BMC Molecular and Cell Biology

In this study, we evaluated the impact of different in vitro 3D culture modelling methods on the activity of doxorubicin (DOX) and 5-fluorouracil (5-FU) in human melanoma spheroids. Human melanoma A375 and IGR39 spheroids were generated using the hanging drop and non-adhesive surface methods. Spheroid growth dynamics were assessed by measuring changes in spheroid diameter. To compare the effects of anticancer drugs in spheroids of different sizes, spheroids of approximately 200 and 400 {micro}m were formed. Drug activity was evaluated based on spheroid growth and cell viability using the MTT assay. A375 spheroids formed using the non-adhesive surface method were more sensitive to DOX than spheroids formed using the hanging drop method. In smaller A375 spheroids, 10 {micro}M 5-FU reduced cell viability more effectively in spheroids formed using the hanging drop method. In contrast, IGR39 spheroids formed by the hanging drop method were more resistant than those formed on a non-adhesive surface. However, in IGR39 spheroids, the effects of DOX and 5-FU on growth and viability did not significantly differ between formation methods. In conclusion, A375 spheroid growth was not significantly influenced by the formation method, whereas IGR39 spheroid growth depended on the method used. A375 spheroids formed on non-adhesive surfaces were more sensitive to DOX, whereas 5-FU activity depended on drug concentration and spheroid size. In IGR39 spheroids, the effects of DOX and 5-FU on growth and viability were largely independent of the spheroid formation method. Based on these results, it can be concluded that the researchers should carefully select the spheroid formation method for their studies, as this may influence the results of the tested compounds effect on their size and viability.

Adenoid cystic carcinoma (ACC) of salivary gland is a "immune-cold" tumour. Annexin A3 (ANXA3) is an apoptotic protein found to be participating in immune cell infiltration in tumour microenvironment (TME) of various cancer cases. Significant low expressions of ANXA3 protein found in adenoid cystic carcinoma. We hypothesized overexpressing ANXA3 transforms ACC "cold" TME to "hot". We cultured UM-HACC-2A and UFH2 spheroids on extracellular matrix and co cultured them with peripheral blood mononuclear cells. We functionalized FDA (The Food and Drug Administration) approved Poly(lactic-co-glycolic acid) PLGA nanoparticles with anti-cMyb antibody and ANXA3 recombinant protein using streptavidin-biotin conjugation. Upon overexpressing ANXA3 in ACC spheroids in immune coculture model using functionalized nanoparticles, significant increase of tumour infiltrating lymphocytes and decrease in the size of the ACC spheroids observed. Apoptotic profiler assay further confirmed significant upregulation of apoptotic proteins, some of them participate in immune infiltration. Overall, this project exhibits promising results showing potential approach to convert ACC into an immune "hot" tumour.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory airway disease associated with impaired mucociliary clearance and persistent inflammation. While prior work has focused on inflammatory and molecular pathways, the physicochemical properties of mucus itself remain poorly characterized. This study aimed to define compositional and biophysical features of CRS mucus that may contribute to dysfunction. Methods: A prospective cross-sectional study was conducted in 15 adults undergoing endoscopic sinus surgery (11 CRS, 4 controls). Mucus was collected from the middle meatus. Hydration was measured by lyophilization. Ionic composition was quantified using mass spectrometry. Viscoelasticity was assessed via oscillatory shear rheology. Total protein, total carbohydrate, sialic acid (Sia) and fucose (Fuc) content were quantified using enzymatic and chemical assays. Statistical comparisons were performed using nonparametric tests. Results: CRS mucus exhibited significantly higher Ca2+; and Mg2+; concentrations (approximately two-fold; p<0.05) and increased variability in hydration and ion content compared to controls. Rheology showed greater heterogeneity and a non-significant trend toward increased viscoelasticity in CRS. Total protein and carbohydrate content were not significantly different; however, the carbohydrate-to-protein ratio was significantly reduced in CRS (p=0.04). Sia content and Sia-to-carbohydrate ratio were significantly elevated in CRS (p=0.04 and p=0.002), particularly in CRS with nasal polyps. Fuc content did not differ between groups. Conclusions: CRS mucus demonstrates coordinated alterations in ionic composition and glycosylation, characterized by increased cation content, hypersialylation, and reduced carbohydrate-to-protein ratios. These changes may contribute to altered mucus properties and impaired mucociliary clearance, highlighting mucus composition as a potential therapeutic target in CRS.

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.

