Biology subjects

Martin, I.

Publications and source records attributed to Martin, I..

At least 19 recordsLinked to original sources

bioRxiv2026

Growth factor-free synthetic matrices reveal intrinsic skeletal progenitor competence

Clinical translation of human skeletal progenitor cells has been limited by the inability to reliably predict their regenerative performance, as current characterization methods rely on phenotypic markers or instructive differentiation assays that obscure intrinsic cell function. Here we demonstrate that human skeletal progenitor competence can only be revealed within a precisely defined and growth factor-free synthetic microenvironment. By independently tuning matrix mechanics, degradability, adhesion, and cell density within a fully synthetic poly(ethylene glycol) hydrogel, we create a permissive niche that enables human bone marrow stromal cells (hBMSCs) to undergo spontaneous bone formation in vivo without osteoinductive supplementation. These exogenous growth factor-free conditions expose donor-specific differences in bone forming capacity that are otherwise masked. Strikingly, supplementation with even low concentrations of BMP-2 abolished inter-donor variability, directly demonstrating that osteoinductive factors override intrinsic progenitor competence. Single-cell transcriptomics, proteomic profiling, and longitudinal in vivo analyses converge to show that high performing donors are defined by chondrocyte-primed transcriptional states and coordinated extracellular matrix (ECM) remodeling programs that establish a pro-osteochondral niche and drive bone formation via endochondral ossification. In contrast, low performing donors fail to initiate this cascade despite comparable phenotypic profiles and proliferation rates. Together, these findings implicate matrix context as a critical regulator of stem cell competence. Within this permissive synthetic niche, intrinsic ECM remodeling capacity emerges as a defining feature of donor-specific osteogenic potential, one that remains invisible to conventional assays. These matrices thus provide a functional platform for prospective stratification of skeletal progenitor cells, with direct implications for donor selection in regenerative medicine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/731042v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@6fa9ceorg.highwire.dtl.DTLVardef@f51dddorg.highwire.dtl.DTLVardef@7bb040org.highwire.dtl.DTLVardef@706a36_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Preserved but not functional: growth biology shapes connectivity resilience in meningioma and glioma

Brain tumors disrupt neural connectivity, but the nature of this disruption depends on tumor growth biology. Here, we analyze pre-operative structural connectivity (SC), functional connectivity (FC), and generalized effective connectivity (GEC) in 14 meningioma patients, 10 glioma patients, and 10 matched controls to characterize how extra-axial and intra-axial tumors differentially affect brain networks. We introduce FC resilience, the relative preservation of functional connectivity in structurally damaged regions, and find that meningioma patients exhibit significantly higher FC resilience than glioma patients, with SC-dominant damage and preserved neural activity in damaged regions. Glioma patients show balanced SC-FC damage and degraded neural activity, consistent with infiltrative destruction of both white matter and neural substrate. Connectivity damage is not localized to the tumor vicinity and is non-randomly distributed across functional networks, with distinct propagation patterns: glioma SC damage clusters along white matter pathways, while meningioma SC damage preferentially targets Limbic and Default networks. Network topology analysis reveals that more segregated functional and effective connectivity, particularly higher modularity, predicts FC resilience in meningioma patients but not in glioma patients, while structural connectivity topology shows no predictive value. Non-equilibrium dynamics, quantified via the Fluctuation-Dissipation Theorem, are elevated in damaged regions of meningioma patients, serving as a dynamical marker of structural damage rather than an independent compensatory mechanism. Clinically, higher FC resilience in glioma patients is associated with worse cognitive outcomes, suggesting that preserved FC without an intact neural substrate does not reflect genuine functional preservation. These findings demonstrate that the interpretation of functional connectivity resilience depends fundamentally on tumor type and its underlying growth biology.

neuroscience↗

bioRxiv2025

Bioengineered human bone-mimetic niche compositions regulatebreast cancer cell quiescence and therapy response

