European Journal of Cell Biology

Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) cell phenotype and extracellular matrix, both processes linked to changes in cytoskeletal contractility and cell shape. Here, we tested whether microenvironment-specific modulation of RhoA signaling can restore NP-like morphology and gene expression in NP cells cultured in 2D and in 3D alginate. In 2D monolayer culture, where cells are spread and mechanically activated, pharmacologic inhibition of RhoA with CT04 reduced RhoA activity, decreased actomyosin contractility gene expression, and shifted morphology toward a smaller, more circular phenotype. Bulk RNA sequencing showed that CT04 treatment increased expression of NP phenotypic and matrix-related genes including ACAN, GDF5, CHST3, and MUSTN1 while decreasing expression of catabolic and fibroblast-associated genes including ADAMTS1/9 and COL1, consistent with enrichment of extracellular matrix pathways. In contrast, RhoA activation with CN03 in 2D culture increased actin and phosphorylated myosin light chain intensity but produced limited phenotypic improvement. In 3D alginate, which minimizes integrin-mediated adhesion, baseline actomyosin markers were reduced relative to 2D culture. In alginate, RhoA activation with CN03 increased the amount of actin, phosphorylated myosin light chain, and actomyosin gene expression, yet also promoted a more compact, circular morphology and increased NP markers, including ACAN and KRT19 with repeated dosing. Across culture conditions, increased cell roundness was consistently associated with increased ACAN expression, indicating strong coupling between cytoskeletal state, morphology, and NP matrix programs. Together, these findings demonstrate that RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D culture, but the direction of optimal modulation depends on the microenvironment, supporting RhoA signaling as a context-dependent therapeutic target for disc regeneration.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

Cells dynamically integrate biochemical and mechanical signals arising from their surrounding microenvironment to regulate morphology and behavior. Mechanical cues like matrix stiffness, surface topography, and other physical perturbations modify biophysical signals. Surface topography, particularly curvature regime acts as any important mediator of mechanotransduction by coordinating cytoskeletal organization, focal adhesion dynamics, and nuclear architecture. Curvature response has been demonstrated at broader length scales and influences nucleus shape change, chromatin organization, and gene regulation, positioning the nucleus as an active mechanosensitive hub. Bone tissue consists of a curvature-rich microenvironment defined by a trabecular architecture at tissue scale and by resorption cavities such as Howships lacunae at cellular scale. While these geometries are essential for homeostasis, their role in pathological context remains poorly understood. Osteosarcoma develops within this mechanically complex multiscale architecture, but how bone-inspired curvature regulates nuclear behavior and signaling in osteosarcoma cells remains unclear. Here, we engineered three-dimensional (3D) concave hemispherical substrates that recapitulate nucleus-scale bone micro-curvature and assessed their effects on human SaOS-2 osteosarcoma cells. In comparison with flat surfaces, concave confinement resulted in pronounced nuclear rounding and softening, accompanied by Lamin A/C reorganization and increased heterochromatin compaction marked by H3K9me3. Curvature-driven nuclear remodeling selectively modulated Hippo pathway main effectors YAP/TAZ without activating NF-{kappa}B mediated canonical inflammatory responses. Furthermore, cells maintained overall viability without elevated pathological DNA damage or apoptotic signaling, suggesting an adaptive, damage-tolerant nuclear response. Overall, these findings indicate nucleus-scale curvature as a critical regulator within the bone microenvironment that governs nuclear modelling and mechanosensitive signaling in osteosarcoma cells. Incorporating physiologically relevant geometry into in vitro models establishes new insight into cancer microenvironment crosstalk and highlights nuclear interior and outer architecture as a key regulator of tumor cell behavior.

