Biology subjects

Schmidt, S.

Publications and source records attributed to Schmidt, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanical stress-induced release of mitochondrial double-stranded RNA activates RIG-I to drive osteogenic differentiation in aortic disease

Background: Gain-of-function mutations in RIG-I and MDA5 (RIG-I-like receptors, RLR) cause Singleton-Merten syndrome, an interferonopathy with early aortic calcification. Here we set out to study a possible involvement of RIG-I-like receptor signaling in the formation of aortic valve stenosis in general. Methods and results: We investigated whether mechanical stress-induced RLR activation promotes osteogenic differentiation and calcification of primary human aortic cells, whether such RLR activation drives the development of aortic valve stenosis in vivo, and whether inhibition of downstream JAK/STAT activation ameliorates aortic valve stenosis. We found that activation of primary human aortic interstitial cells by exogenous RIG-I- and MDA5 agonists induced a type I interferon response and an osteogenic program including increased expression of BMP-2 and BSP2, and promoted calcification. These effects were RIG-I/MDA5 or MAVS-dependent, as assessed by CRISPR/Cas9 knockouts. The stress-granule protein G3BP1 and the Wnt receptor FZD5 were induced downstream of RLR activation and were required for osteogenic gene expression and calcification. To assess a potential endogenous source of RIG-I activation, mechanical stretch mimicking valvular tissue stress was applied in cell cuture, which induced a significant RIG-I-dependent interferon response. Sequencing of highly purified preparations of double-stranded RNA from stretched cells demonstrated a predominant mitochondrial origin of double-stranded RNA, and depleting mitochondrial RNA abolished the stretch-dependent response. The JAK/STAT inhibitor ruxolitinib suppressed the RIG-I-induced response including the expression of osteogenic factors in vitro. In a murine wire-injury model of aortic stenosis, MAVS deficiency attenuated valvular morphological changes and improved hemodynamic parameters. Ruxolitinib attenuated hemodynamic progression, prevented aortic stenosis development, and improved survival. Conclusions: Altogether, our results demonstrate that the mechanical-stress-induced release of mitochondrial double-stranded RNA activates RIG-I signaling in aortic interstitial cells, thereby driving osteogenic differentiation and calcification via a MAVS- and G3BP1-dependent pathway. JAK/STAT inhibition with ruxolitinib mitigates this process in a mouse model of aortic valve stenosis, identifying a candidate therapeutic strategy.

cell biology↗

bioRxiv2026

Low-dose ketamine tilts the cortical excitation--inhibition balance irrespective of systemic physiological response

Non-invasive proxies of cortical excitation--inhibition (E:I) balance, are increasingly used to understand human neural dynamics; especially so the aperiodic (1/f) electroencephalographic (EEG) exponent. Ketamine, an NMDA-receptor antagonist, is thought to shift this balance toward excitation. Propofol, a GABAA-receptor agonist, should behave oppositely. Here we show that these pharmacological manipulations at low doses tilt the noninvasive read-out of E:I balance as predicted, and we demonstrate this effect to be robust against potential mediation by the ketamine-related systemic cardiovascular response. In a single-blind, placebo-controlled crossover study, 25 healthy adults received low, subanesthetic doses of ketamine, propofol, or placebo by target-controlled infusion during resting-state EEG. We further analyzed electrocardiogram (ECG) and dissociating ratings. Ketamine flattened the aperiodic exponent (i.e., a shift toward excitation) and raised heart rate and blood pressure; propofol exerted its effects in the opposite direction, while both agents reduced alpha oscillatory power. Critically, the cardiovascular response did not mediate the cortical E:I shift. Controlling for heart rate left the ketamine effect not only intact but numerically stronger, suggesting that E:I balance findings might even be underestimated when ignoring the systemic physiological response.