Disseminated tumor cells (DTCs) in the bone are widely regarded as the cellular seeds of late metastatic relapse in estrogen receptor-positive (ER+) breast cancer (BC). However, how distinct bone compartments influence BC cell quiescence and endocrine therapy response remains unclear. Mechanistic insight has been hindered by limited access to human bone samples and by the lack of relevant human models that permit controlled manipulation of stromal compartments. Here, we developed a fully human, 3D bone-mimetic system for modular, controllable assembly of engineered osteoblastic (eON), vascularized (eVN), and vascularized osteoblastic (eVON) niche compositions. These niches were generated by perfusion culture of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and/or human adipose tissue-derived stromal vascular fraction (hAT-SVF) cells within porous ceramic scaffolds. The resulting tissue microenvironments were then used as a substrate for the culture of an ER+ BC cell line, expressing a mutant reporter of p27 to monitor the quiescent status. We found that the eON enhanced BC cell proliferation, whereas the eVN was enriched in quiescent BC cells positive for NR2F1, a dormancy-associated transcription factor, and located near perivascular elements. Treatment with the selective ER degrader fulvestrant reduced BC cell numbers in vascularized niches (eVN and eVON) but not in eON, despite comparable receptor degradation. In summary, we developed a modular human platform for dissecting niche-specific regulation of BC quiescence, proliferation, and endocrine therapy response. The system can be further used to investigate perivascular niche-dependent mechanisms of BC cell dormancy and to guide the development of therapeutic strategies preventing recurrence in ER+ BC patients.

bioengineering↗

bioRxiv2025

The Lipid-binding PX Domain of RRC-1 (ARHGAP32/33) is Required for Optimal Assembly and Function of Integrin Adhesion Complexes at the Muscle Cell Boundary in C. elegans

Integrin adhesion complexes (IACs) are a network of many proteins that serve as anchors of the cell to the extracellular matrix (ECM). In muscle, IACs located at costameres, also serve to transmit the force of muscle contraction to the outside of the cell. We have reported that IACs, which are found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the RacGEF PIX-1 for their proper assembly or maintenance. We have reported that a RacGAP for the PIX pathway is RRC-1, is in a complex with PIX-1, and that RRC-1 is required for assembly or maintenance of IACs at MCBs. Our previous studies suggested that RRC-1 might be associated with the muscle cell membrane, and here we present evidence that this occurs via its PX domain, a domain that is known to bind to membrane phosphoinositides (PIPs). We predict the existence of a PX domain based on bioinformatic analysis and AlphaFold3, which includes conserved residues characteristic of most PX domains and a PIP binding site. This region of RRC-1 binds to phosphoinositides in vitro. Analysis of a nematode strain that has an in-frame deletion of the PX domain, indicates that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffold protein GIT-1. Lastly, we show that the overexpression of the full length RRC-1, but not RRC-1 with an in-frame deletion of its PX domain, results in reduced accumulation of IAC components and reduced whole animal movement. Our study highlights the importance of RRC-1s lipid interactions at the cell membrane for proper assembly and function of IACs in C. elegans muscle. Article SummaryIntegrin adhesion complexes (IACs) facilitate the transmission of force of muscle contraction to the outside of the cell. IACs, found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the PIX-1 (a RacGEF) signaling pathway for their assembly or maintenance. A RacGAP for the PIX pathway is RRC-1. Here, we show that RRC-1 has a PX domain that binds to membrane phosphoinositides. We also show that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffolding protein GIT-1.

cell biology↗

bioRxiv2025

Rapid identification of microbial pathogens and antimicrobial resistance from bloodstream infections using long-read sequencing

