Biology subjects

Smith, K.

Publications and source records attributed to Smith, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Biomineralization from platelet δ-granules as the origin of cardiovascular calcification in humans and other animals.

Cardiovascular calcification is present in practically all cardiac diseases, which are the top killers in the world today1, and is particularly associated with atherosclerosis2, aortic stenosis3 and rheumatic fever4. If not the direct cause of death, calcification contributes considerably to complications that can lead to heart failure5. Nonetheless, the origins and mechanisms of cardiovascular calcification are still strongly debated3,6-12. Just over a decade ago, it has been reported that nano and micron-sized calcified spherical particles, formed from a single crystal of magnesium-containing calcium phosphate, were the first calcified structure that could be detected in cardiovascular tissue13. These particles were found even before any sign of cardiac disease was present and were present in all stages of cardiac diseases13. The ubiquity of these particles suggests their importance for the origins and development of cardiovascular calcific diseases. Here, we show that these particles originate from platelet {delta}-granules and are present in mammals, birds and lizards. Based on our results, we suggest a new mechanism for the origins of these particles, complementing existing models of cardiovascular calcification7,14, and bringing a new, early, and hitherto unaccounted key event in the process of cardiovascular calcification. This new mechanism model, along with a better understanding of the early stages of cardiovascular calcification, could open the path for the development of pharmacological prevention and treatment solutions for several cardiac diseases.

pathology↗

bioRxiv2026

APP Dosage and Extracellular Domain Variants Drive Distinct Defects in Neurogenesis modeled in Down Syndrome iPS cells

The amyloid precursor protein (APP) is heavily studied as the source of amyloid beta in Alzheimers disease (AD), however, the complex functions of APP remain poorly understood, as does the impact of APP dosage on neurodevelopment. Here we study APP specifically in the context of Trisomy 21. In an effort to reduce APP dosage in trisomy 21 iPSCs, we generated trisomic isogenic lines which vary in APP dosage, including a full APP knock-out line, as well as lines carrying mutations of the APP extracellular domain. We used a panel of these lines to study potential impacts of APP dosage or structure on two distinct steps of neurogenesis in trisomic cells: 1) terminal differentiation of human neuro-progenitor cells (NPC) to post-mitotic neurons and 2) neuron structure as reflected in neurite outgrowth. Complete loss of APP causes marked defects in each of these two distinct steps, reducing both the terminal differentiation of NPCs to neurons, and proper neurite development for extended neuron structure. Hence, APP is necessary for both aspects of normal neurogenesis. Further analyses of the null and other mutant lines indicate that APP likely impacts these two distinct steps by two different mechanisms. Collective results suggest that the reduced terminal differentiation of NPCs reflects an effect of APP dosage, whereas the defects in neurite extension are due to structural mutation of the APP extracellular domain. Absence of APP or reduced (monosomic) APP dosage prolonged the cycling of trisomic NPCs, which is known to be regulated by Notch signaling. APP and Notch are the main targets of gamma-secretase cleavage, hence we hypothesized that APP dosage may impact neurogenesis indirectly, potentially via effects on Notch signaling. To test this, we treated NPCs with Compound E which inhibits gamma-secretase (and Notch signaling); results show this restored levels of neurogenesis in APP depleted lines, supporting an indirect effect of APP dosage. In contrast, results indicate that disruption of APP extracellular domain integrity impacts neurite extension via a more direct role of APP in neuron structural maturation. This study describes a resource of well-characterized APP mutant isogenic DS iPSC lines, implicates a dynamic interplay between APP dosage and Notch signaling, and raises new questions about the impact of APP dosage in orchestrating neural progenitor fate decisions during human brain development, specifically in the context of trisomy 21.