neuroscience↗

bioRxiv2026

Kelch13 stochastics determine drug survival in resistant malaria parasites

As resistance to the frontline antimalarial artemisinin (ART) in the deadliest malaria species Plasmodium falciparum spreads, it threatens the gains made in reducing global malaria burden over the last decades. Mutations in the gene encoding Kelch13 (K13) hold a central role in ART resistance. Yet even in clonal populations harbouring a resistance conferring k13 mutation, only a portion of parasites survive drug exposure. Here we show that stochastic cell- to-cell variation of cellular K13 levels determines survival of individual parasites. Using isogenic parasite lines, we establish that decreased cellular K13 levels correlate with resistance and in addition a fitness cost through an increased cell cycle length. As resistance increases, parasites show a shift of the stochastic range towards lower K13 levels, increasing the proportion of drug survivors. While this range was inherited, individual parasites with differing K13 levels gave rise to progeny across the full spectrum. Hence, while the resistance fitness profile of each parasite line is defined by the range of K13 levels, the fate of individual parasites within that range is determined by stochastics. These findings provide an explanation why only a portion of genetically identical parasites survive drug exposure and reveal a system where the stochastics of a single protein determine individual parasite drug survival and fitness.

microbiology↗

bioRxiv2026

Rhythmic replay of short-term memory neural patterns revealed by time-resolved error prediction

Theta oscillations are hypothesized to provide a temporal scaffold for short-term memory (STM). In this model, memory representations are organized into successive theta phases, reducing conflict between competing representations during encoding and maintenance. Previous studies have shown that sensory representations of memorized information are rhythmically reactivated at theta frequency. Whether this theta-rhythmic mechanism is shared across encoding and maintenance, traditionally viewed as temporally distinct processes, remains unclear. It is also unknown whether this rhythmic reactivation predicts subsequent memory fidelity rather than merely reflecting the reinstatement of sensory representations. We recorded EEG from 21 participants performing STM with colored, oriented items under low and high memory load. Using time-resolved multivariate pattern analysis, we predicted subsequent STM error from encoding- and maintenance-period activity. We found that distributed neural patterns predicted STM error and that this prediction fluctuated at theta frequency, independently of memory load and feature. Cross-temporal generalization indicated that a shared neural pattern recurred across encoding and into maintenance, consistent with periodic reactivation of a common neural code rather than a succession of distinct states. Prediction was temporally offset across spatial positions and item features, indicating that the features of a multi-feature object are encoded by separate, rhythmically interleaved processes. These findings characterize STM encoding and maintenance as a rhythmic, recurrent process and link theta-rhythmic fluctuations in memory fidelity to distributed cortical activity spanning multiple brain regions and frequency bands.

neuroscience↗

bioRxiv2026

Preoperative biliary stenting is associated with functional dysbiosis and impaired bile acid metabolism in pancreatic cancer - Impact of preoperative biliary stenting on gut microbiota

Recent evidence suggests that the gut microbiome plays a role in the development and treatment response of pancreatic ductal adenocarcinoma (PDAC). However, the functional impact of tumor location and preoperative biliary stenting (PBS) on microbial composition and metabolism remains poorly understood. In this prospective study, preoperative stool specimens were collected from patients undergoing surgery for PDAC at Heidelberg University Hospital, Germany, between March 2020 and July 2021. Whole-genome shotgun metagenomic sequencing was performed to characterize microbial composition and functional pathways. A total of 63 preoperative stool samples were analyzed, including 40 patients with pancreatic head tumors (63.5%) and 23 with body/tail tumors (36.5%). Microbial community composition differed significantly according to tumor location (Bray-Curtis, p=0.005), with enrichment of Ruminococcus bromii in body/tail tumors. Among patients with pancreatic head tumors, PBS was associated with reduced alpha diversity (Shannon index, p=0.04), depletion of taxa including members of the Eubacteriales and Clostridiales orders as well as the genera Raoultella and Prevotella, and reduced abundance of selected genes involved in secondary bile acid metabolism. PBS was also associated with a higher rate of major postoperative complications according to Clavien-Dindo >3a (28.6% vs 3.8%; p=0.04). These findings suggest that biliary intervention may induce functional dysbiosis characterized by reduced microbial diversity and impaired bile acid metabolism, potentially disrupting host- microbiome crosstalk and contributing to adverse postoperative outcomes in pancreatic cancer.