The gold standard for bloodstream infection (BSI) diagnostics involves culturing positive blood cultures (BC) using phenotypic methods for organism identification and antimicrobial resistance (AMR) testing, which can take up to five days. However, it is crucial to optimize antimicrobial therapy as soon as possible to reduce morbidity and mortality. We present a novel laboratory and bioinformatic workflow to rapidly identify bacterial and fungal organisms and AMR determinants from positive BCs using Oxford Nanopore Technologies long-read sequencing. Using a robust clinical sample size (n=307), after a BC has flagged positive, our average turnaround time from DNA extraction to determination of species identity was 4.4 h for a multiplex run of 12 BCs, and 3.7 h for a single sample run. We demonstrated that our pipeline taxonomic species identification results agreed with conventional MALDI-TOF identification for almost all positive BCs (97.7%, 300/307). Most species were accurately identified within the first hour of sequencing (93.7 %, 281/300). We explored AMR detection for clinically relevant antimicrobials and observed that assembly-based tools had higher agreement to conventional AST (81.2% after 1 h of sequencing, 89.6% after 5 h of sequencing) than read-based tools. Finally, we developed a publicly available analysis pipeline (venae) that generates a clinician-friendly HTML report, is quick to run, and can dynamically update as more sequencing data is acquired. This study demonstrates how applying rapid, real-time genomics to BSI diagnostics can support clinical decision-making and improve patient outcomes by reducing turnaround times. IMPACT STATMENTEarly pathogen detection and administration of appropriate antimicrobial therapy for BSIs has major impacts on patient survival; early administration of effective antimicrobials reduces mortality, morbidity, length of hospital stay, and development of antimicrobial resistance. Rapid real-time genomics has high potential to improve clinical decision-making and patient outcomes by reducing turnaround times (TATs) while providing high-resolution data for organism identification, AMR determination and pathogen typing. Here, we present a laboratory and bioinformatic workflow that accurately identifies species and AMR determinants in positive blood cultures within several hours, which is quicker than conventional methods which can take days. This workflow is a step forward on the path towards point-of-care diagnostics and applying real-time genomics to characterize microbial infections in clinical settings. DATA SUMMARYIllumina sequencing data for matching pure isolates were deposited in National Centre for Biotechnology Information Sequence Read Archive (NCBI SRA) BioProject PRJNA1380445. Bioinformatic analysis pipeline is available here: https://github.com/phac-nml/venae.

genomics↗

bioRxiv2025

Dual-lineage human 3D bone niche model reveals osteoclast-driven osteomimicry in prostate cancer

Bone is the predominant site of metastasis in advanced prostate cancer (PCa), yet the mechanisms governing tumor-bone interactions remain incompletely understood. A modular human three-dimensional (3D) in vitro bone niche model is developed that integrates osteoblasts and osteoclasts within a mineralized scaffold, recreating an endosteal-like microenvironment for co-culture with PCa cell lines and patient-derived organoids (PDOs). The engineered construct maintains osteoblastic differentiation and supports osteoclastogenesis, confirmed by lineage markers including osteocalcin, osteopontin (OPN), and tartrate-resistant acid phosphatase (TRAP). Co-culture with PCa cells downregulates osteoblast- and osteoclast-associated genes (IBSP, OPN, TRAP) in bone cells, suggesting tumor-mediated suppression of bone remodeling. Conversely, co-cultured PCa cells exhibit niche-dependent osteomimicry, characterized by upregulation of osteoblastic (SPARC, BGLAP) and osteoclastic (TRAP) markers and strongly regulated by the presence of osteoclasts. The platform also supports engraftment and proliferation of PDOs without PCa-specific exogenous growth factors, underscoring its translational relevance. This osteoblastic-osteoclastic niche model thus provides a human system capturing PCa-bone cell interactions, with potential utility to investigate therapeutic responses in a clinically relevant context. Table of ContentsA human 3D bone niche integrating osteoblasts and osteoclasts enables co-culture with prostate cancer cell lines and patient-derived organoids. The engineered niche (i) models reciprocal phenotypic regulation between bone and cancer cells, (ii) captures osteoclast-enhanced osteomimicry in tumor cells and (iii) establishes a biomimetic platform for mechanistic studies of metastatic prostate cancer cells in bone. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/689049v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@b16ee9org.highwire.dtl.DTLVardef@e8a013org.highwire.dtl.DTLVardef@6543f6org.highwire.dtl.DTLVardef@50fceb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

An organotypic in vitro model of human papillomavirus-associated precancerous lesions allowing automated cell quantification for preclinical drug testing