developmental biology↗

bioRxiv2026

Remodeling of the hepatic circadian transcriptome across the estrous cycle

The circadian clock system drives rhythms in gene expression, tailoring an organism's behavior and physiology to the ~24-hour day-night environmental cycle. The mechanisms underlying this system are believed to be largely the same between adult males and females, but recent findings are starting to challenge this notion. Menstrual/estrous cycles (e-cycles) in females modulate a variety of circadian-controlled behaviors, but how they may interact with rhythmicity at the system level are an unknown but vital dimension in circadian biology in females. To start bridging this gap, we explored the circadian transcriptome of the mouse liver across the four phases of the free-running e-cycle. Much to our surprise, we found that the e-cycle imposes a ~4-day rhythm in the 24-hour rhythmicity of a considerable proportion of the circadian transcriptome, with the largest differences in rhythmicity occurring between pre- and post-ovulation phases. These differences also appear highly time-of-day dependent, and gene promotor analysis suggests that both e-cycle and sex dependent rhythmicity may be due to relatively simplistic mechanisms, possibly involving the action of core circadian transcription factors on output genes. Taken together, this study establishes that the estrous cycle modulates circadian gene expression in the liver, suggesting that circadian system has evolved specific mechanisms tailored to the different needs of males and females in their normal daily environments.

Systems Biology↗

bioRxiv2026

Human CD8-iTreg are potent GVHD suppressors and tumoricidal effectors by release of Granzyme-K+ Supramolecular Attack Particles

Regulatory CD8+ T-cells (CD8+ Treg) are a distinct yet understudied T-cell subset capable of simultaneous immunosuppression and cytolysis. Here, we characterized induced human CD8+ Treg (CD8-iTreg) generated from peripheral blood CD8+CD25- T-cells using anti-CD3e mAb-loaded artificial antigen presenting cells, IL-2, TGF{beta}, and Rapamycin. These CD8-iTreg differentiated into a stable, highly proliferative bifunctional population with suppressive activity comparable to CD4-iTreg while retaining cytolytic capacity similar to conventional CD8 cytotoxic T lymphocytes (CTL). Multi-parameter spectral flow cytometry and single-cell RNA-seq revealed a distinct immunoregulatory signature: a predominantly Treg-like profile marked by tissue-residency marker CD103 with increased canonical Treg markers (FoxP3, HELIOS, CD25, CD39, CTLA-4, CCR4, and IL-10) and reduced pro-inflammatory cytokines. A unique cytotoxic program was marked by elevated Granzyme-K (GzmK) and Thrombospondin-4 (Tsp-4), a thrombospondin family extracellular matrix glycoprotein upregulated in activated CD8+ T-cells. Cytolysis was primarily mediated by Perforin (Prf) and multiple Granzymes packaged into Tsp-4 supramolecular attack particles (SMAPs), with GzmK contributing to both cytotoxic and suppressive functions. After anti-CD19scFv CAR (CAR19) transduction, CAR19+ CD8-iTreg showed superior in vivo anti-tumor efficacy compared with CAR19-CTLs, significantly reducing tumor burden and prolonging survival in a CD19+ Nalm-6 human leukemia xenograft model while maintaining low pro-inflammatory cytokine production. In a xenogeneic graft-versus-host disease (GVHD) model with residual human leukemia, CAR19 CD8-iTreg inhibited GVHD lethality and controlled tumor growth without increasing systemic inflammation. Together, these findings support CD8-iTreg-based CAR therapies as a strategy to retain potent anti-leukemic activity while limiting inflammatory toxicities of conventional CAR T-cells, properties particularly beneficial in treating auto- and allo-immune diseases. One sentence summaryCD8-iTreg drive parallel tumoricidal and immunoregulatory functions mediated by releasing Tsp-4+ SMAPs containing granzyme K.

immunology↗

bioRxiv2026

Modular Assembly of an Infectious Clone of the Akata strain of EBV using Synthetic Genomics Methods in Yeast