microbiology↗

bioRxiv2026

Decoding and Reprogramming Redox Partner Specificity in Rieske Oxygenases for Enhanced Catalytic Activity

Multicomponent Rieske oxygenases catalyze diverse oxidative transformations but require precisely matched redox partners to sustain efficient electron transfer, severely limiting their modularity and biocatalytic application. Yet, the molecular logic underlying this specificity remains poorly defined. Here we decode the molecular principles governing redox partner specificity in representative three-component Rieske oxygenase systems. Through systematic mutagenesis analysis and cross-component reconstitution assays, we identify a single ferredoxin residue that acts as a class-defining determinant of oxygenase recognition. Guided by this insight, we reprogram electron transfer between non-cognate components by complementary engineering of the oxygenase interface, creating an unnatural redox chain with substantially enhanced catalytic turnover compared to the native system. Spectroscopic, binding and computational analyses reveal that productive electron transfer arises from optimized electrostatic complementarity and redox potential alignment rather than maximal binding affinity. Extending this strategy to another oxygenase system demonstrates its generality. Together, these results establish transferable design rules for rationally engineering electron transfer pathways in multicomponent oxygenases, enabling their predictable adaptation as customizable biocatalysts.

biochemistry↗

bioRxiv2026

Scalable computation of ultrabubbles in pangenomes by orienting bidirected graphs

MotivationPangenome graphs are increasingly used in bioinformatics, ranging from environmental surveillance and crop improvement to the construction of population-scale human pangenomes. As these graphs grow in size, methods that scale efficiently become essential. A central task in pangenome analysis is the discovery of variation structures. In directed graphs, the most widely studied such structures, superbubbles, can be identified in linear time. Their canonical generalization to bidirected graphs, ultrabubbles, more accurately models DNA reverse complementarity. However, existing ultrabubble algorithms are quadratic in the worst case. ResultsWe show that all ultrabubbles in a bidirected graph containing at least one tip or one cutvertex--a common property of pangenome graphs--can be computed in linear time. Our key contribution is a new linear-time orientation algorithm that transforms such a bidirected graph into a directed graph of the same size, in practice. Orientation conflicts are resolved by introducing auxiliary source or sink vertices. We prove that ultrabubbles in the original bidirected graph correspond to weak superbubbles in the resulting directed graph, enabling the use of existing lineartime algorithms. Our approach achieves speedups of up to 25xover the ultrabubble implementation in vg, and of more than 200x over BubbleGun, enabling scalable pangenome analyses. For example, on the v2.0 pangenome graph constructed by the Human Pangenome Reference Consortium from 232 individuals, after reading the input, our method completes in under 3 minutes, while vg requires more than one hour, and four times more RAM. AvailabilityOur method is implemented in the BubbleFinder tool github.com/algbio/BubbleFinder, via the new ultrabubbles subcommand. Contactalexandru.tomescu@helsinki.fi

bioinformatics↗

bioRxiv2026

Vegetation increases CH4 emissions and methanotroph diversity in marine sediments

Seagrass meadows are key blue carbon (C) ecosystems, storing large amounts of organic C over centuries. Their climate benefits may be reduced by methane (CH) emissions, whose microbial and environmental descriptors in Zostera noltei meadows, dominant seagrass in North-Western Europe, remain poorly understood. We studied CH fluxes, CH-producing and consuming microbial communities and sediment physico-chemical parameters in Z. noltei meadows and adjacent bare sediments across seven sites in Arcachon Bay, France. In situ CH fluxes were measured at low tide and microbial communities were characterised using targeted metagenomics of three functional genes (mcrA, mmoX, pmoA) and quantitative PCR. CH fluxes were higher in vegetated than bare sediments (24.4 {+/-} 2.6 vs. 9.4 {+/-} 0.7 {micro}mol m-{superscript 2} d-{superscript 1}). Mixed linear models and random forest analyses identified C accumulation rate and CO2 flux as the strongest positive descriptors of CH fluxes. Vegetated sediments hosted more diverse methanotrophs, while methanogens showed no habitat differences. Four genera (mcrA-Methanolobus, mmoX-Methylocella, pmoA-Methylococcus, Methyloglobulus) emerged as abundant, seagrass-associated, correlated with CH fluxes, and highlighted by models. Functional diversity, especially pmoA richness, was a stronger microbial descriptor of CH fluxes than gene abundance or a specific genus. Findings indicate Z. noltei meadows enhance C burial and CH emission, with methanotroph diversity potentially mitigating CH emissions. Our results provide the first integrated assessment of CH fluxes and their descriptors in Z. noltei meadows, highlighting the intertwined nature of C burial and CH emissions and the need to account for both in blue C climate assessments.