Summary and graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/661539v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@11e0c27org.highwire.dtl.DTLVardef@1affaf8org.highwire.dtl.DTLVardef@85c10forg.highwire.dtl.DTLVardef@145d491_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIA durable organotypic epithelial raft culture was established as a model of cervical precancer. C_LIO_LIPlausible time- and dose-dependent effects of cisplatin, 5-FU, and sinecatechins treatment were observed on keratinocytes and HPV-transformed cells. C_LIO_LITreatment effects were reliably quantified using machine learning-based cell classification. C_LIO_LIThis model may serve as a platform for preclinical investigation of topical and systemic treatment. C_LI Oncogenic human papillomaviruses (HPV) are causally responsible for invasive cancers and precancerous lesions. These lesions represent a considerable disease burden worldwide, yet no causally effective treatments are available. The development of HPV tumor models realistically reflecting the in vivo treatment situation is necessary for finding new effective and tissue-sparing treatments. This study aimed to establish an in vitro model of HPV-induced precancerous lesions for preclinical drug testing and to provide an automated method for quantifying cell counts to assess treatment effects in this model. To establish organotypic epithelial raft cultures (OTCs) as a model of HPV-induced precancerous lesions, we cultivated HPV-transformed cervical cancer cell lines SiHa, CaSki, HeLa, and SW756 in conjunction with primary human keratinocytes on a dermal equivalent and evaluated the impact of different cultivation variables. To demonstrate suitability of our model for preclinical drug application studies, we applied 5-fluorouracil, sinecatechins, and cisplatin either onto the air-exposed surface or to the growth medium, mimicking topical and systemic drug administration routes. We then developed a machine learning-based approach to quantify cell counts reflecting treatment effects in the established OTCs. We successfully optimized a durable in vitro model of HPV-induced precancerous lesions. The model enabled monitoring of treatment effects in a three-dimensional context with differentiated consideration of the targeted HPV-transformed cells and surrounding normal epithelium. We demonstrated that extreme gradient boosted tree classifiers (XGBoost) can be successfully used to distinguish the number of tumor cells, normal keratinocytes, and degraded cells with high accuracy (81.8{+/-}8%; P=2x10-6). Critically, quantification results plausibly reflected microscopical observations and gave a fine-grained picture of time- and dose-dependent treatment effects. The established model in combination with automated cell quantification can serve as a valuable tool in analyzing major preclinical endpoints in in vitro studies, such as target cell treatment efficacy, potential side effects, and treatment schedule optimization.

cancer biology↗

bioRxiv2025

Antimicrobial Susceptibility and Genomic Determinants of Resistance and Virulence in Mycoplasma cynos and Mycoplasma felis

Mycoplasma cynos and Mycoplasma felis are important respiratory pathogens in dogs and cats. Due to the challenges of culturing these fastidious bacteria, little is known about their antimicrobial susceptibility or mechanisms of pathogenicity. Treatment is typically empirical, as in vitro antimicrobial activity has not been evaluated, and therapeutic efficacy remains unclear. This study aimed to assess in vitro susceptibility and identify genetic markers of antimicrobial resistance (AMR) and virulence in M. cynos and M. felis clinical isolates. Minimum inhibitory concentrations (MICs) for doxycycline, tetracycline, minocycline, enrofloxacin, marbofloxacin, and azithromycin were determined using a broth microdilution assay developed for this study. Hybrid genomes were generated using Oxford Nanopore and Illumina sequencing. AMR-associated single-nucleotide polymorphisms (SNPs) in the gyrA gene correlated with high MICs to enrofloxacin and marbofloxacin in both species. Mutations in 23S rRNA were associated with reduced susceptibility to azithromycin. In M. felis, novel variants in gyrA and the 50S ribosomal protein L4 were linked to decreased susceptibility to fluoroquinolones and azithromycin, respectively. The data also suggest potential intrinsic resistance to azithromycin in M. felis. All isolates required low MICs of tetracyclines, and resistance mutations were not identified in the 16S rRNA gene, supporting tetracyclines as effective first-line treatment options. Virulence genes, particularly those associated with adhesion and immune evasion, were detected in both M. cynos and M. felis. This study presents the first comprehensive genomic and phenotypic analysis of AMR and virulence in M. cynos and M. felis, providing new insights into their pathogenicity and informing evidence-based therapeutic strategies.