BackgroundThe use of autologous or allogeneic cell therapies has now entered to the clinical practice in several fields of medicine, especially in oncology and hematology. From this regard, 2D-cell manufacturing is complex and costly and bioreactors have attracted major interest for efficient and cost-effective mass production of cells. Bioreactors have several advantages such as homogeneous repartition of nutrients and gas, control of all culture parameters and increased yield. However, the important shear stress generated by those bioreactors is an important disadvantage as it can affect cell survival or cell quality. This important shear stress is the result of the mixing method using either blades (used in stirred-tanked bioreactors) or gas bubbles (used in airlift bioreactors). Another downside of the use of bioreactors is the difficulty to scale-up. As the volume increases, the shear stress generated by blades radically increases leading to cell death and a decrease of cell quality. DescriptionIn this study, we describe a bioreactor developed using a different mixing method effectively reducing the shear stress and facilitating scale-up. This bladeless method uses an inclination of the bioreactor as well as rotation to mix fluids in a container. Here we described different steps that led to the adaptation of this bioreactor, initially developed for fragile microalgae culture, for mammalian cell culture amplification. The bioreactor was tested to amplify a natural killer (NK) cell line NK92 which is an IL-2 dependent cell line used in clinical trials for cancer therapy. We have tested the influence of 1-The number of cells seeded; 2-The influence of the rotation speed on cell growth and viability; 3-The influence of the bioreactor angle on the above parameters; 4-The duration of the culture. ResultsCells were initially seeded at 2.5.105 / ml in a volume of 380 ml. According to the rotation speed of 15, 30, 45 and 60 rpm, we have observed an increase of cell numbers at day 3 (3-fold), day 5 (7-fold) and day 7 (10-fold) compared to seeding, the best expansion being obtained at day 7 with a rotation speed of 45 rpm. The optimal angle of rotation was found to be 3 degree, with an optimal amplification at day 7 versus day 3 (p < 0.01). The viability was also found to be optimal in the latter condition. ConclusionsThese preliminary results demonstrate that NK92 cells could be amplified using this bioreactor. In the best tested condition, neither cell viability nor cell growth was impacted. These results strongly suggest the potential use of this device in future clinically applicable conditions.

Acidification of the oral environment has been implicated in the initiation and progression of oral pathologies including oral cancer, but how acidic environments modulate normal oral epithelial cell (OEC) responses to microbial ligands is not understood. This study examined the impact of acidic stress on OEC morphological, molecular, and functional responses to toll-like-receptor ligand engagement in vitro. OEC cultures were exposed to either normal (pH:=:8.0) or acidified growth media (pH:=:3.0) for 24 hours prior to machine-learning-guided morphological analysis and exposure to either toll-like receptor (TLR)5 (flagellin) or TLR2/TLR1 (Pam3CSK4) agonists. Multiplex gene expression technology was used to quantify the transcriptional responses of metabolic-and immune-related genes at 6 hours post-TLR agonist exposure. OEC-mediated production of transforming growth factor-beta (TGF-{beta}) was assessed by enzyme-linked immunosorbent assay at 2-, 6-, and 24-hours post-agonist exposure. Results showed that acid exposure induced significant changes to OEC morphology resembling epithelial-mesenchymal transition, the differential expression of n=197 metabolic-and n=43 immune-related genes and significantly increased OEC TGF-{beta}1 production. The results demonstrate that acid stress skews normal OECs towards pro-inflammatory and pro-oncogenic phenotypes when faced with concomitant microbial ligand challenge and provide key molecular clues to OEC survival strategies with potential implications for elucidating the early molecular events in the development of epithelial dysplasia. Article HighlightsO_LIAcute acid exposure reduces survival of OECs C_LIO_LIA subpopulation of OECs is resistant to acid-mediated cell loss and undergo morphometric changes consistent with epithelial-mesenchymal transition C_LIO_LIConcurrent acid stress and TLR stimulation modulates transcription of immune and metabolic genes in OECs C_LIO_LIAcid stress increases TGF-{beta}1 protein production of OECs following TLR agonist stimulation C_LI

Intestinal organoids are three-dimensional in vitro structures derived from stem cells and serve as a valuable model for studying intestinal biology and pathophysiology. This study optimized the isolation, expansion, and differentiation of canine intestinal organoids from duodenum and colon. Organoids were generated from canine intestinal crypts and cultured in Matrigel with a growth factor cocktail. The impact of prostaglandin E2 (PGE2) concentration on organoid growth was evaluated, and a two-phase differentiation protocol--comprising patterning and differentiation media--was implemented, including interleukin (IL)-22 in the duodenal differentiation phase. Organoids cultured with 100 nM PGE2 exhibited increased crypt budding and organoid-forming efficiency, indicative of enhanced stem cell proliferation. Differentiated organoids expressed key intestinal markers (VIL1, SI, CHGA, MUC2), and forskolin-induced swelling demonstrated functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activity. Although the sample size (n=2) limits generalizability, this optimized protocol provides a relevant in vitro model for studying canine intestinal function. The model can be used in future research for disease modelling and translational applications, supporting downstream studies in gastrointestinal disease, drug permeability, and comparative One Health research.

