Inducing Cellular Senescence in Mouse Embryonic Fibroblasts (MEFs).

Inducing cellular senescence in mouse embryonic fibroblasts (MEFs) is a robust tool to study the molecular mechanisms underlying senescence establishment and their heterogeneity. This protocol provides a detailed guide to generate MEFs and routinely induce senescence in MEFs using several DNA damage-dependent and DNA damage-independent induction methods.

Investigation of Protein Lysine Acetylation in Antiviral Innate Immunity.

Protein lysine acetylation involved in the antiviral innate immunity contributes to the regulation of antiviral inflammation responses, including type 1 interferon production and interferon-stimulated gene expression. Thus, investigation of acetylated antiviral proteins is vital for the complete understanding of inflammatory responses to viral infections. Immunoprecipitation (IP) assay with anti-targeted-protein antibody or with acetyl-lysine affinity beads followed by immunoblot provides a classical way to determine the potential modified protein in the antiviral innate pathways, whereas mass spectrometry can be utilized to identify the accurate acetylation lysine residues or explore the acetyl-proteomics. We demonstrate here comprehensive methods of protein lysine acetylation determination in virus-infected macrophages and embryonic fibroblast cells or proteins-overexpressed HEK 293 T cells in the context of antiviral innate immunity.

Metformin represses the carcinogenesis potentially induced by 50 Hz magnetic fields in aged mouse fibroblasts via inhibition of NF-kB.

Aging is a risk factor for various human disorders, including cancer. Current literature advocates that the primary principles of aging depend on the endogenous stress-induced DNA damage caused by reactive oxygen species 50 Hz low-frequency magnetic field was suggested to induce DNA damage and chromosomal instability. NF-kB, activated by DNA damage, is upregulated in age-related cancers and inhibition of NF-kB results in aging-related delayed pathologies. Metformin (Met), an NF-kB inhibitor, significantly reduces both NF-kB activation and expression in aging and cancer. This in vitro study, therefore, was set out to assess the effects of 5mT MF in 50 Hz frequency and Met treatment on the viability and proliferation of aged mouse NIH/3T3 fibroblasts and expression of RELA/p65, matrix metalloproteinases MMP2 and MMP9, and E-cadherin (CDH1) genes. The trypan blue exclusion assay was used to determine cell viability and the BrdU incorporation assay to determine cell proliferation. The MMP-2/9 protein analysis was carried out by immunocytochemistry, NF-kB activity by ELISA and the expressions of targeted genes by qRT-PCR methods. Four doses of Met (500 uM, 1 mM, 2 mM and 10 mM) suppressed both the proliferation and viability of fibroblasts exposed to the MF in a dose-dependent pattern, and the peak inhibition was recorded at the 10 mM dose. Met reduced the expression of NF-kB, and MMP2/9, elevated CDH1 expression and suppressed NF-kB activity. These findings suggest that Met treatment suppresses the carcinogenic potential of 50 Hz MFs in aged mouse fibroblasts, possibly through modulation of NF-kB activation and epithelial-mesenchymal transition modulation.

Decellularized extracellular matrix derived from dental pulp stem cells promotes gingival fibroblast adhesion and migration.

BACKGROUND: Decellularized extracellular matrix (dECM) has been proposed as a useful source of biomimetic materials for regenerative medicine due to its biological properties that regulate cell behaviors. The present study aimed to investigate the influence of decellularized ECM derived from dental pulp stem cells (DPSCs) on gingival fibroblast (GF) cell behaviors. Cells were isolated from dental pulp and gingival tissues. ECM was derived from culturing dental pulp stem cells in growth medium supplemented with ascorbic acid. A bioinformatic database of the extracellular matrix was constructed using Metascape. GFs were reseeded onto dECM, and their adhesion, spreading, and organization were subsequently observed. The migration ability of the cells was determined using a scratch assay. Protein expression was evaluated using immunofluorescence staining. RESULTS: Type 1 collagen and fibronectin were detected on the ECM and dECM derived from DPSCs. Negative phalloidin and nuclei were noted in the dECM. The proteomic database revealed enrichment of several proteins involved in ECM organization, ECM-receptor interaction, and focal adhesion. Compared with those on the controls, the GFs on the dECM exhibited more organized stress fibers. Furthermore, cultured GFs on dECM exhibited significantly enhanced migration and proliferation abilities. Interestingly, GFs seeded on dECM showed upregulation of FN1, ITGB3, and CTNNB1 mRNA levels. CONCLUSIONS: ECM derived from DSPCs generates a crucial microenvironment for regulating GF adhesion, migration and proliferation. Therefore, decellularized ECM from DPSCs could serve as a matrix for oral tissue repair.