microbiology↗

bioRxiv2025

Genetic basis of maneb-induced dopaminergic neurodegeneration in Drosophila

Parkinsons disease (PD) is a complex neurodegenerative disease driven by combined genetic and environmental factors. Human studies support increased PD risk following exposure to the pesticide maneb yet animal studies generally report subtle or no dopaminergic phenotypes unless maneb is combined with additional pesticides. Consequently, it is unclear whether exposure to maneb alone promotes dopamine (DA) neurodegeneration and if so, what the underlying mechanisms are. We hypothesized that gene-environment interactions are major determinants of maneb-mediated neurodegeneration and in support of this find that DA neuron viability is quantitatively divergent among 186 maneb-exposed genetically varying fly strains from the Drosophila Genetic Reference Panel (DGRP). Through genome-wide association analysis we identify several candidate genetic modifiers of maneb-induced DA neurodegeneration and further validate two candidate genes, fz2 and CG14186 which we find potentiate maneb-induced DA neurodegeneration when knocked-down. fz2 and the mammalian ortholog of CG14186 (TMEM237) are both thought to be necessary for intact Wnt pathway signaling in nervous system development and maintenance. Accordingly, we find that adult-specific perturbation of Wnt signaling is sufficient to promote maneb-induced DA neuron loss. Collectively, these results support a role for gene-environment interactions in PD etiology and reveal candidate mediators of maneb-related DA neurodegeneration in vivo. ARTICLE SUMMARYExposure to the pesticide maneb has been linked to increased PD risk although animal models of neurodegeneration have produced mixed results. We developed a Drosophila model for delayed onset dopamine neuron loss following maneb exposure and find that maneb-related neurodegeneration is quantitatively divergent across a large collection of Drosophila strains. We use a genome-wide association approach with follow-up validation to identify candidate modifier genes, uncovering roles for fz2 and CG14186. Both genes are implicated in Wnt signaling and we further show that deregulated Wnt signaling in adults promotes maneb-induced dopamine neuron loss. These studies support a role for gene-environment interactions in maneb neurotoxicity and yield insight into the underlying genes involved.

genetics↗

bioRxiv2025

Genome-wide analysis reveals genes mediating resistance to paraquat neurodegeneration in Drosophila

Parkinsons disease (PD) is thought to develop through a complex interplay of genetic and environmental factors. Epidemiological studies have linked exposure to certain pesticides such as paraquat with elevated PD risk, although how a persons genetic makeup influences disease risk upon exposure remains unknown. Here, we used a genome-wide approach to uncover genes that play a role in resistance to paraquat-induced dopaminergic neurodegeneration in Drosophila. We developed a paraquat exposure model displaying delayed-onset dopaminergic (DA) neurodegeneration to recapitulate this aspect of human disease. We reveal that genetic background is a strong determinant of paraquat-induced DA neurodegeneration susceptibility across a series of nearly 200 fly strains called the Drosophila genetic reference panel (DGRP). Through unbiased genome-wide analysis and follow-up validation, we identify two candidate paraquat resistance genes, luna and CG32264. In gene-level studies, decreased expression of luna or CG32264 is associated with paraquat-induced DA neuron loss while overexpression of either gene prevents neurodegeneration in vivo. The mammalian ortholog of CG32264 is Phactr2, which has previously been linked to human idiopathic PD risk in several populations. Hence, our results reveal genes regulating paraquat-induced DA neuron loss that intersect with human PD risk variants, supporting the potential relevance of our findings to PD and underscoring a role for gene-environment interactions in pesticide-related DA neurodegeneration. ARTICLE SUMMARYParaquat is a widely used herbicide linked to increased PD risk and to dopaminergic neurodegeneration in animal studies. Gene-environment interactions likely influence whether an individual exposed to paraquat eventually manifests PD and presents a major opportunity to yield insight into PD genetics. We developed a paraquat-induced neurodegeneration model in Drosophila, applied this model to nearly 200 fly strains belonging to the Drosophila Genetic Reference Panel and used a genome-wide association approach to identify candidate modifier genes of paraquat-induced dopamine neuron loss which we subsequently validated through RNAi and overexpression functional testing. Through this approach, we reveal two novel paraquat resistance genes, luna and CG32264. Strikingly, the putative mammalian ortholog of CG32264 (Phactr2) was previously linked to human PD, supporting the potential relevance of our findings to human disease.

