

The Candidate Phyla Radiation (CPR) represents a bacterial superphylum estimated to include between 15-50% of all bacterial species, yet CPR bacteria remain challenging to culture and have been primarily identified through metagenomic approaches. Candidatus Mycosynbacter amalyticus is a parasitic CPR bacterium that specifically targets Gordonia amarae, an actinobacterium with a hydrophobic, mycolic acid-rich cell envelope. Previous cryo-electron tomography indicated that Ca. M. amalyticus assembles thin extracellular filaments that are important for host interaction, yet their molecular identity remains unknown. Here, we applied single-particle cryo-electron microscopy to determine high-resolution structures of these filaments (2.8 and 3.6 [A]), enabling the unambiguous identification of two previously uncharacterized pilins from the experimental density maps. These pilins, designated PamA and PamB, assemble into unique helical filaments distinct from all previously characterized filaments in both domain architecture and assembly mechanism. Despite low sequence identity, both PamA and PamB share conserved structural principles, including Ig-like folds and donor-strand exchange-mediated assembly. Phylogenetic analysis indicates that Pam pilins are exclusive to CPR bacteria, with homologues distributed predominantly across the classes Saccharimonadia and Microgenomatia. Analysis of the conserved pam operon identifies putative chaperones (PamC and PamD) and assembly factors structurally homologous to chaperone-usher pili components, suggesting an analogous but distinct assembly pathway. These findings expand the known diversity of bacterial pilus systems and demonstrate the power of structural approaches for characterizing uncharacterized proteins encoded within CPR genomes.

microbiology↗

bioRxiv2026

SPrOUT: A computational and targeted sequencing approach for mixed plant DNA identification with Angiosperms353

PremiseThe identification of plant species from mixed samples is crucial in various fields, including ecological surveys, conservation efforts, and food and dietary supplement safety. Traditional methods face potential challenges due to the high costs of DNA sequencing, inefficiencies in computational workflows, and incomplete sequence databases. Methods and ResultsThis study introduces a novel approach using the Angiosperms353 target sequencing kit for efficient taxonomic identification of angiosperm DNA in mixed samples. Our method assembles short pair-end reads for each mixed sample. Using gene sets of Angiosperms353 from 871 species, we apply phylogenetic inference to categorize the variance in phylogenetic distance across genes to identify the presence of taxa in mixed plant samples. The pipeline reaches 98.1 to 99.6% accuracy, 92.9 to 100% precision for identifying unknown taxa in in-silico mixes, and 90.7% accuracy and 98.0% precision for mock supplement mixtures. We explored the parameter cutoffs of the pipeline to offer an empirical range for different applications. ConclusionsThe Angiosperms353 and HybPiper assembly proved effective in sorting mixed plant DNA samples. Our method offers a framework for scientific and practical applications in plant species identification in both single and mixed samples.

bioinformatics↗

bioRxiv2026

Objective Quality Assessment for Precision Functional MRI Data

Precision functional mapping (PFM) enables individual-level characterization of brain network organization but requires substantially more and higher-quality fMRI data than is standard. Despite its growing use, objective criteria for data sufficiency and quality needed to ensure interpretable and replicable individual-level results remain unclear. Here, we introduce the Network Similarity Index (NSI), an objective measure of the extent to which functional connectivity (FC) patterns in an individual dataset express the large-scale network structure required for PFM. NSI captures the integrity of low-spatial-frequency, coherent network organization and denoising fidelity, and aligns closely with blinded expert assessments of PFM usability. NSI also accounts for variability in the rate at which FC becomes reliable across individuals. Here, we provide an open-source framework for NSI-based data quality evaluation and models for linking NSI values with expert-judged PFM suitability. This framework can also inform expected returns from additional data collection, enabling principled decisions about data sufficiency and replication in precision fMRI research.

neuroscience↗

bioRxiv2026

Environmental DNA reveals hidden eukaryotic diversity and fine-scale community patterns across seascape areas in the Northern Red Sea

Understanding how reef-associated biodiversity responds to seascape features is essential for monitoring and conserving coral reef ecosystems. Environmental DNA (eDNA) from seawater provides access to benthopelagic eukaryotic diversity but its relationship with benthic structure remains poorly understood. We conducted simultaneous assessment of benthopelagic eDNA derived from near-reef seawater and benthic photoquadrat surveys across 12 coral reef sites in the northern Red Sea, spanning three seascape regions: the Gulf of Aqaba, nearshore Northern Red Sea (NRS), and offshore NRS. We examined whether spatial patterns in benthopelagic eDNA communities were structured across regions and whether variation in benthic cover explained differences in eDNA-derived assemblages obtained from water samples. Benthopelagic eDNA revealed fine-scale spatial structuring across regions but showed non-significant whole-community correlation with benthic composition. When examined by major taxonomic groups, taxon-specific relationships emerged, with some taxa (i.e., Micromonas sp.) showing increasing relative abundances in reefs characterized by lower benthic complexity. While traditional photoquadrat surveys captured 72 benthic sessile taxa including dominant benthic groups (e.g. hard corals and algae) across four eukaryotic phyla, benthopelagic eDNA documented a broader range of eukaryotic diversity, including planktonic, cryptic, and low abundant taxa spanning 35 phyla. Notably, eDNA detected cryptic organisms overlooked by visual surveys, such as the giant clam Tridacna sp., even where present but not recorded in photoquadrats. Our results demonstrate that benthopelagic eDNA and visual surveys provide complementary perspectives on reef biodiversity. Rather than serving as a direct proxy for benthic structure, benthopelagic eDNA captures spatial and taxonomic patterns that may be overlooked by visual transects, supporting its use in seascape-scale biodiversity assessments and conservation planning efforts in dynamic and understudied reef systems.