ecology↗

bioRxiv2025

PEG-free, Triphosphate-Stabilized LNPs Enable Potent RNA Delivery

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/695515v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@d919d8org.highwire.dtl.DTLVardef@12fe9aforg.highwire.dtl.DTLVardef@96e2d2org.highwire.dtl.DTLVardef@54f73_HPS_FORMAT_FIGEXP M_FIG C_FIG Polyethylene glycol (PEG)-lipids have long enabled lipid nanoparticle (LNP) formulations by providing steric stabilization and prolonged circulation. However, anti-PEG immune responses may impede repeated systemic dosing in mRNA therapeutics due to the phenomenon known as accelerated blood clearance (ABC). Here, we introduce a PEG-free, polyanionic alternative in which sodium triphosphate (3P) electrostatically associates with ionizable surface lipids, conferring long-term colloidal stability through charge repulsion rather than steric shielding. 3P-LNPs maintained size uniformity, morphology, and mRNA integrity for over nine months at 4 {degrees}C. In a proof-of-concept study involving a single intravenous injection of LNPs in mice carrying a luciferase and a GFP transgene, early expression was approximately two-fold higher compared to PEG-LNPs, while expression levels at 24 hours and hepatic tolerability remained comparable. Serum enzyme and cytokine profiles indicated no necrosis or inflammation. These findings establish 3P-LNPs as a stable, biocompatible platform free of PEG, with potential for repeat systemic mRNA administration while avoiding PEG-related effects.

biochemistry↗

bioRxiv2025

Optimization of a T7 RNA polymerase expression system for high-yield protein production in Cupriavidus necator H16

Many chemical manufacturing routes are being replaced with enzymatic processes to improve sustainability. In biocatalysis, enzyme production is often the main bottleneck. Bacterial proteins are frequently produced using the workhorse E. coli BL21(DE3) and its derivatives. However, other bacteria with beneficial characteristics can also be engineered for this purpose. Cupriavidus necator H16 (C. necator), for example, is a Gram-negative bacterium well-known for its chemolithoautotrophic metabolism and high polyhydroxybutyrate (PHB) accumulation. Previous studies have demonstrated protein production without inclusion body formation, which is one of the main challenges when producing enzymes in E. coli. Nevertheless, high-yield protein production in C. necator remains an understudied field. Here, we investigated the bottlenecks limiting protein production in C. necator. We optimized a T7 RNA polymerase genetic system and quantified the impact of several genetic elements such as T7 promoter, RBS strength and codon usage towards GFP production. Codon usage was the main factor limiting protein production in C. necator. Tuning the RBS strength and selecting a different T7 promoter strongly influenced expression leakiness. We then produced the ene-reductase YqjM from Bacillus subtilis in C. necator, analyzing the performance of the engineered C. necator strain and our previously developed pMVRha expression plasmid. C. necator produced high amounts of soluble protein and outperformed the gold standard, E. coli BL21(DE3) in producing FMN-loaded enzyme. This result highlights the potential of non-model bacteria to achieve high-yield enzyme production and promote the transition to biocatalysis-driven chemical synthesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/688765v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1a6584corg.highwire.dtl.DTLVardef@1ef094dorg.highwire.dtl.DTLVardef@4bb4c3org.highwire.dtl.DTLVardef@489c85_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Pilot-scale production of leucine from CO2