genetics↗

bioRxiv2025

Root exudation facilitate water infiltration and rewetting of dry soil

The way plant roots facilitate water infiltration in soil may be just as important as the efficiency with which the root system in turn extracts it from the soil. Here we studied the mechanisms through which the root system facilitates water infiltration through a dry soil layer. Dye tracing experiments were conducted in model soil microcosms to characterise how root growth and exudation affects the permeability of dry layers of the model soil. Results showed that dissolved root exudates may be the primary facilitator of water infiltration, which may be linked to water surface tension. We conclude that in dry soil, root architecture and root exudation may combine to facilitate the infiltration of water and decrease the water lost by evaporation. These findings could enhance our understanding of the traits that provide drought resistance in crops.

plant biology↗

bioRxiv2025

FULL-LENGTH HYBRID TRANSCRIPTOME OF THE OLFACTORY ROSETTE IN SENEGALESE SOLE (Solea senegalensis): AN ESSENTIAL GENOMIC RESOURCE TO IMPROVE REPRODUCTION AT FARMS

Senegalese sole is a promising European aquaculture species whose main challenge is that captive-born males (F1) are unable to reproduce in farms, hindering breeding programs. Chemical communication through the olfactory system is hypothesized to stem this issue. Although significant advancement in genomic resources has been made over the past decade, scarce information exists on the genomic basis of olfaction, a special sensory system for demersal species like flatfish, which could play a prominent role in reproduction, social and environmental interactions. A full-length transcriptome of the olfactory rosettes including females, males, juveniles and adults, of both F1 and wild origins, was generated at the isoform-level by combining Oxford Nanopore long-read and Illumina short-read sequencing technologies. A total of 20,670 transcripts actively expressed were identified: 13,941 were known transcripts, 5,758 were novel transcripts from known genes, and 971 were novel genes encoding novel transcripts. Special attention was paid to the olfactory receptor gene families (OlfC, OR, ORA and TAAR) expression. Our comprehensive olfactory transcriptome of Senegalese sole provides a foundation for delving into the functional basis of this complex organ in teleost and flatfish. Furthermore, it provides a valuable resource for addressing reproductive management challenges in Senegalese sole aquaculture.

genomics↗

bioRxiv2025

Impact of meningioma and glioma on whole-brain dynamics

Brain tumors, particularly meningiomas and gliomas, can profoundly affect neural function, yet their impact on brain dynamics remains incompletely understood. This study investigates alterations in normal brain function among meningioma and glioma patients by assessing dynamical complexity through the Intrinsic Ignition Framework. We analyzed resting-state fMRI data from 34 participants to quantify brain dynamics using intrinsic ignition and metastability metrics. Our results revealed distinct patterns of disruption: glioma patients showed significant reductions in both metrics compared to controls, indicating widespread network disturbances. In contrast, meningioma patients exhibited significant changes predominantly in regions with substantial tumor involvement. Resting-state network analysis demonstrated strong metastability and metastability/ignition correlations between regions in controls, which were slightly weakened in meningioma patients and severely disrupted in glioma patients. These findings highlight the differential impacts of gliomas and meningiomas on brain function, offering insights into their distinct pathophysiological mechanisms. Furthermore, these results show that brain dynamics metrics can be effective biomarkers for identifying disruptions in brain information transmission caused by tumors.

neuroscience↗

bioRxiv2024

Phenotypic diversity and shared genomic determinants among isolates causing a large incidence of disseminated gonococcal infections in Canada