Intervertebral disc (IVD) injury is a major cause of low-back pain and can lead to structural deficits and mechanical instability. When the IVD is compromised, neuromuscular compensation by paraspinal muscles, such as the multifidus (MF) and longissimus (ML), is critical for maintaining spine stability. However, it is unknown how IVD injury and its interaction with nociception affect neuromuscular control. This study assessed the effects of IVD injury and additional muscle-derived nociception on trunk motor control during locomotion in a rat model. IVD injury was induced via needle puncture at L4/L5. One week later, hypertonic saline was injected into the lumbar MF to induce nociception. Trunk and pelvic kinematics, bilateral EMG activity of MF and ML were recorded during treadmill locomotion at baseline, one week after IVD injury, and immediately following hypertonic saline injection. Trunk and pelvic kinematics and bilateral muscle activation patterns remained largely consistent across conditions. No significant changes were found in stride duration, pelvic, lumbar and spine angle changes, variability, or movement asymmetry. MF activation was bilaterally synchronized, whereas ML showed left-right alternating activation patterns. Following IVD injury, right MF mean activation and EMG variability increased significantly compared to baseline. When muscle-derived nociception was added in the unstable spine (IVD injury) condition, left MF minimum amplitude was significantly reduced, and instability-related increases in right MF mean activation and variability were attenuated, but not fully reversed. These findings suggest that IVD injury, alone or in combination with muscle-derived nociception, elicits localized neuromuscular adaptations without disrupting the global locomotor patterns.

Osteosarcoma (OS) is an aggressive bone cancer that most commonly affects children and young adults. OS exhibits a high degree of genomic complexity, as well as cellular plasticity, and dynamic transcriptional regulation is suggested to contribute to treatment resistance and metastasis. Cell lines are well characterized as models to advance our knowledge on OS biology. HOS and U2OS cells have increased invasiveness and higher migratory ability compared with MG63. In this study, we employed a tandem array of consensus transcription factor response elements (catTFREs) proteomic approach to characterize transcription factor (TF) regulatory networks related to OS aggressiveness. We mapped 7,594 proteins and enriched 352 transcription factors and coregulators. When we integrated proteomics with cell line specific gene expression and chromatin accessibility we classified the proteins into different OS cell line dependent sub-clusters and identified TFs and coregulators common for all cell lines and specific for individual cell lines. We demonstrate that RUNX2, MYBL2 and HMGA2 are specifically enriched in HOS and U2OS and may be linked to the cell aggressiveness. ETV5, JUNB, NFIX and ZEB1 were among TFs specific to MG63. Our analysis provides a more comprehensive understanding of the transcriptional drivers that shape OS regulatory landscapes and may have future therapeutic implications.

Bone morphogenetic proteins (BMPs) play an important role in dorsal spinal cord patterning. Their presence in the roof plate of the midbrain indicates a role in its development. We examined whether the BMP signaling contributes to dorsal midbrain size expansion in chick embryos by missexpressing pathway activators and inhibitors. Overactivation of BMP4 did not affect midbrain development, whereas GDF7 reduced midbrain growth. In contrast, expression of a truncated dominant-negative BMP receptor type 1b or the extracellular inhibitor Chordin had no detectable effect. Ectopic expression of SMAD6, the intracellular inhibitor of the BMP/ TGF-{beta} pathway, significantly reduced midbrain size, which correlated with decreased proliferation rates of SMAD6-overexpressing cells. In some cases, SMAD6 also disrupted MTN axon trajectory. These results indicate an important role for SMAD-dependent signaling pathways in early dorsal midbrain growth.

Osteoblasts generate bone by secreting collagen and mineralizing it in response to various signaling cues. We have previously shown that the majority of ATP generated by differentiated osteoblasts in response to glucose is through glycolysis in contrast to undifferentiated cells that are more dependent on oxidative phosphorylation. To confirm our previous findings, metabolomics was performed for unlabeled polar metabolites, revealing elevated glycolytic metabolites at the later stages of differentiation. Krebs cycle (TCA cycle) metabolites were also changed confirming metabolic rerouting with differentiation. We hypothesized that an increase in mitophagy shifts ATP generation towards glycolysis resulting in the observed bioenergetic and metabolic changes. Utilizing calvarial osteoblasts isolated from a mitophagy reporter mouse model (MitoQC), an increase in mitophagy and the mitophagy receptor, Bnip3, was observed with osteoblast differentiation. KD of Bnip3 in osteoblasts inhibited differentiation and mineralization arising from impaired mitochondrial function. In vivo, male Bnip3 null mice exhibited a significant decrease in osteoblast numbers resulting in lower bone mass. Mechanistically we identified decreased fusion and increased fission factors, impaired stress signaling and increased proapoptotic factors in the absence of Bnip3. These data demonstrate for the first time that BNIP3 expression and mitophagy during osteoblast differentiation are necessary for relieving mitochondrial stress to maintain optimal bone mass.