SULF1 expression is increased and promotes fibrosis through the TGF-ß1/SMAD pathway in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology. Despite the increasing global incidence and poor prognosis, the exact pathogenic mechanisms remain elusive. Currently, effective therapeutic targets and treatment methods for this disease are still lacking. This study tried to explore the pathogenic mechanisms of IPF. We found elevated expression of SULF1 in lung tissues of IPF patients compared to normal control lung tissues. SULF1 is an enzyme that modifies heparan sulfate chains of heparan sulfate proteoglycans, playing a critical role in biological regulation. However, the effect of SULF1 in pulmonary fibrosis remains incompletely understood. Our study aimed to investigate the impact and mechanisms of SULF1 in fibrosis. METHODS: We collected lung specimens from IPF patients for transcriptome sequencing. Validation of SULF1 expression in IPF patients was performed using Western blotting and RT-qPCR on lung tissues. ELISA experiments were employed to detect SULF1 concentrations in IPF patient plasma and TGF-ß1 levels in cell culture supernatants. We used lentiviral delivery of SULF1 shRNA to knock down SULF1 in HFL1 cells, evaluating its effects on fibroblast secretion, activation, proliferation, migration, and invasion capabilities. Furthermore, we employed Co-Immunoprecipitation (Co-IP) to investigate the regulatory mechanisms involved. RESULTS: Through bioinformatic analysis of IPF transcriptomic sequencing data (HTIPF) and datasets GSE24206, and GSE53845, we identified SULF1 may potentially play a crucial role in IPF. Subsequently, we verified that SULF1 was upregulated in IPF and predominantly increased in fibroblasts. Furthermore, SULF1 expression was induced in HFL1 cells following exposure to TGF-ß1. Knockdown of SULF1 suppressed fibroblast secretion, activation, proliferation, migration, and invasion under both TGF-ß1-driven and non-TGF-ß1-driven conditions. We found that SULF1 catalyzes the release of TGF-ß1 bound to TGFßRIII, thereby activating the TGF-ß1/SMAD pathway to promote fibrosis. Additionally, TGF-ß1 induces SULF1 expression through the TGF-ß1/SMAD pathway, suggesting a potential positive feedback loop between SULF1 and the TGF-ß1/SMAD pathway. CONCLUSIONS: Our findings reveal that SULF1 promotes fibrosis through the TGF-ß1/SMAD pathway in pulmonary fibrosis. Targeting SULF1 may offer a promising therapeutic strategy against IPF.

SNX27:Retromer:ESCPE-1-mediated early endosomal tubulation impacts cytomegalovirus replication.

Introduction: Cytomegaloviruses (CMVs) extensively reorganize the membrane system of the cell and establish a new structure as large as the cell nucleus called the assembly compartment (AC). Our previous studies on murine CMV (MCMV)-infected fibroblasts indicated that the inner part of the AC contains rearranged early endosomes, recycling endosomes, endosomal recycling compartments and trans-Golgi membrane structures that are extensively tubulated, including the expansion and retention of tubular Rab10 elements. An essential process that initiates Rab10-associated tubulation is cargo sorting and retrieval mediated by SNX27, Retromer, and ESCPE-1 (endosomal SNX-BAR sorting complex for promoting exit 1) complexes. Objective: The aim of this study was to investigate the role of SNX27:Retromer:ESCPE-1 complexes in the biogenesis of pre-AC in MCMV-infected cells and subsequently their role in secondary envelopment and release of infectious virions. Results: Here we show that SNX27:Retromer:ESCPE1-mediated tubulation is essential for the establishment of a Rab10-decorated subset of membranes within the pre-AC, a function that requires an intact F3 subdomain of the SNX27 FERM domain. Suppression of SNX27-mediated functions resulted in an almost tenfold decrease in the release of infectious virions. However, these effects cannot be directly linked to the contribution of SNX27:Retromer:ESCPE-1-dependent tubulation to the secondary envelopment, as suppression of these components, including the F3-FERM domain, led to a decrease in MCMV protein expression and inhibited the progression of the replication cycle. Conclusion: This study demonstrates a novel and important function of membrane tubulation within the pre-AC associated with the control of viral protein expression.