Lymphocyte-extracellular matrix (ECM) interactions occur intermittently throughout the lymphocytes life cycle. Alterations in blood insulin levels following feeding modulates naive lymphocyte trafficking and adhesion to fibronectin via a pathway involving insulin-like growth factor-1 receptor (IGF-1R), phospholipase C gamma 1 (PLC-{gamma}1) and {beta}2 integrin activation. Lymphocytes exert traction forces, on the ECM during the process of extravasation. While these forces are essential for several homeostatic processes, the role of insulin in modulating lymphocyte-derived traction forces upon ECM adhesion is unknown. The aim of the current study was to investigate the effect of insulin on the traction generated by lymphocytes when adhered onto a fibronectin-coated substrate. Jurkat T-cells were placed on a fibronectin layer (50{micro}g/ml, 100{micro}m thickness) coated on polyacrylamide gels of stiffness 400Pa with red fluorescence beads as fiduciary markers. The cellular force generated by Jurkat T-cells was mapped using traction force microscopy. To elucidate the role of PLC-{gamma}1 in cellular force generation, the traction of Jurkat T-cells lacking PLC-{gamma}1, as well as those of a knockout cell where PLC-{gamma}1 was restored were quantified and compared with wild-type Jurkat T-cells. Lack of PLC-{gamma}1 attenuated adhesion when compared to wild-type Jurkat T-cells. Additionally, the traction force generated by each cell type decreased with increasing concentration of extracellular calcium. Treatment of adherent Jurkat T-cells with insulin increased traction in lower extracellular calcium condition while a dip was observed when a high extracellular calcium was present, in comparison to the untreated cells. However, the effect of insulin treatment was lost in the case of Jurkat T-cells lacking PLC-{gamma}1. Together these results indicate that insulin regulates traction force generated by adherent Jurkat T-cells via a process involving PLC-{gamma}1, in a calcium dependent manner.

In mice, oral epithelial stem cells (OESCs) are essential for oral mucosal homeostasis and repair. Less is known regarding the role of OESCs in the human oral mucosa. Here, we studied the behaviour of OESCs and their contribution to tissue maintenance and repair in oral lichen planus (OLP). OLP is a chronic T cell-mediated disease characterized by basal keratinocyte degeneration, epithelial atrophy, acanthosis, and hyperkeratosis. Using immunohistological techniques and semi-automated image analysis, we observed that in OLP proliferative activity was increased in the normally largely quiescent basal cell compartment. In areas of OLP mucosa with intact basal cell layer, expression of NGFR, KRT15, and KRT19-markers of slowly cycling reserve OESCs, was strongly reduced or absent. In contrast, expression of CSPG4, a marker for actively cycling stem cells, was increased in OLP basal cells. Tissue compartmentalization, as evaluated by keratin expression, was strongly disturbed. Taken together, our findings indicate that the inflammation in OLP leads to activation and proliferation of OESCs that give rise to a population of cells with an aberrant differentiation programme. Along with the well-documented epithelial up-regulation of anti-apoptotic proteins in OLP, this likely reflects an attempt by the epithelium to avoid overt ulceration.

Stress Granules (SGs) are cytoplasmic biomolecular condensates that form in response to a variety of stress conditions, though their function remains unclear. "Canonical" SGs - caused by stressors like sodium arsenite - are dynamic and cytoprotective, allowing cells to evade cell death during periods of stress. Ultraviolet (UV) irradiation is known to elicit a "non-canonical" SG subtype, lacking canonical SG components such as eukaryotic initiation factor 3 and polyadenylated mRNAs. The exact function of UV SGs, and the mechanisms driving their formation, remain unknown. Here we report the findings of a comparative analysis of UVA, UVB and UVC exposures on SG formation in three cell types: osteosarcoma (U2OS), keratinocytes (HaCaT), and mouse embryonic fibroblasts (MEF). We observed that SG formation in response to UV is highly cell type dependent. UVB and UVC induce robust SG formation in U2OS cells. However, only UVC exposure induced modest SG formation in MEFs, and none of the wavelengths caused SGs in HaCaT. While UVC-induced SGs in U2OS cells appear to be cell cycle dependent and specific to G1, UVB induced SG formation regardless of cell cycle stage. We tested the hypothesis that oxidative stress triggered by UV may be driving UV SG formation, and that keratin may buffer this effect, by overexpressing keratin in U2OS. Interestingly, we found that keratin and antioxidant treatment efficiently suppressed arsenite-induced SGs but had no effect on UV SGs. Our work confirms that UV SG formation is cell type specific and is not driven by oxidative stress.