We have used synthetic biology recombination methods in yeast to build herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) genomes from multiple fragments. The genomes were built using transformation-associated recombination (TAR) in yeast, by virtue of overlapping sequences between the different fragments. This study demonstrates the successful assembly of the Epstein-Barr virus (EBV) genome. We used as the model genome, the Akata Burkitts lymphoma genome, specifically the BX1 genome which encodes a neomycin selectable marker and a GFP expression cassette in the BXLF1 region. The 171.3 kb genome was first deconstructed into 11 fragments in silico, each having 80 bp overlapping sequence between the fragments. The 11 fragments (TAR 1 to TAR 11) were cloned using TAR in yeast, analyzed by restriction enzyme analyses and Nanopore sequencing to validate the cloned fragment. The EBV genome was built in two stages: TAR fragments 1 to 6 and TAR fragments 7 to 11 were assembled to generate two half-genomes. The whole genome (TAR 1-11) was then assembled by joining TAR 1-6 with TAR 7-11. Complete EBV genomes were examined by PCR assays and restriction enzyme analyses and then transfected into HEK-293 cells to generate virus producer cell lines. The HEK-293 cell clones were tested for virus production following lytic induction using baculovirus transduction of Zta, Rta and glycoprotein B (BALF4). The supernatants from these induced cells were harvested and used to infect Raji cells. This analysis revealed a significant number of cells displaying strong GFP fluorescence indicative of infectious virus. We used this supernatant virus to infect primary B cells and were able to derive lymphoblastoid cell lines (LCL) indicative of the ability of this virus to transform B cells. We tested this method for engineering different mutations. Two mutations were made, one in Zta and the other in the small capsid protein (BFRF3). Mutations were engineered in the TAR plasmid in which the genes reside and after sequence validation, assembled into the TAR 1-6 half genome and then the TAR 1-11 genome, which was used to generate HEK-293 cell clones. For the {Delta}Zta cell lines, we could detect virus in the supernatants only if baculovirus expressing Zta in trans was included, this {Delta}Zta EBV virus could transform B cells. The small capsid protein (BFRF3) decorates the capsid shell and is required for capsid assembly in a self-assembly system. When the HEK-293 cell clones were induced using co-expression of Zta, Rta and gB, no virus was detected in the culture supernatants. However, if we provided BFRF3 in trans using baculovirus expressing this protein, virus was detected in the supernatants. This provides the first report of the essential role of the small capsid protein in EBV-infected cells.

Synthetic Biology↗

bioRxiv2026

Integrative multi-omic analysis identifies tumor-intrinsic p38 as a driver of immune exclusion in human epithelial cancers

Patients with tumors not responding to immune-checkpoint inhibition (ICI) often harbor a non-T cell-inflamed tumor microenvironment, characterized by the absence of IFN-{gamma}-associated CD8+ T cells and dendritic cell activation. While the role of p38 mitogen-activated protein kinases (MAPKs) in regulating dendritic and myeloid cells is established, the tumor-intrinsic immunomodulatory function of p38 remains underexplored. Here, we identify tumor cell-intrinsic p38 signaling as a target candidate associated with immune exclusion and reduced immunotherapy response. In human papillomavirus-negative head and neck squamous carcinoma (HNSCC), molecular analysis of 395 tumor tissues revealed a p38-centered network enriched in non-T cell-inflamed tumors. Multi-cancer single-cell RNA sequencing analysis of over 200,000 cells further identifies p38 activation as a potential immune-exclusion program across multiple epithelial tumor types, including HNSCC and lung squamous cell carcinoma (LUSC), supported by tissue validation in [~]250 human biospecimens using multispectral imaging and digital spatial profiling. Functional studies demonstrate that p38 knockdown or pharmacologic inhibition in HNSCC and LUSC cell lines increases T cell migration, with CXCL16 identified as a chemokine mediator in vitro; neutralization of CXCL16 attenuated this effect. Together, these findings identify tumor-intrinsic p38 activation as a driver of immune exclusion in epithelial cancers and support its potential as a therapeutic target to overcome immunotherapy resistance.