N-glycoproteomic and proteomic alterations in SRD5A3-deficient fibroblasts.

SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify N-glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. N-glycoproteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique N-glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (P < 0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder.

Key Clinical and Histopathological Features of a Pterygium-Like Induced Lesion in a Rabbit Model.

Purpose: Surgery is the definitive treatment for pterygium; therefore, reliable animal models are required for translational research. The goal of this investigation was to establish a standardized preclinical model of pterygium-like lesion. Methods: A subconjunctival injection of fibroblasts (NIH3T3) and extracellular matrix was administered to 22 New Zealand rabbits. Clinical evaluation was assessed at different points, the severity of the lesion was scored according to four grades and correlated with the area of hyperemia and the histopathological findings on day 23. Results: Thirteen of 22 eyes (60%) developed pterygium-like lesions after 7 days and progressed through different grades. Initially, grade 3, characterized by an elevated and fleshiness conjunctiva with tortuous hyperemia, was evident on day 7. By day 15, lesion decreased to grade 2, with less elevation and hyperemia. Subsequent improvement was noted, with grade 1 on day 18. Finally, day 23 was marked by a white‒yellow lesion, classified as grade 4. The area of hyperemia increased from grade 2 to grade 3 (P < 0.05) and decreased from grade 3 to grade 4 (P ≤ 0.05). Histopathological analysis revealed a tendency toward increasing inflammation at grades 2, 3, and 4. There was a correlation between clinical features and the degree of inflammation. Conclusions: Subconjunctival injection of NIH3T3 and extracellular matrix induces a pterygium-like lesion that progresses across four grades, beginning with an acute inflammatory process that evolve a chronic form. This study provides a replicable model for simulating pterygium. Translational Relevance: The development of a standardized preclinical model of pterygium to evaluate new pharmacological or surgical treatments.

Modification of transvaginal polypropylene mesh with co-axis electrospun nanofibrous membrane to alleviate complications following surgical implantation.

BACKGROUND: Surgeries for treating pelvic organ prolapse involving the utilization of synthetic mesh have been associated with complications such as mesh erosion, postoperative pain, and dyspareunia. This work aimed to reduce the surgical implantation-associated complications by nanofibrous membranes on the surface of the polypropylene mesh. The nanofiber of the nanofibrous membrane, which was fabricated by co-axial electrospinning, was composed of polyurethane as fiber core and gelatin as the fiber out layer. The biocompatibility of the modified mesh was evaluated in vitro by cell proliferation assay, immunofluorescence stain, hematoxylin-eosin (HE) staining, and mRNA sequencing. Polypropylene mesh and modified mesh were implanted in a rat pelvic organ prolapse model. Mesh-associated complications were documented. HE and Picro-Sirius red staining, immunohistochemistry, and western blotting were conducted to assess the interactions between the modified mesh and vaginal tissues. RESULTS: The modified mesh significantly enhanced the proliferation of fibroblasts and exerted a positive regulatory effect on the extracellular matrix anabolism in vitro. When evaluated in vivo, no instances of mesh exposure were observed in the modified mesh group. The modified mesh maintained a relatively stable histological position without penetrating the muscle layer or breaching the epidermis. The collagen content in the vaginal wall of rats with modified mesh was significantly higher, and the collagen I/III ratio was lower, indicating better tissue elasticity. The expression of metalloproteinase was decreased while the expression levels of tissue inhibitor of metalloproteinase were increased in the modified mesh group, suggesting an inhibition of collagen catabolism. The expression of TGF-ß1 and the phosphorylation levels of Smad3, p38 and ERK1/2 were significantly increased in the modified mesh group. NM significantly improved the biocompatibility of PP mesh, as evidenced by a reduction in macrophage count, decreased expression levels of TNF-α, and an increase in microvascular density. CONCLUSIONS: The nanofibrous membrane-coated PP mesh effectively reduced the surgical implantation complications by inhibiting the catabolism of collagen in tissues and improving the biocampibility of PP mesh. The incorporation of co-axial fibers composed of polyurethane and gelatin with polypropylene mesh holds promise for the development of enhanced surgical materials for pelvic organ prolapse in clinical applications.