ecology↗

bioRxiv2026

EpiBrain: the brain's epigenetic landscape in a snapshot

Epigenetics connect nature with nurture and can help explain how genes, experiences, and the environment influence gene expression to promote individual differences in behavior and mental health. However, how these perturbations induce epigenetic changes across various regions of the brain remains largely unknown. To visualize changes in brains epigenetic landscape at a glance, we develop EpiBrain, a method utilizing whole-brain imaging and registration to capture epigenetic changes in the entire zebrafish brain in a snapshot. Using this method, we uncovered brain-wide epigenetic changes induced by brain activity, chemical exposures, and genetic mutations. We found that gadd45b, an immediate early gene with epigenetic modulatory activity, mediates experience-induced epigenetic changes in the brain, and that these changes contribute to brain activity and behavioral alterations. EpiBrain enables rapid and unbiased assessment of whole-brain epigenetic changes following physiological and disease-relevant perturbations and facilitates mechanistic discoveries at the molecular level.

neuroscience↗

bioRxiv2026

Applying PCR cycle autonormalization to improve PacBio full-length 16S rRNA sequencing

The bacterial 16S rRNA gene is widely used to characterize host-associated and environmental microbiomes, most commonly through sequencing short hypervariable regions. Recent improvements in PacBio sequencing chemistry and concatenation approaches can now enable high-throughput, full-length 16S rRNA gene sequencing with high accuracy and depth. However, PCR-induced errors remain a major limitation, particularly for full-length amplicons where error accumulation may be elevated due to a five-fold increase in sequence lengths. These challenges are exacerbated when samples vary widely in microbial biomass, making it difficult to select a single optimal number of PCR cycles. Here, we evaluated the effects of PCR cycle autonormalization on PacBio Kinnex full-length 16S rRNA gene sequencing across seven agriculturally relevant specimen types. We compared conventional PCR protocols (20, 24, and 30 cycles) with an autonormalization approach in which individual reactions were terminated during exponential amplification based on real-time fluorescence thresholds. Autonormalized reactions consistently retained a higher proportion of sequences following denoising and chimera removal, exhibited fewer substitution-based errors, and produced more even read distributions across samples. Meanwhile, overamplified reactions (30 cycles) showed elevated error rates and higher sequence removal, particularly in samples with greatest microbial biodiversity. Importantly, PCR protocol had minimal effects on overall community composition relative to specimen types. These results demonstrate that PCR cycle autonormalization improves data yield for full-length 16S rRNA sequencing while maintaining robust inferences, while facilitating streamlined workflows for heterogeneous samples. ImportanceAmplicon-based sequencing of the 16S rRNA gene is foundational tool in microbiome research, yet PCR amplification remains a major source of technical error. This challenge is magnified for full-length 16S rRNA sequencing and for workflows that process specimen types with widely varying microbial biomass. Selecting a single PCR cycle number can result in under-amplification of samples with low 16S rRNA copies or over-amplification and error accumulation in high-titer samples. Here, we show that PCR cycle autonormalization, implemented using iconPCR, substantially improves PacBio full-length 16S rRNA sequencing by reducing PCR-induced substitution errors and chimeras while maintaining consistent biological inference. Autonormalization also enables blind pooling of amplicons without post-PCR quantification or equimolar normalization, reducing hands-on time and sample loss. These benefits make cycle autonormalization particularly valuable for high-throughput and production-scale sequencing applications handling diverse specimen types.

microbiology↗

bioRxiv2025

Suppression of non-canonical autophagy induces endothelial and cardiac dysfunction

BackgroundWhile roles for canonical autophagy in the pathophysiology of cardiovascular disease have been established, we have limited understanding of the non-canonical functions of autophagy proteins in this context. LC3-asssociated endocytosis (LANDO) is a novel non-canonical function of autophagy proteins, in which LC3 (microtubule-associated protein light chain 3) is conjugated to early endosome membranes using a portion of the canonical autophagy machinery, and functions in the endocytic recycling of several plasma membrane proteins. Here we ask whether perturbation of LANDO can promote cardiovascular pathogenesis. MethodsCardiac and endothelial functions were assessed by echocardiography and flow-mediated dilatation in mice lacking Rubicon (Rubcn-/-) or the WD domain of ATG16L1 (Atg16l1{Delta}WDki), two known effectors of LANDO. Mice with conditional depletion of Rubicon in the endothelial, myeloid and cardiomyocyte compartments were used as well. Three-dimensional murine cardiac vasculature leakiness was investigated by light sheet fluorescence microcopy. Endothelial activation induced by shear stress was characterized in vitro in primary endothelial cells isolated from murine lungs and human aortic endothelial cells. Associations between genetically predicted expression of candidate genes involved in LANDO and human cardiovascular parameters were studied in the Young Finns Study and the UK Biobank. ResultsCompared to littermate controls, young Rubcn-/- and Atg16l1{Delta}WDki mice showed a decrease in cardiac and endothelial functions, as did mice with endothelium-specific deficiency. VEGFR2 recycling to the plasma membrane and nitric oxide pathway during shear stress were disrupted in LANDO-deficient primary murine and human endothelial cells. Proteomic analysis in primary human aortic endothelial cells revealed an upregulation of intracellular hemoglobin subunit alpha (Hb-) upon shear stress, which was blunted when RUBCN was ablated. Genetic expression studies uncovered several candidate genes related to LANDO that correlated with cardiovascular parameters. These included the retromer complex subunit VPS29, disruption of which decreased Hb- expression levels in human endothelial cells. ConclusionsOur data support a pivotal role of non-canonical functions of autophagy proteins in recycling VEGFR2 upon shear stress activation in endothelial cells together with Hb- expression that may contribute to the etiology of cardiovascular diseases.

physiology↗