Cancer Biology↗

bioRxiv2026

An Integrating Sphere Based Method for Quantifying RNA Encapsulation Efficiency in Lipid Nanoparticles without Lysis

Encapsulation efficiency (EE) is a critical quality attribute of lipid nanoparticle (LNP)-RNA formulations, determining the fraction of active RNA payload available for delivery and influencing therapeutic potency, dose accuracy, and product consistency. Conventional fluorescence-based EE assays, such as RiboGreen (RG), require detergent-mediated LNP lysis, which can introduce systematic bias arising from incomplete particle disruption and additional sample handling steps. Here, we report a lysis-free method for direct EE determination using Scatter-Free Absorption Spectroscopy (SFAS) within an integrating sphere. In this approach, RG fluorescence appears as a quantitative negative absorbance feature in the SFAS spectrum, enabling direct quantification of free RNA from intact LNP suspensions in a single measurement. Applied to SM-102 LNPs loaded with polyA, the method yielded an EE of 97%, in close agreement with conventional plate-reader measurements (98%). This approach provides a practical, formulation-independent, and potentially more robust strategy for standardized routine EE analysis and LNP quality-control workflows.

molecular biology↗

bioRxiv2026

Scalable machine learning improves resistance prediction and identifies novel determinants in Mycobacterium tuberculosis

Multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (MTB) represents a growing global health crisis, characterized by limited treatment options and high mortality rates. Rapid and accurate prediction of resistance profiles is critical to guide effective therapy and curb transmission. Whole-genome sequencing (WGS) offers promise for individualized resistance profiling, yet existing computational tools remain constrained by predefined mutation catalogs and prohibitive resource requirements for large-scale analyses. Here, we present AURA, a GPU-accelerated, pangenome-scale machine learning framework for de novo resistance prediction. Trained on 12,185 globally diverse MTB isolates, AURA predicts resistance to 13 first-line, second-line, and repurposed antibiotics with high precision and identifies 59 novel resistance-associated loci, including variants in katG, pncA, rpoC, and members of the PE/PGRS gene family. By enabling model training on an unprecedented genomic scale, AURA provides new insights into the genetic architecture of resistance and establishes a scalable platform for precision-guided therapy and global surveillance of MTB.

bioinformatics↗

bioRxiv2026

Selective conservation of symbiont cell-surface glycans across generations in a vertically transmitting coral

Coral resilience under climate change depends on the stability of coral-Symbiodiniaceae symbioses. While vertically transmitting corals inherit symbionts directly from parental colonies, the extent to which symbiont cellular traits are conserved across life stages remains unclear. Here, we examined cell-surface glycan profiles of Symbiodiniaceae in parental colonies and eggs of the coral Montipora capitata. Glycan signatures were structured by symbiont genus and differed between life stages, with mannose/glucose- and galactose-containing glycoproteins as primary drivers of variation. Despite life-stage differences, parent-offspring comparisons revealed significant conservation of glycan profiles, indicating intergenerational transmission of symbiont cellular traits that differed between Cladocopium and Durusdinium and were driven by distinct glycan classes. These results suggest that vertical transmission preserves key recognition-relevant glycans while allowing flexibility in other symbionts surface traits, providing a mechanistic basis for symbiosis stability.

cell biology↗

bioRxiv2026

Whole genome-based Characterization of Virulence and Antimicrobial Resistance Determinants in Clinical Campylobacter jejuni Isolates from Minnesota, 2018-2021

Campylobacter jejuni is a leading cause of foodborne gastroenteritis globally and is classified by the CDC as a serious public health threat due to increasing resistance to fluoroquinolones and macrolides. This study used whole-genome sequencing to characterize the virulence and antimicrobial resistance profiles of 2,783 clinical C. jejuni isolates collected from Minnesota residents from 2018 through 2021. More than 90% of the isolates had genes related to stress defense (rpoN and htrB), cytolethal distending toxin (cdtA, cdtB, and cdtC), and cell adhesion and invasion (ciaB, cadF, and flaC). A diverse array of antimicrobial resistance genes was detected, with beta-lactam resistance genes having a particularly high prevalence. The gyrA point mutation associated with quinolone resistance was present in 29% of isolates. To evaluate the correlation between genotypic and phenotypic antimicrobial resistance profiles, the antimicrobial susceptibility testing results from a subset of isolates were compared with genotypic resistance profiles. Results showed a strong overall correlation, particularly for tetracycline and quinolones, though 24 discrepancies were detected. In the majority of discrepancies (n=21), genomic antimicrobial resistance markers were absent in isolates that were phenotypically resistant, suggesting possible unknown resistance mechanisms or limitations in current sequencing methods. The remaining three discrepancies occurred in isolates that had the tet(O) resistance gene but were susceptible to tetracycline phenotypically. These findings highlight the value of whole genome sequencing in improving antimicrobial resistance surveillance and understanding virulence factors in C. jejuni, supporting its integration into routine monitoring practices to better manage and understand antimicrobial resistance in foodborne pathogens.