The incidence of disseminated gonococcal infection (DGI) has remained low since the advent of antibiotics, however recent surge in DGI have inexplicably emerged within several regions during the past decade. In an effort to understand whether Neisseria gonorrhoeae that cause disseminated disease can be differentiated from non-invasive strains, we have performed a phenotypic and genotypic analysis on a selection of isolates obtained from invasive and uncomplicated infections in Canada. Phenotypic analysis of a matched subset of 19 isolates obtained since 2013 found that these varied in their capacity to aggregate in suspension and in their association with serum complement proteins, however these interactions did not discriminate between the invasive and mucosal isolates. Sequence typing of 360 Canadian isolates revealed that two porB alleles are significantly associated with the DGI strains, one of these being present throughout the past decade whereas the other became associated more recently. A PopNet-based population dynamics analysis, which instead establishes relationships based upon variance among discrete chromosomal segments, found that DGI isolates were restricted in their phylogenetic distribution. While this implies a genetically-linked potential to cause invasive disease, it cannot distinguish between an inherent difference in the phenotype of these populations or the horizontal exchange of some virulence factor among closely related strains. Regardless, a large number of genetic determinants are enriched in the DGI strains, making these enticing candidates for future work to understand how they might either promote the gonococcal capacity to cause systemic infection or reduce the presentation of clinical symptoms from localized infection so that it remains untreated. AUTHOR SUMMARYNeisseria gonorrhoeae is a sexually transmitted bacteria that causes over 82 million cases of gonorrhea each year. With its re-emergence, rising incidence rates and the prevalence of multidrug resistant strains increasing, the bacteria is considered to be a high burden threat to global public health. While disseminated gonococcal disease arising from untreated infections are uncommon, there have recently been regions with high incidence invasive disease. Here, we take advantage of the active ongoing collection of gonococcal isolates in Canada to perform a combined phenotypic and genotypic analysis that aims to understand whether certain strains are more often linked to invasive infections. We found that all disseminated isolates bound to the complement regulatory factors factor H and/or C4 binding protein to facilitate their resistance to the bactericidal activity of serum, but this was not sufficient to explain the heightened instance of invasive disease. While classical genome-based phylogenetic analysis displayed little association between invasive strains, PopNet-based analysis revealed that invasive isolates fell within defined sub-populations and indicated variant alleles enriched among the disseminating bacteria, and a broader pan-genome approach revealed genes more likely to be present in invasive strains. Our study thereby provides support for a genetic contribution to the invasive potential of gonococcal isolates and provides candidate drivers of this virulent outcome.

microbiology↗

bioRxiv2024

miRNA-29-CLIP uncovers new targets and functions to improve skin repair

MicroRNAs (miRNAs) control organogenesis in mammals but their role in specific cell types is not fully explored. miRNAs exert their function by binding mRNAs and inhibiting translation. Skin is an excellent model to study the role of miRNAs in primary cells of epidermal (keratinocytes) and mesodermal (fibroblasts) origin, because the growth of these cells is tightly controlled at translation. Previous research demonstrated that miRNA-29 family functions during skin repair, however, the exact mRNA targets and the downstream mechanisms of miRNA-29-mediated regulation of cell growth is missing. Here, we use miRNA crosslinking and immunoprecipitation (miRNA-CLIP) method to find the direct targets of miRNA-29 in keratinocytes and fibroblasts from human skin. We uncover previously unrecognized roles of miRNA-29 in protein folding and RNA processing, common to all cell types tested, and determine the cell-specific role of miRNA-29. Using modified anti-sense oligonucleotides (ASO) in 2D and 3D cultures of keratinocytes and fibroblasts, we enhanced cell-to-matrix adhesion and found an autocrine and paracrine mechanism of miRNA-29-dependent cell growth. Our results include a comprehensive list and functional analyses of mRNAs directly bound by miRNA-29 keratinocytes and fibroblasts, determined by miRNA-CLIP and ASO-mediated inhibition of miRNA-29 followed by RNA-seq. We reveal a full transcriptome of human keratinocytes with enhanced adhesion to the wound matrix, which supports regeneration of the epidermis and is regulated by miRNA-29. The functions of miRNA-29 identified in this study can provide a new approach to improve cutaneous repair by restoring and enhancing the endogenous mechanisms through the stage-specific delivery of miRNA-29 ASO.