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

Congenital heart defects frequently arise from alterations in the elongation of the cardiac outflow tract (OFT). Proper elongation of the OFT depends on the coordinated deployment of progenitor cells from the second heart field (SHF) and on dynamic interactions with the extracellular matrix (ECM). Among ECM components, fibronectin (Fn1) and tenascin-C (TnC) have emerged as key regulators of cardiac morphogenesis. Studies in mouse embryos have shown that mesodermal Fn1 is required to maintain proper TnC localization within SHF cells. To study heart development, mammalian models are challenging to use because of their in utero development. This limitation highlights the need for alternative models with external development, where direct observation is possible; however, in these systems, the cellular organization of the SHF and the dynamics of its ECM environment remain poorly characterized Here, we investigated the cellular and extracellular architecture of SHF cells localized to the dorsal pericardial wall (DPW) during heart development in Xenopus laevis. We show that SHF cells undergo a stage-dependent transition from a predominantly monolayered organization at NF35 to a multilayered structure at NF42. This transition is accompanied by dynamic remodeling of the ECM, characterized by increased expression of Fn1, TnC, and Collagen I (ColI) and by redistribution of ECM components within the DPW. Functional experiments revealed that depletion of Fn1 disrupts cardiac morphogenesis, leading to shortening of the OFT and reduced ventricular size. Moreover, loss of Fn1 decreases TnC and ColI levels and alters the spatial organization of TnC within the DPW, indicating that Fn1 is required for proper ECM assembly within the SHF cells. These findings identify Fn1 as a key regulator of ECM assembly within the DPW and highlight how ECM remodeling contributes to the organization of SHF progenitor cells during OFT elongation. Altogether, we demonstrated that Xenopus laevis is a powerful model for studying ECM-driven mechanisms of cardiac morphogenesis.

ObjectiveTo validate SERPINB2 and SERPINA9 as chondrogenic biomarker candidates across independent transcriptomic platforms and cell sources, to characterise the complete SERPIN expression landscape during kartogenin (KGN)-induced chondrogenic differentiation of human mesenchymal stem cells (hMSCs), and to identify novel SERPIN biomarker candidates and their signalling context during cartilage lineage commitment. DesignMulti-platform transcriptomic analysis across three independent datasets: (i) Affymetrix HGU133+2 microarray of KGN-induced chondrocytes versus undifferentiated hMSCs (ATCC source); (ii) Affymetrix Clariom D whole-transcriptome array of KGN-treated versus control hMSCs from an independent Mexican source (SINREG Laboratories); and (iii) previously published qPCR validation. Differential expression was computed using limma with Benjamini,Hochberg correction. SERPIN-focused cross-platform correlation and targeted pathway analysis were performed. ResultsThe Clariom D dataset yielded 1,869 differentially expressed genes (925 upregulated, 944 downregulated; FDR < 0.05) from 29,124 transcripts tested. SERPINB2 was concordantly upregulated across all three platforms (Clariom D: fold-change [FC] +3.54, FDR = 0.006; HGU133+2: log2FC = +3.29, nominal P = 0.027; qPCR confirmed), establishing it as one of the most reproducible transcriptomic signals in chondrogenic differentiation. In the direct Bone versus Cart comparison, SERPINB2 showed [~]45-fold chondrogenic enrichment (log2FC = -5.45, adjusted P < 0.0001). Cross-platform SERPIN correlation was significant (Pearson r = 0.54, P = 0.0025; n = 29 shared genes). Four additional SERPINs reached genome-wide significance on Clariom D: SERPINE2 (FC +2.57), SERPING1, SERPIND1, and SERPINE1. SERPINA9 was not replicated in the independent SINREG source, identifying it as a context-dependent marker. ConclusionsSERPINB2 is a robust, cross-platform chondrogenic biomarker with translational potential for osteoarthritis (OA) monitoring. The coordinated SERPIN programme activates a multi-layered proteolytic and signalling network during cartilage lineage commitment, positioning SERPINB2 as a functional regulator of the chondro-osteogenic lineage decision.