Cells are mechanosensitive, responding to external mechanical stimulation. Nanovibrational stimulation has been shown to enhance cell contractility and actin stress fibre formation. These changes in morphology occur quickly, alongside associated mechanical changes. Here, the relationship between acute morphological and mechanical changes in NIH 3T3 fibroblastic cells in response to nanovibrational stimulation is presented. A 1 kHz, 30 nm vibration is applied continuously for 72 hours. Atomic force microscopy (AFM) quantifies mechanical properties of the nucleus and cytoplasm at multiple timepoints, while immunofluorescence tracks morphological changes. Within 3 hours of stimulation, both nuclear and cytoplasmic stiffness increase significantly, accompanied by a decrease in the cellular fluid exponent, suggesting a shift of the cell towards more solid-like behaviour. These changes correlate with increased nuclear area. Actin polymerisation also increases within 24 hours, although variably. To understand the role of the cytoskeleton, actin polymerisation and contraction are inhibited using cytochalasin D and blebbistatin. Results show that inhibition prevents stiffness increases and results in a higher fluid exponent, indicating a more fluid-like state. These findings demonstrate that actin-myosin dynamics mediate cell stiffening under nanovibrational stimulation. Interestingly, prolonged stimulation appears to reverse this effect, suggesting that temporal optimisation of stimulation may enhance long-term mechanotransducive responses.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Purine moieties are essential for many functions within the eukaryotic cell, including energy, signaling and nucleic acid synthesis. While purine starvation is known to induce stress resistance in eukaryotic model organism budding yeast Saccharomyces cerevisiae, it remains unclear whether the physiological response is related to disruption of synthesis pathway in particular position or it is uniform across all genetic deficiencies within the de novo adenine biosynthesis pathway. It is also not known how purine starved cells perceive purine shortage - weather they share the same signaling elements with nitrogen starvation or not. MethodsWe characterised physiology of strains with deletions in adenine biosynthesis pathway when cultivated in full or purine deficient and compared to cell physiological parameters when cultivated in nitrogen deficient media. We tested stress tolerance, carbon flux, cell cycle arrest and did transcription profiling (RNA-seq). ResultsOur findings demonstrate that purine starvation-induced stress resistance is significantly modulated by the specific step at which the pathway is interrupted. Transcriptional analysis revealed that purine starvation in many aspects phenocopies nitrogen starvation, particularly - in both starvations strong downregulation of ribosome related genes occurs. In the same time several metabolic features which differ from N- and ade- starvations: pentose phosphate pathway is specifically upregulated within ade4{Delta}-ade2{Delta} and downregulated in N-cells. Notably, the expression of stress-responsive genes such as HSP12, HSP26, and GRE1 varied between mutants, suggesting that the accumulation of pathway intermediates (e.g., AIR in ade2{Delta}) or the absence of downstream precursors (AICAR) alters the perception of starvation especially in the case of ade16{Delta}ade17{Delta} strain. ConclusionsMetabolic and stress-tolerance phenotypes of purine auxotrophs are not merely a result of purine depletion but seems that the response is signalled via the same pathways, like TOR1. The results suggest that strains having mutations within various positions of the purine pathway "perceive" purine limitation a bit differently - especially when we compare the end of the pathway with the other mutants. Different phenotypic outcomes of the occasional purine depletion might give preferences for organisms which have mutations in the beginning rather at the end of the pathway. Besides, our findings might have implications in the design of synthetic pathways and the use of auxotrophic markers in yeast research.