microbiology↗

bioRxiv2026

Physiologically relevant media are associated with overlapping metabolic responses in primary human hepatocytes and Huh7 cells

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is challenging to study in vivo in humans and in vitro models are limited. Although primary human hepatocytes (PHHs) are considered the gold-standard, immortalized hepatic cell lines are utilised due to scalability. This study compared the metabolic responses of PHHs with our Huh7-based model cultured in physiologically-relevant fatty acid (FA) mixtures. MethodsPHH and Huh7 cells were treated with 2% human serum, sugars and FAs enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 4 or 7 days, respectively. Stable isotope tracers investigated basal metabolic changes in response to treatment. Cell viability, media biochemistry, intracellular metabolism, lipid droplet morphology and gene expression were quantified. ResultsHuh7 cells had greater viability than PHHs, while NEFA uptake and triglyceride secretion were similar. OPLA and POLA increased large lipid droplets in Huh7 cells, whereas only OPLA produced comparable effects in PHHs. Despite higher baseline TG in PHHs, both models showed similar lipid composition, de novo lipogenic responses, and glycogen levels. Compared to Huh7 cells, PHHs exhibited higher 3-hydroxybutyrate, lower lactate, reduced glucose uptake, and donor-dependent transcriptomic variability. ConclusionsHuh7 cells are metabolically adaptable and when cultured in physiologically-relevant media, produce metabolic readouts similar PHH cells.

cell biology↗

bioRxiv2026

GC-MS Based Comparative Metabolomics of Host Plants and Insect Gut Extracts

Herbivorous insects exhibit pronounced metabolic plasticity, enabling adaptation to diverse host plants which complicates pest management strategies. Understanding how plant metabolites are transformed during insect digestion is critical for elucidating plant-insect interactions. We combined gas chromatography-mass spectrometry (GC-MS)-based untargeted metabolomics with UV-Vis quantification of total phenols and flavonoids to compare host plant tissues and insect gut extracts in three systems: fall armyworm (Spodoptera frugiperda) larvae on maize (Zea mays), silkworm (Bombyx mori) on mulberry (Morus alba) and desert locust (Schistocerca gregaria) on wheatgrass (Triticum aestivum). UV-Vis analysis revealed consistent enrichment of total phenols in insect gut relative to host plants ([~]1.4-.35-fold), while flavonoids were reduced ([~]2-7-fold). GC-MS analyses showed clear separation of gut and plant metabolomes, with <35% shared metabolites and the majority unique to insect guts. Insect extracts were enriched in hydrocarbons, fatty acids, sterols, and terpenoid derivatives, reflecting extensive biochemical transformation. Sex-specific metabolite differences were observed in silkworm and desert locust guts despite identical diets. These findings indicate that herbivorous insect guts act as dynamic biochemical reactors, selectively restructuring plant metabolomes through flavonoid turnover, phenolic enrichment, sterol bioconversion, and lipid assimilation. This conserved metabolic strategy across phylogenetically distinct insects underscores adaptive mechanisms for nutrient acquisition and detoxification, a pathway that can be exploited in plant pest control.

plant biology↗

bioRxiv2026

Immune checkpoint inhibitors amplify type 2 immune mediated repair bypro-regenerative scaffolds