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f059d9org.highwire.dtl.DTLVardef@9aeffforg.highwire.dtl.DTLVardef@27cd1org.highwire.dtl.DTLVardef@150b7d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

This retrospective study aims to explore the interactive effects of biological maturation and relative age effect (RAE) on talent identification. 56 male elite soccer players matched for chronological age (15.08{+/-}0.41 years) were studied. Test items included anthropometry (height, body mass, sitting height, leg length, BMI and Quetelet index), physiology (power, speed, agility, speed endurance and aerobic performance), soccer-specific skills (passing, shooting and dribbling), psychology (achievement motivation, orientation and resilience) and biological maturation (APHV) tests. The test results were analyzed independent sample t-test, Pearson correlation analysis, and stratified regression. Conclusion: Biological maturation significantly influences anthropometry (height, weight and Quetelet index), lower limb explosive, and speed (single-leg jump, standing triple jump, and 30-m sprint) in U16 male elite soccer players in Shanghai. The relative age effect shows no significant impact on talent selection indicators, which is attributed to the accumulated training load effect. The mechanisms of biological maturation and RAE in youth soccer talent selection are distinct and operate independently.

BackgroundPeri-synaptic astrocyte processes (PAPs) play a fundamental role in synapse formation and function. Central afferent synapse loss and astrocyte dysfunction greatly impede sensory-motor circuitry in spinal muscular atrophy (SMA) disease progression, however mechanisms underpinning tripartite synapse dysfunction remains to be fully elucidated. The aims of this study were to further define PAP and motor neuron synaptic defects in human SMA disease pathology and implement a therapeutic intervention strategy to improve motor neuron function. MethodsWe derived astrocyte monocultures and motor neuron astrocyte co-cultures from healthy and SMA patient induced pluripotent stem cell (iPSC) lines to assess intrinsic astrocyte filopodia defects and phenotypes occurring at the synapse-PAP interface, respectively, using cell surface capture mass spectrometry proteomics, confocal and super resolution microscopy, synaptogliosome isolation, and electrophysiology. ResultsSMA astrocytes demonstrated intrinsic filopodia actin defects featuring low abundance of actin-associated cell surface N-glycoproteins, and decreased filopodia density and CDC42-GTP levels after actin remodeling stimulation. This phenotype is likely driven by the significant reduction of CD44 and phosphorylated ezrin, radixin and moesin ERM proteins (pERM) within SMA astrocyte filopodia. The dual combination of SMN1 gene therapy and forskolin treatment, an adenylyl cyclase activator leading to increased cyclic adenosine monophosphate (cAMP) levels and actin signaling pathway stimulation, led to extensive branching and increased filopodia density of SMA astrocytes during actin remodeling. SMA patient-derived motor neuron and astrocyte co-cultures, particularly samples derived from male patient iPSC lines, demonstrated a significant decrease in synapse number, actin-associated pre-synaptic neurotransmitter release protein, synapsin I (SYN1), and PAP-associated expression of pERM and glutamate transporter, EAAT1. Our astrocyte-targeted SMN1 augmentation and forskolin treatment paradigm restored SYN1 protein levels within the SMA synaptogliosome, resulting in significant increases in motor neuron synapse formation and function, but did not fully restore PAP-associated proteins levels at the synapse. ConclusionsSMA astrocytes demonstrate intrinsic actin-associated defects within filopodia, which correlates with decreased pERM levels at tripartite motor neuron synapses. We also define a SMN- and cAMP-targeted treatment paradigm that significantly increases pre-synaptic neurotransmitter release protein levels to improved SMA motor neuron synapse formation and function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/714618v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1257ab8org.highwire.dtl.DTLVardef@19c0010org.highwire.dtl.DTLVardef@c84552org.highwire.dtl.DTLVardef@3f1e62_HPS_FORMAT_FIGEXP M_FIG C_FIG