cell biology↗

bioRxiv2024

Evolutionary trajectories of IDH-mutant astrocytoma identify molecular grading markers related to cell cycling

1To study the evolutionary processes that drive malignant progression of IDH-mutant astrocytomas, we performed multi-omics on a large cohort of matched initial and recurrent tumor samples. The overlay of genetic, epigenetic, transcriptomic and proteomic data, combined with single-cell analysis, have identified overlapping features associated with malignant progression. These features are derived from three molecular mechanisms and provide a rationale of the underlying biology of tumor malignancy: cell-cycling, tumor cell (de-)differentiation and remodeling of the extracellular matrix. Specifically, DNA-methylation levels decreased over time, predominantly in tumors with malignant transformation and co-occurred with poor prognostic genetic events. DNA-methylation was lifted from specific loci associated with DNA replication and was associated with an increased RNA and protein expression of cell cycling associated genes. All results were validated on samples of newly diagnosed IDH-mutant astrocytoma patients included the CATNON randomized phase 3 clinical trial. Importantly, malignant progression was hardly affected by radio- or chemotherapy, indicating that treatment does not affect the course of disease. Our results culminate in a DNA-methylation based signature for objective tumor grading.

cancer biology↗

bioRxiv2024

Natural Variation in Age-Related Dopamine Neuron Degeneration is Glutathione-Dependent and Linked to Life Span

Aging is the biggest risk factor for Parkinsons disease (PD), suggesting that age-related changes in the brain promote dopamine neuron vulnerability. It is unclear, however, whether aging alone is sufficient to cause significant dopamine neuron loss and if so, how this intersects with PD-related neurodegeneration. Here, through examining a large collection of naturally varying Drosophila strains, we find a strong relationship between life span and age-related dopamine neuron loss. Strains with naturally short-lived animals exhibit a loss of dopamine neurons but not generalized neurodegeneration, while animals from long-lived strains retain dopamine neurons across age. Metabolomic profiling reveals lower glutathione levels in short-lived strains which is associated with elevated levels of reactive oxygen species (ROS), sensitivity to oxidative stress and vulnerability to silencing the familial PD gene parkin. Strikingly, boosting neuronal glutathione levels via glutamate-cysteine ligase (Gcl) overexpression is sufficient to normalize ROS levels, extend life span and block dopamine neurons loss in short-lived backgrounds, demonstrating that glutathione deficiencies are central to neurodegenerative phenotypes associated with short longevity. These findings may be relevant to human PD pathogenesis, where glutathione depletion is reported to occur in idiopathic PD patient brain through unknown mechanisms. Building on this, we find reduced expression of the Gcl catalytic subunit in both Drosophila strains vulnerable to age-related dopamine neuron loss and in human brain from familial PD patients harboring the common LRRK2 G2019S mutation. Our study across Drosophila and human PD systems suggests that glutathione synthesis and levels play a conserved role in regulating age-related dopamine neuron health.

neuroscience↗

bioRxiv2023

Modelling Osteoarthritis pathogenesis through Mechanical Loading in an Osteochondral Unit-on-Chip

A cure for osteoarthritis (OA), the most prevalent musculoskeletal disease, remains an unmet need. Investigating the molecular and cellular processes leading to OA is challenged by the absence of human models that capture the complex interplay among different tissues in the joint under pathophysiological mechanical loading. In this study, we have engineered an OsteoChondral Unit (OCU)-on-chip system where composite hyaline cartilage - mineralized osseous microtissue analogues are exposed to controlled, tissue-specific compression regimes akin to those of the OCU in vivo. Through single-cell transcriptomic analysis, we demonstrate the critical relevance of the mineralized layer in inducing chondrocyte subpopulations implicated in the progression of OA. Upon exposure to hyperphysiological loading, the OCU-on-chip captures early phenotypic traits of OA pathogenesis, comprising alterations of subchondral mineral content and acquisition of previously described OA genetic signatures. This system enabled to identify novel upstream drivers of OA metabolic changes, including mechanically induced ribosomal alterations, as well as associated molecular targets towards the development of disease-modifying OA therapies.

bioengineering↗