Extracellular matrix (ECM) scaffolds induce type 2 immunity to promote repair. Here, we show that immune cells recruited to ECM-treated murine muscle injuries and clinical soft tissue defects express immune checkpoints. Specifically, TH2 cells and regulatory T cells (Tregs) increase LAG3 expression, while macrophages express PDL2. TCR analysis and a triple-reporter strain for interleukin (IL)-13 and Treg fate-mapping suggest that Tregs in ECM-treated wounds transition into TH2-like exTregs that express LAG3. Immune checkpoint inhibition (ICI) significantly stimulated type 2 immunity in ECM-treated wounds, including increased TH2 cells, Treg transition to TH2-like exTregs, and pro-regenerative macrophages. Moreover, ICI enhanced muscle repair and reduced fibrosis in ECM-treated wounds. Collectively, these findings show Treg/TH2 plasticity in wound healing and introduce a novel ICI application to enhance immune-mediated regeneration.

bioengineering↗

bioRxiv2026

From SCUBA to spectra: Broadly applicable methods for coral metabolomics research

Corals represent a complex assemblage consisting of a host cnidarian, symbiotic dinoflagellate microalgae, and associated microbiomes and viromes, collectively called the coral holobiont. Corals are foundational to tropical reefs, yet their global decline due to climate change and other stressors creates an uncertain future for this valuable ecosystem. Metabolomics is a powerful means to unravel biochemical interactions within the holobiont that underpin coral resilience and adaptation. However, the remote nature of reefs and the analytical demands of this technique often limit its application. Untargeted metabolomics presents analytical challenges that are amplified in complex samples like corals, such as identifying the biological source of metabolites. Here, we evaluate how different sample fixation methods and time delays before storage--unavoidable in field contexts--affect coral metabolome profiles. We further present a framework for mapping metabolites in holobiont samples to their coral host and algal symbiont origins and introduce a spectral library to improve and automate annotation of coral lipids. Additionally, we demonstrate how single samples can be used concurrently for metabolomics, DNA amplification, and proteomics. Together, our study provides a streamlined, field-adaptable workflow for coral metabolomics that enables larger-scale studies and broader adoption of metabolomics in coral reef research and conservation.

biochemistry↗

bioRxiv2026

SCALPEL: A pipeline for processing large-scale spatial transcriptomics data

Spatial transcriptomics enables the precise mapping of gene expression patterns within tissue architecture, offering unprecedented insights into cellular interactions, tissue heterogeneity, and disease pathology that are unattainable with traditional transcriptomic approaches. We present a tool for processing spatial transcriptomics data, SCALPEL (Spatial Cell Analysis, Labeling, Processing, and Expression Linking). SCALPEL is specifically designed to support the analysis of large, atlas-level datasets. Our new workflow features advanced 3D segmentation optimized for dense and heterogeneous tissues, refined filtering criteria, and transcriptome-based doublet detection to remove low-quality or artifactual cells. Cell type label transfer from existing taxonomies is further improved through updated filtering thresholds. Spatial domain detection is incorporated to capture local transcriptomic organization, and tissue sections are registered to the Allen Mouse Brain Common Coordinate Framework version 3 (CCFv3) for precise anatomical alignment. Genome-wide expression imputation from single-cell RNA-sequencing (scRNAseq) further enriches the dataset. Crucially, we benchmark the performance of this updated pipeline against a previously published version of our whole-mouse-brain (WMB) dataset (Yao et al., 2023b), demonstrating substantial improvements in cell number, expression profile clarity, and spatial registration. These advances provide a robust foundation for downstream spatial analyses and set a new standard for large-scale spatial transcriptomics studies.

bioinformatics↗

bioRxiv2026

Characterizing cellular subpopulations critical to treatment response in autoimmune diseases