Ergosterol has multiple functions in filamentous fungi and yeasts, although only a part of the functions seems to be understood. An antifungal peptide, theonellamide A (TNM-A) induces drastic morphological changes in fission yeast cells by targeting plasma membrane ergosterol. TNM-A induces overproduction and ectopic accumulation of cell wall glucan at both growing tips and septum through a yet unknown mechanism. Here we show that TNM-A treatment causes accumulation of 1,3-{beta}-glucan at cell-polarity sites, not by increased activity of 1,3-{beta}-glucan synthase, but by an increased, persistent localization of the glucan synthase enzymes. Screening based on subcellular localization of proteins at periphery or polarity sites suggested the involvement of the Rho family GTPase Cdc42. In agreement, TNM-A induced both activation of Cdc42 and enhancement of membrane trafficking of glucan synthase enzymes. In conclusion, our chemical genetics analyses using TNM-A suggest that membrane ergosterol regulates the activity of Cdc42, which further regulates the localization of glucan synthases and cell wall biosynthesis. Highlights (four sentences)- Thenoellamide A (TNM-A) induces an ectopic overproduction of cell wall glucan. - TNM-A treatment causes increased, persistent localization of glucan synthases at the cell tips and septum. - TNM-A activates Cdc42 and upregulates membrane trafficking of glucan synthases. - Ergosterol is involved in proper activation/inactivation of Cdc42.

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

BackgroundSkin is constantly exposed to mechanical forces such as pressure and friction, which need to be sensed and buffered to ensure tissue homeostasis and barrier function. Desmosomes are essential for epidermal integrity, but their role in converting mechanical cues into cellular signaling responses are not well understood. MethodsHere, we combine proteomics and shear-stress assays with live-cell reporters to investigate how desmosomes modulate stress-kinase pathways in keratinocytes. ResultsWe show that the desmosomal adhesion molecule DSG3 is essential not only for cell-cell adhesion but also for modulating p38MAPK and ERK signaling. Loss of DSG3 disrupts mechanotransduction-related protein networks, including the expression of the mechanosensitive channel Piezo1. Under static conditions, DSG3 dampens ERK activity via Piezo1-dependent mechanisms, whereas DSG3 suppresses p38MAPK activity through an independent mechanism. In contrast, DSG3 is required to trigger an activation of both ERK and p38MAPK in response to shear stress in a Piezo1-dependent manner. Experiments with domain-specific DSG3 mutants demonstrate that cell cohesion and signaling responses are partially uncoupled, while maintaining DSG3 tail integrity was crucial for p38MAPK and ERK responses. ConclusionThese findings demonstrate that DSG3 independently coordinates adhesion and mechanotransduction in a domain-specific manner, providing novel insights into how DSG3 contributes to epithelial integrity under dynamic mechanical environments.

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

We present a novel protocol for intranasal colonization of germ-free mice with human nasal microbiota, coupled with an optimized DNA extraction method for murine nasal samples compatible with 16S rRNA sequencing. This protocol may facilitate investigation of the functional roles and causal effects of nasal microbial communities in chronic rhinosinusitis and antimicrobial resistance.

ObjectiveTo 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel. MethodsThe presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGF{beta}1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability. ResultsPDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGF{beta}1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD. ConclusionUrethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD. HighlightsO_LIPDE4 isozymes A, B, and D are expressed in adult anterior urethras C_LIO_LIPDE4 is expressed equally from proximal bulbar to meatal urethra C_LIO_LIEpithelial cells and fibroblasts can be cultured from healthy and stricture urethra C_LIO_LITGF{beta}1 may not be an optimal method to model aUSD in vitro C_LIO_LIUnlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells C_LI

Mesenchymal stem cells (MSCs) have garnered significant attention over the past three decades due to their robust regenerative potential, primarily mediated by their paracrine activity by releasing soluble bioactive factors and extracellular vesicles (EVs). The MSC secretome plays a pivotal role in wound healing by influencing cellular migration, inflammation, angiogenesis, extracellular matrix (ECM) remodeling, and re-epithelialization. SPARC (Secreted Protein Acidic and Rich in Cysteine), a multifunctional ECM glycoprotein involved in tissue repair and remodeling, regulates key processes such as cell migration, proliferation, angiogenesis, and survival. Despite its known role in ECM dynamics, the impact of SPARC expression on the regenerative properties of MSCs remains underexplored. In this study, we hypothesized that SPARC overexpression in MSCs enhances their secretomes regenerative capacity. Using lentiviral systems, we generated SPARC-overexpressing (+SPARC) and SPARC-knockdown (KD-SPARC) MSCs to investigate SPARCs role in wound healing. Conditioned media (CM) derived from these MSCs were analyzed in vitro for their effects on human skin keratinocytes and fibroblasts. Our results revealed that SPARC expression significantly influences cell-specific migration and cell cycle. Furthermore, in an in vivo wound healing model, CM from +SPARC MSCs accelerated regeneration, while SPARC absence in MSCs CM delayed the healing process. These findings underscore the critical role of SPARC in modulating MSC secretome composition and enhancing its regenerative efficacy. This study highlights SPARC as a promising therapeutic target for the development of advanced regenerative therapies aimed at improving cutaneous wound healing outcomes.