On a cellular level, proteinogenic amino acids (AAs) are the building blocks of proteins. On a global scale, AAs serve as important nutrients for humans and animals. Beyond their nutritional value, AAs are of relevance in medicine, and, due to their chemical properties, they are indispensable for applications in many different realms, such as pharmaceutics, cosmetics, animal feed, food, and the beverage industry. Here, we report the first archaeal cell factory for leucine production from CO2 that has been generated by rational design, random mutagenesis and pathway engineering. The cell factory has been bioprocess-technologically examined and successfully scaled-up with regard to productivity, product quality, and operational stability. In a 2-day fed-batch campaign, we produced 181 g of leucine from CO2 at 150 L pilot-plant scale at a mean volumetric leucine productivity of 65 mg L-1 h-1. A thorough techno-economic analysis indicates that the roll-out of leucine production from CO2 is nearly economically feasible on a global scale.

bioengineering↗

bioRxiv2025

A sulfatide-centered ultra-high resolution magnetic resonance MALDI imaging benchmark dataset for MS1-based lipid annotation tools

Spatial omics techniques are indispensable for studying complex biological systems and for the discovery of spatial biomarkers. While several current matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) instruments are capable of localizing numerous metabolites at high spatial and spectral resolution, the majority of MSI data is acquired at the MS1 level only. Assigning molecular identities based on MS1 data presents significant analytical and computational challenges, as the inherent limitations of MS1 data preclude confident annotations beyond the sum formula level. To enable future advancements of computational lipid annotation tools, well-characterized benchmark - or ground truth - datasets are crucial, which exceed the scope of synthetic data or data derived from mimetic tissue models. To this end, we provide two sulfatide-centered, biology-driven magnetic resonance MSI (MR-MSI) datasets at different mass resolving powers that characterize lipids in a mouse model of human metachromatic dystrophy. This data includes an ultra-high-resolution (R [~]1,230,000) quantum cascade laser mid-infrared imaging-guided MR-MSI dataset that enables isotopic fine structure analysis and therefore enhances the level of confidence substantially. To highlight the usefulness of the data, we compared 118 manual sulfatide annotations with the number of decoy database-controlled sulfatide annotations performed in Metaspace (67 at FDR < 10%). Overall, our datasets can be used to benchmark annotation algorithms, validate spatial biomarker discovery pipelines, and serve as a reference for future studies that explore sulfatide metabolism and its spatial regulation.

biochemistry↗

bioRxiv2025

Direct encoding of new visual concepts in early visual cortex

Neocortical memories are assumed to rely on slow systems consolidation through hippocampal offline reactivation. However, recent studies show rapid neocortical integration of new memories for information associated with prior knowledge. Testing the limits of rapid neocortical learning, we tracked memory formation across 24h with functional and diffusion-weighted MRI in subjects acquiring either conceptual or detailed knowledge of novel objects from identical visual stimulation. Concept learning-induced rapid, 24h-stable changes in strength and pattern of functional early visual cortex responses. These were co-localized with microstructural changes, correlated positively with categorization performance and negatively with context recognition. Detailed item-context learning elicited analogous changes in posterior parietal areas. Results show that the neocortex can rapidly learn without prior knowledge and that concept learning can occur in early sensory regions that process corresponding visual features. Thus, we substantiate the possibility of direct or parallel neocortical encoding without offline abstraction from individual episodes.

neuroscience↗

bioRxiv2025

Globally unified analysis of riverine eDNA reveals common associations of fish biodiversity with drainage characteristics