Single-cell RNA sequencing provides a powerful approach for characterizing cell types, states, and lineages within heterogeneous tissues. However, identifying cell subpopulations that drive phenotypes, particularly treatment responses, remains a challenge. In this study, we performed comprehensive analyses to identify treatment response-associated cell subpopulations in autoimmune diseases by mapping bulk response information onto single-cell data. We integrated single-cell and bulk biopsy data from 314 responders and 619 non-responders treated with six therapeutics targeting tumor necrosis factor (TNF), integrin, or interleukin pathways in inflammatory bowel diseases (IBD) and psoriasis (PsO). Our analyses captured 128,428 interactions among 3,617 differentially expressed genes (DEGs), 852 pathways, and nine cell types spanning immune, stromal, and epithelial compartments. The importance of epithelial barrier integrity and enterocyte-mediated permeability in responses and inflammatory signaling in macrophages in non-responses in all tested therapies were highlighted by the presence of shared DEGs and pathways in both Crohns disease (CD) and ulcerative colitis (UC). In PsO, keratinocytes drove non-response to integrin-targeting therapies via disrupted adhesion, migration, and sustained epidermal inflammation. Additionally we introduce SCTRAD (https://immbioinfoabbv.shinyapps.io/SCTRAD/), an online web-based platform that allows exploration of mechanisms that influence the heterogeneity of cellular responses through DEGs and pathways at the single-cell level, as well as analysis of the cell-type-specific drug-related gene network. Our findings provide a comprehensive analysis of the role of cellular heterogeneity in treatment outcomes and for advancing precision medicine strategies in autoimmune diseases.

immunology↗

bioRxiv2025

PKN2 regulates cell-junctions to limit colitis and colon tumour formation

Intestinal barrier failure is a defining feature of inflammatory bowel disease and a key driver of inflammation-associated colorectal cancer. However, the epithelial mechanisms that preserve barrier stability during inflammatory stress remain incompletely defined. Here, we identify the Rho-regulated kinase PKN2 as a non-redundant safeguard of tight junction integrity and a candidate tumour suppressor in colitis-associated colorectal cancer. Conditional PKN2 deletion in mice sensitised the colon to inflammatory injury and significantly increased adenoma formation in an inflammatory cancer model, with tumour burden tightly correlating with colitis severity. Mechanistically, PKN2 localises to tight junctions and is required to stabilise barrier integrity during injury in mouse and organoid models. PKN2 loss induces transcriptional programs conserved in human inflammatory bowel disease, where reduced PKN2 expression associates with increased disease severity and altered therapeutic response. Notably, PKN2 is encoded within genomic loci previously linked to intestinal inflammation susceptibility and tumour suppression in both humans and mice. Our findings identify PKN2 as a functional effector underlying these phenotypes and demonstrate that even heterozygous loss is sufficient to confer heightened sensitivity to inflammatory injury and tumour initiation, positioning PKN2 as a central regulator of tight junction stability that shapes disease severity, treatment response and cancer risk.

cell biology↗

bioRxiv2025

Human C. difficile-specific memory B cells encode protective IgG1 despite predominance of non-neutralizing antibodies.

Clostridioides difficile remains a common source of nosocomial infection resulting in a wide range of clinical outcomes and for which there is no vaccine and limited therapeutic options. C. difficile Toxin B (TcdB)-specific IgG is the best correlate of protection against severe and recurrent disease. However, there are very few therapeutic IgG fully human monoclonal antibodies (hmAbs) that have shown efficacy thus far. We therefore hypothesized that the memory B cell (Bmem) compartment of individuals who have recovered from infection may be dominated by non-protective IgG molecules but encode some antibodies that are protective. We therefore produced hmAbs from a library of Bmem-encoded IgG1 sequences. Most TcdB-specific hmAbs displayed low affinity binding and poor neutralization of TcdB in vitro while a few hmAbs had high affinity binding and good TcdB neutralization. The results correlated with in vivo studies in which high affinity, TcdB-neutralizing hmAbs provided moderate protection of C57Bl/6 mice against C. difficile disease. Similar protection was observed in Tg32 mice expressing the human FcRn transgene, indicating that gut delivery of hmAb did not account for limited efficacy. Although non-protective antibody sequences dominate the repertoire, human Bmem cells may be a good source of therapeutic hmAbs for C. difficile treatment.

immunology↗