Influence of light-polymerizing units and zirconia on the physical, chemical and biological properties of self-adhesive resin cements.

BACKGROUND: Self-adhesive resin cements (SARCs) are widely used for fixed prostheses, but incomplete cleaning near the gingival margin can cause inflammation. However, the factors influencing cement properties and the biological response of gingival fibroblasts to cement eluates are not well understood. This study examines the impact of two light-polymerizing units (LPUs) on the physical and chemical properties of two SARCs under simulated clinical conditions, as well as the subsequent response of human gingival fibroblasts (hGFs) to these eluates. METHODS: Dental cement discs of SARCs were polymerized using Kerr DemiPlus and 3 M Elipar DeepCure-S LED LPUs with or without a 2-mm thick zirconia screen. Physical properties (microhardness, surface roughness, residual monomers) were evaluated. hGFs' cell viability, wound healing potency, and gene expression were assessed. RESULTS: Both Maxcem and RelyX exhibited reduced microhardness and increased surface roughness when polymerized through zirconia or with DemiPlus LPU. Higher residual monomers (HEMA and GDMA in Maxcem; TEGDMA in RelyX) concentration was observed with DemiPlus and zirconia polymerization. Maxcem polymerized with DemiPlus exhibited lower cell viability, impaired healing, and altered gene expression in hGFs compared to those polymerized with Elipar LPU. Gene expression changes included downregulated NRF2 and HO-1 and upregulated CCR-3. CONCLUSIONS: Light-polymerizing Maxcem through zirconia with DemiPlus LPU compromised SARCs' properties, leading to higher residual monomers and negatively impacting hGFs' viability, healing, and gene expression. Careful material selection and polymerization techniques are crucial to minimize adverse effects on surrounding tissues. CLINICAL SIGNIFICANCE: Clinicians should exercise caution when using LPUs and SARCs, especially when polymerizing through zirconia. This will help optimize the physical and chemical properties of SARCs and minimize potential adverse effects on the surrounding gingival soft tissues.

The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification.

OBJECTIVE: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). METHODOLOGY: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 µM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. RESULTS: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 µM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 µM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 µM Dynasore. CONCLUSION: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.

Inhibition of intestinal inflammation and fibrosis by Scutellaria Baicalensis georgi and Boswellia serrata in human epithelial cells and fibroblasts.

OBJECTIVE AND RATIONALE: Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, manifests with chronic intestinal inflammation and frequent sequential fibrosis. Current pharmacological therapies may show harmful side effects and are not useful for prevention or resolution of fibrosis. Thus, the use of alternative therapies is emerging as a novel useful approach. Previous results suggest that Scutellaria baicalensis Georgi (SBG) and Boswellia serrata (BS) display anti-inflammatory properties. The aim of this study was to investigate in intestinal epithelial cells and fibroblasts the anti-inflammatory and anti-fibrotic potential of SBG and BS, alone or in combination. METHODS: Human colorectal adenocarcinoma cells (HT29), human intestinal epithelial cells (HIEC6) and human colon fibroblasts (CCD-18Co) were used. Cells were pretreated with SBG and BS and then exposed to pro-inflammatory and pro-fibrotic cytokines. RESULTS: SBG and BS extracts significantly decreased pro-inflammatory cytokine expression and improved epithelial restitution in HT29 and HIEC6 cells. Besides, fibrotic marker expression, including SNAIL, ACTA2, ZNF281, was strongly reduced. Colon myofibroblasts treated with SBG and BS showed a significant decrease of fibrotic markers as well. CONCLUSIONS: SBG and BS extracts significantly reduce inflammation and impair fibrosis in intestinal epithelial cells and colon myofibroblasts. No cooperative effect is observed.

SUMOylation of MFF coordinates fission complexes to promote stress-induced mitochondrial fragmentation.