The association between higher levels of physical activity and lower cancer risk and mortality is well established. However, a causal link is yet to be proven. Recent studies showed a decrease in the proliferation rates of cultured human cancer cells when the human serum employed to stimulate them was conditioned by acute exercise. Here, we tested the hypothesis that serum mediates some of the putative benefits of exercise on cancer through alterations to the growth pattern and susceptibility to chemotherapy agents of cancer cells. To this end, human non-small cell lung cancer (NSCLC) cells were exposed to serum from two cohorts that differed significantly on their levels of physical activity and, accordingly, cardiorespiratory fitness, but were otherwise identical (master athletes and non-exercisers), collected before and after an acute exercise intervention. Serum levels of glucose, lipids, albumin, C-reactive protein and cytokines were determined and the impact of the serum responses to acute and lifelong exercise on the above-mentioned parameters were analyzed. We found that acute exercise decreased the cells proliferation rate, yet shortened the cells lag phase after detachment, whereas lifelong exercise had the opposite effects. Significantly, we showed, for the first time, that lifelong exercise increased susceptibility to a chemotherapy agent (cisplatin), which may contribute to the decreased cancer mortality rates found among those who exercise regularly. Similar to the cellular effects, changes to serum cytokine levels - several of them linked to the senescence-associated secretory phenotype - depended on whether serum was conditioned by acute or by chronic exercise. Key pointsChronic exercise increased the in vitro susceptibility of lung cancer cells to cisplatin. Acute and chronic exercise modulated the in vitro tumorigenic potential of lung cancer cells. Effects were mediated by serological changes produced by exercise. Acute and chronic exercise had distinct impacts on serological cytokine levels.

Development of motoneurons from stem cells is characterized by a change from glycolytic to oxidative metabolism. Since this transition remains poorly understood, we examined it at five distinct differentiation stages from hiPSC to motoneuron. While a direct comparison of hiPSCs and mature motoneurons confirmed the expected glycolytic-to-oxidative shift, the intermediate stages showed that the conversion was not monotonic. After an initial drop of glycolysis at the hiPSC-to-neuroepithelial transition, late neuroepithelial cells showed intermittent peaks of the glycolytic marker lactate dehydrogenase A and the metabolic regulator TIGAR. Furthermore, the lactate-produced-to-glucose-consumed ratio remained elevated. A fully oxidative phenotype was only assumed upon progress from neural progenitors to motoneurons, portrayed by a definitive drop of the lactate-produced-to-glucose-consumed ratio, an increase of mitochondrial membrane charging, and shifts from lactate dehydrogenase A to B, from pyruvate dehydrogenase to anaplerotic pyruvate carboxylase, and from Mitofusin 1 to 2. Together, our data show that metabolic maturation in human motoneurons does not occur as a simple switch. Instead, it unfolds through distinct stages in a directional yet nonlinear manner.