Freshwater biodiversity is declining at a pace that outstrips the capacity of existing monitoring approaches both in temporal and spatial dimensions, highlighting the urgent need for rapid and scalable assessment and attribution of biodiversity states and changes. Here, we present one of the first global assessments and unified analyses of riverine fish biodiversity using environmental DNA (eDNA) collected from 1818 sites across 113 river systems. We quantified species richness, functional redundancy, phylogenetic diversity, and genetic sequence diversity, and related them to drainage characteristics. Our results showed that eDNA effectively captured global patterns of multi-faceted riverine fish biodiversity and disentangled the roles of climate and human activities in shaping biodiversity-area relationships. Catchments in warmer climates consistently enhanced biodiversity accumulation with area, while higher human activity intensity weakened this scaling. Species richness, functional, and genetic sequence diversity exhibited stronger negative responses to human activities in larger catchments. In contrast, phylogenetic diversity showed the strongest negative effects in smaller catchments with these impacts diminishing as catchment area increased, highlighting the facet-dependent nature of biodiversity responses to environmental gradients. Our findings demonstrate the power of eDNA-based datasets for harmonized, multi-faceted biodiversity assessments, offering a scalable approach for detecting and attributing biodiversity change and informing conservation strategies under accelerating global change.

ecology↗

bioRxiv2025

Integrated reanalysis of global riverine fish eDNA datasets shows robustness and congruence of biodiversity conclusions

The analysis of environmental DNA (eDNA) has revolutionized biodiversity assessments in aquatic ecosystems, enabling non-invasive monitoring of fish communities across diverse regions. However, the global comparability of these eDNA datasets remains ambiguous due heterogeneous sampling protocols and bioinformatic workflows across studies, particularly regarding the robustness of their conclusions on biodiversity assessments. Here, we conducted a meta-analysis of 58 riverine fish eDNA metabarcoding datasets, covering 1,818 sampling sites worldwide, to evaluate the robustness of eDNA-derived biodiversity patterns. We found that species richness estimates and metrics of community structure derived under a common bioinformatic workflow were overall consistent with those of original analyses, despite the relatively high variability in bioinformatic analyses in the respective original studies. Contrastingly, congruence of species identity varied more extensively across datasets, mostly reflecting different completeness and regional relevance of reference databases. Restricting taxonomic assignment to basin-specific species pools improved species identification accuracy, while datasets lacking publicly accessible or well-curated reference data were more prone to mismatches. Year of sampling had a positive effect on taxonomic congruence, such that more recent studies showed increased robustness, also reflecting improved reference database coverage and enhanced species-level identification over time and overall method congruence in more recent years. Overall, the suitability and potential of eDNA for global biodiversity monitoring is corroborating overall robust biodiversity estimates, irrespective of the bioinformatic approaches. Our study underlines the effectiveness and need of further harmonization of bioinformatic workflows and strengthened region-specific reference databases for improved taxonomic resolution and comparability across studies.

ecology↗

bioRxiv2025

Structural analysis of HERC2/UBE3A and HERC2/DOCK10 complexes provides new insights into the molecular basis of Angelman, Angelman-like and Dup15q Syndromes

UBE3A and HERC2 are two mutually interacting HECT E3 Ubiquitin ligases whose genes are altered in 15q11.2-13.1 Duplication (Dup15q) Syndrome, Angelman Syndrome (AS) and other phenotypically-related mental retardation syndromes. Using quantitative binding assays, X-ray crystallography and sequence conservation analysis, we show that the HERC2/UBE3A complex occurs in probably most animals with a central nervous system, via a conserved interface involving the RLD2 domain of HERC2 and a "DxDKDxD" motif of UBE3A. We found that HERC2 also recognizes and binds to similar DxDKDxD motifs within a handful of other proteins relevant to brain development (DOCK10, PCM1, USP35, BAZ2B, ARID4A, ARIP4, RERE and MYT1). We further investigated the interaction of HERC2 with DOCK10, a RAC1- and CDC42-GEF protein that regulates dendritic spine morphogenesis in hippocampal neurons. Both disruption of the HERC2-binding motif in DOCK10 and knockdown of HERC2 affected the GEF activity of DOCK10. We also show that the DOCK10-induced dendritic spine formation is dependent on its ability to bind HERC2. Structural modeling of full-length DOCK10, free or bound to either RLD2, CDC42 or RAC1 indicates that the GEF activation of full-length DOCK10 requires a conformational change that is stimulated by binding to HERC2. Based on our data, we propose that under pathological conditions, in developing brains with an abnormal dosage of either HERC2 or its dominant partner UBE3A, increased or decreased amounts of HERC2/DOCK10 complexes could lead to altered GTPase activation. This in turn could affect dendritic spine formation and neurodevelopment.