Mitochondria undergo fragmentation in response to bioenergetic stress, mediated by dynamin-related protein 1 (DRP1) recruitment to the mitochondria. The major pro-fission DRP1 receptor is mitochondrial fission factor (MFF), and mitochondrial dynamics proteins of 49 and 51 kilodaltons (MiD49/51), which can sequester inactive DRP1. Together, they form a trimeric DRP1-MiD-MFF complex. Adenosine monophosphate-activated protein kinase (AMPK)-mediated phosphorylation of MFF is necessary for mitochondrial fragmentation, but the molecular mechanisms are unclear. Here, we identify MFF as a target of small ubiquitin-like modifier (SUMO) at Lys151, MFF SUMOylation is enhanced following AMPK-mediated phosphorylation and that MFF SUMOylation regulates the level of MiD binding to MFF. The mitochondrial stressor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) promotes MFF SUMOylation and mitochondrial fragmentation. However, CCCP-induced fragmentation is impaired in MFF-knockout mouse embryonic fibroblasts expressing non-SUMOylatable MFF K151R. These data suggest that the AMPK-MFF SUMOylation axis dynamically controls stress-induced mitochondrial fragmentation by regulating the levels of MiD in trimeric fission complexes.

Comparative transcriptome of normal and cancer-associated fibroblasts.

BACKGROUND: The characteristics of a tumor are largely determined by its interaction with the surrounding micro-environment (TME). TME consists of both cellular and non-cellular components. Cancer-associated fibroblasts (CAFs) are a major component of the TME. They are a source of many secreted factors that influence the survival and progression of tumors as well as their response to drugs. Identification of markers either overexpressed in CAFs or unique to CAFs would pave the way for novel therapeutic strategies that in combination with conventional chemotherapy are likely to have better patient outcome. METHODS: Fibroblasts have been derived from Benign Prostatic Hyperplasia (BPH) and prostate cancer. RNA from these has been used to perform a transcriptome analysis in order to get a comparative profile of normal and cancer-associated fibroblasts. RESULTS: The study has identified 818 differentially expressed mRNAs and 17 lincRNAs between normal and cancer-associated fibroblasts. Also, 15 potential lincRNA-miRNA-mRNA combinations have been identified which may be potential biomarkers. CONCLUSIONS: This study identified differentially expressed markers between normal and cancer-associated fibroblasts that would help in targeted therapy against CAFs/derived factors, in combination with conventional therapy. However, this would in future need more experimental validation.

Electroless silver plating on fabrics for antimicrobial coating: comparison between cotton and polyester.

In the past few years, due to the Covid-19 pandemic, the interest towards textiles with antimicrobial functionalities faced a significant boost. This study proposes a rapid and convenient method, in terms of reactants and equipment, for fabricating antimicrobial coatings on textiles. Through the electroless silver plating reaction, silver coatings were successfully applied on cotton and polyester, rapidly and at room temperature. Functionalized samples were characterized by morphological (optical and scanning electron microscopies) and chemical tests (X-ray photoelectron spectroscopy, XPS) to investigate the nature of the silver coating. Although distinct nanoparticles did not form, XPS analysis detected the presence of silver, which resulted in an increased surface roughness and hydrophobicity of both cotton and polyester textiles. Ag-coated samples exhibited approximately 80% biocompatibility with murine L929 fibroblasts or human HaCaT cells, and strong antibacterial properties against Escherichia coli in direct contact tests. In antiviral experiments with SARS-CoV-2 virus, treated cotton showed a 100% viral reduction in 30 min, while polyester achieved 100% reduction in 1 h. With a human norovirus surrogate, the Feline Calicivirus, both treated textiles have a faster antiviral response, with more than 60% viral reduction after 5 min, while achieving a 100% reduction in 1 h. In conclusion, this study presents a fast, efficient, and low-cost solution for producing antimicrobial textiles with broad applications in medical and healthcare scenarios.

Evaluation of bacteriophages for the alleviation of potential bacterial contamination risks in developmental engineering.

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE.

Cromolyn sodium and masitinib combination inhibits fibroblast-myofibroblast transition and exerts additive cell-protective and antioxidant effects on a bleomycin-induced in vitro fibrosis model.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic lung disease. While recent studies have suggested the potential efficacy of tyrosine kinase inhibitors in managing IPF, masitinib, a clinically used tyrosine kinase inhibitor, has not yet been investigated for its efficacy in fibrotic lung diseases. In a previous study on an in vitro neurodegenerative model, we demonstrated the synergistic antitoxic and antioxidant effects of masitinib combined with cromolyn sodium, an FDA-approved mast cell stabilizer. This study aims to investigate the anti-fibrotic and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro model of pulmonary fibrosis. Fibroblast cell cultures treated with bleomycin and/or hydrogen peroxide (H2O2) were subjected to masitinib and/or cromolyn sodium, followed by assessments of cell viability, morphological and apoptotic nuclear changes, triple-immunofluorescence labeling, and total oxidant/antioxidant capacities, besides ratio of Bax and Bcl-2 mRNA expressions as an indication of apoptosis. The combined treatment of masitinib and cromolyn sodium effectively prevented the fibroblast myofibroblast transition, a hallmark of fibrosis, and significantly reduced bleomycin / H2O2-induced apoptosis and oxidative stress. This study is the first to demonstrate the additive anti-fibrotic, cell-protective, and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro fibrosis model, suggesting its potential as an innovative therapeutic approach for pulmonary fibrosis. Combination therapy may be more advantageous in that both drugs could be administered in lower doses, exerting less side effects, and at the same time providing diverse mechanisms of action simultaneously.