Macrophages/osteoclasts are highly fusogenic cells that interact closely with bone-metastatic breast cancer cells. These cancer cells adapt to bone microenvironments by undergoing osteomimicry, acquiring bone-like phenotypes. Exploration using human breast cancer-bone metastases dataset revealed that a small population of epithelial breast cancer cells express osteoclast-like and osteomimicry genes at the single-cell level. Cell fusion and cell-in-cell (CIC) processes are two uncommon yet prognostically significant mechanisms in cancer. We showed that co-culture between murine breast cancer cells and osteoclasts yielded a unique osteoclast phenotype through dynamic cell-in-cell (CIC) interactions and fusion-like behaviours between pre-osteoclasts/mature osteoclasts and breast tumor cells, resulting in osteoclast-tumor hybrid-like cells. These tumor cell interactions characterized by membrane retention and nuclear adjacency to host nuclei were consistently observed throughout osteoclast differentiation. Single-cell sequencing analysis and interpretative assays on hybrid-like cells revealed altered extracellular matrix (ECM) modification processes, immunoregulatory, and cancer-associated pathways compared to unfused osteoclasts. Tumor cells co-cultured with osteoclasts expressed hematopoietic and osteoclast-lineage factors more strongly than tumor cells cultured alone with their effects amplified under direct cell-cell contact. The presence of these hybrid-like cells was validated in human breast cancer-bone metastases. We propose that disseminated bone-tropic breast cancer cells were stimulated by osteoclasts to undergo a non-canonical, dynamic osteoclast differentiation and CIC formation to form hybrid-like cells that may facilitate bone metastatic lesions.

Postnatal growth of the mandibular condyle requires coordinated expansion of fibrocartilage and production of chondrocytes, yet the cellular populations that organize this process remain incompletely defined. Here we identify a Wnt-responsive fibrocartilage progenitor population that contributes to postnatal mandibular condylar cartilage growth. Using a direct Wnt activity reporter (R26-WntVis), inducible genetic lineage tracing (Axin2CreERT2), and single-cell transcriptomics, we define a Wnt-enriched progenitor-like cluster localized predominantly within the fibrocartilage zone. Lineage tracing demonstrates that Axin2-lineage cells expand laterally within fibrocartilage and generate vertically aligned chondrocytes in the chondrocartilage compartment, indicating bidirectional growth contribution in vivo. Conditional ablation of {beta}-catenin in Axin2-lineage cells results in depletion of the fibrocartilage compartment and premature activation of chondrogenic differentiation programs, whereas constitutive {beta}-catenin activation disrupts compartmental organization without enhancing proliferation. Mechanistically, we identify Foxm1 as a Wnt-associated proliferative mediator enriched in fibrocartilage, and genetic reduction of Foxm1 cooperates with {beta}-catenin deficiency to impair condylar growth. In parallel, {beta}-catenin loss derepresses TGF-{beta}-Smad signaling and enhances chondrogenic differentiation, indicating that canonical Wnt activity coordinates proliferative maintenance while restraining lineage commitment within the same cellular compartment. Together, these findings identify a Wnt-responsive fibrocartilage progenitor system that regulates postnatal mandibular condylar cartilage growth by coupling Foxm1-associated proliferative maintenance with suppression of TGF-{beta}-dependent chondrogenic differentiation during temporomandibular joint development. Graphical abstractWnt-responsive fibrocartilage progenitors coordinate postnatal mandibular condylar cartilage growth through Foxm1-dependent proliferative maintenance and suppression of TGF-{beta}-driven chondrogenic differentiation.

IntroductionThe exploration of alternative strategies for neural tissue regeneration and repair is giving rise to a novel paradigm in neurosurgery: fusogenic therapy. This approach promises rapid restoration of peripheral nerve and spinal cord function by circumventing Wallerian degeneration and eliminating the delay associated with axonal regrowth. Its potential stems from the capacity of fusogens to induce axonal fusion and achieve immediate membrane sealing, complemented by their pronounced neuroprotective properties. However, experimental data on fusogens and their effects are inconsistent, often contentious, and derived using heterogeneous methodologies. MethodsWe present the first comprehensive systematic review covering nearly four decades of research on fusogens for axonal membrane repair and 26 years of their experimental and clinical application in mammalian and human models for peripheral and central nervous system restoration. The review includes a meta-analysis of fusogen efficacy following traumatic spinal cord and peripheral nerve injuries. ResultsConducted in accordance with the PRISMA 2020 flow protocol and PICO criteria, our analysis incorporates 86 sources, 20 of which were included in the meta-analysis. DiscussionIn summary, we have systematized the prevailing approaches and methods for fusogen application, delineated key contentious issues, and identified promising directions for the development of axonal fusion technology.