biochemistry↗

bioRxiv2025

Dichotomous SMAD2/3 regulation and selective anti-hypertrophic activity of heparin during in vitro chondrogenesis of mesenchymal stromal cells

BackgroundEndochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin-polyethylene glycol (PEG) hydrogels loaded with transforming growth factor beta (TGF-{beta}) instructed stable chondral MSC development in vivo. We here assessed this approach in vitro, utilizing heparin-PEG hydrogels or soluble heparin supplementation of chondrogenic medium. MethodsHuman MSCs were cultured in heparin-PEG hydrogels (22.4 mg/mL crosslinked heparin, 120ng TGF-{beta}1) or as hydrogel-free pellet cultures treated with soluble heparin (0, 10, 100, 700 g/mL) in TGF-{beta}1-containing (10 ng/mL) chondrogenic medium. Chondral and endochondral signaling (1-3 h, 4 weeks) and cartilage matrix formation (4 weeks) were analyzed using Western blot, histology, qPCR, ELISA, and enzyme activity. ResultsUnlike in vivo, human MSCs differentiated in heparin-PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treatment with soluble heparin (10-700 {micro}g/mL) revealed reduced TGF-{beta}-small mother against decapentaplegic (SMAD)3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/{beta}-catenin pathway in vitro but insufficiently silenced TGF-{beta}-SMAD1/5/9 activation and unfortunately reduced anti-hypertrophic prostaglandin E2 (PGE2) levels. Ultimately, treatment with 10 {micro}g/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP mRNAs, alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous anti-hypertrophic interventions (parathyroid-hormone related protein (PTHrP)-pulses, wingless-int (WNT)-inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation. ConclusionsTaken together, heparin-TGF-{beta} showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic power and context-dependent lineage-instructive properties: permitting endochondral differentiation in vitro but chondral development in vivo. Thus, environmental contributions are mandatory to allow heparin-PEG-guided chondral versus endochondral lineage commitment of MSCs in vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/673657v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@140313aorg.highwire.dtl.DTLVardef@160049dorg.highwire.dtl.DTLVardef@cee22corg.highwire.dtl.DTLVardef@62e961_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2025

Modeling the role of urokinase plasminogen activator, uPA, and circulating Cancer-Associated Fibroblasts (cCAFS) in breast cancer cell extravasation

Circulating Cancer-Associated Fibroblasts (cCAFs) have been discovered in circulating tumor cell clusters from all stages of disease progression in breast cancer patients. We have shown that CAFs promote lung metastases in the mouse tail vein model when they are clustered with triple negative breast cancer (TNBC) MDA-MB231 cells. Following on this observation, we saw that MDA-MB231-luciferase labeled cells persist at higher levels when present in CAF23/MDA-MB231 co-clusters compared to MDA-MB231 mono-clusters within the first 3 days after tail vein injection. This prompted us to investigate whether CAFs aid cancer cell extravasation from capillary venules into the lung parenchyma, which would impart better survival and faster seeding of metastases. Ex vivo lung extravasation assays showed that within the first 8-24 hrs after tail vein injection, more cells from CAF23/MDA-MB231 co-clusters extravasated than cells from MDA-MB231 mono-clusters. Using in vitro endothelial binding assays, we determined that CAF/TNBC co-clusters bind to HUVEC endothelial cells better than TNBC mono-clusters. Single Cell RNA-seq identified several genes in the fibrinolysis pathway whose expression increases in TNBC cells when they are clustered with CAFs. One of these genes is PLAU, which encodes the urokinase-type plasminogen activator, uPA. siRNA knockdown of PLAU decreased in vitro TNBC-endothelial cell interactions and ex vivo extravasation of MDA-MB231 mono-clusters, revealing a role for uPA/PLAU in breast cancer cell extravasation. Our data helps to define the role of CAFs in breast cancer extravasation and highlights the importance of our previous work showing that CAFs promote tumor cell dissemination and metastasis.

cancer biology↗