Cytotoxicity assessment of eluates from vacuum-forming thermoplastics.

OBJECTIVES: This study aimed to evaluate possible cytotoxic effects of thermoplastic materials commonly used for occlusal splints and orthodontic appliances. METHODS: Seven thermoplastics were included: three variants of the Essix sheets (C+, Plus, and Tray Rite; Dentsply Sirona), three thermoplastics (Bleach Heavy, Splint, and X-Heavy; Cavex Holland) and Invisalign (Align Technology). Cylindrical specimens (n = 24; 10 mm diameter) were incubated in cell culture medium for 24 h and 14 days. After incubation, the medium was collected, serially diluted, and dosed to primary human gingival fibroblasts in triplicate. Medium processed like the samples was used as negative control. Cell viability was evaluated by XTT and LDH assay to assess metabolic activity and membrane integrity, respectively. Next, cell cycle was assessed with flow cytometry after exposing HGFs to undiluted extracts. RESULTS: The 24-hour and 14-day extracts did not evoke cytotoxicity after 24-hour incubation. No significant differences in cell viability (one-way ANOVA, p > 0.05 ) in the XTT and LDH assays or in cell cycle distribution between the different materials (two-way ANOVA, p > 0.05 ). CONCLUSION: The thermoplastics tested in the study showed no evident in-vitro cytotoxic effects. Further investigation should focus on determining which compounds are released from thermoplastic materials and assessing potential toxicity related to exposure to these compounds. CLINICAL SIGNIFICANCE: Our study adds to the growing body of evidence on the biocompatibility of dental thermoplastics. This can aid clinical decision-making, as thermoplastics are expected to be safe to use in terms of cytotoxicity.

Inhibition of Ubiquitin C-Terminal Hydrolase L1 Facilitates Cutaneous Wound Healing via Activating TGF-ß/Smad Signalling Pathway in Fibroblasts.

Ubiquitin C-terminal hydrolase L1 (UCHL1) plays vital roles in cell proliferation, angiogenesis, inflammation and oxidative stress. Nevertheless, it is unclear whether UCHL1 could regulate the biologic behaviour of cells and ultimately influences wound healing. We aim to illustrate the roles and the underlying mechanism of UCHL1 in cutaneous wound healing. Murine full-thickness excisional wound model was utilised to study the effects of UCHL1 on wound healing through topical administration of the UCHL1 inhibitor LDN57444, followed by assessment of wound areas and histological alterations. Subsequently, ethynyldeoxyuridine, scratch and transwell assays were performed to examine fibroblast migration and proliferation. The extracellular matrix (ECM)-related genes expression and transforming growth factor-ß (TGF-ß)/Smad signalling pathways activation were investigated by immuno-fluorescent staining, Western blots and quantitative reverse transcription polymerase chain reaction. We identified elevated UCHL1 expression in non-healing wound tissues. The UCHL1 expression displayed a dynamic change and reached a peak on Day-7 post-wounding during the healing process in mice. Cutaneous administration of LDN57444 promoted wound healing by facilitating collagen deposition, myofibroblast activation and angiogenesis. In vitro experiments demonstrated that UCHL1 concentration dependently inhibited migration, ECM synthesis and activation of human dermal fibroblasts, which was mechanistically related to downregulation of TGF-ß/Smad signalling. Furthermore, these effects could be reversed by TGF-ß inhibitor SB431542. Our findings reveal that UCHL1 is a negative regulator of cutaneous wound healing and considered as a novel prospective therapeutic target for effective wound healing.

NPC1 links cholesterol trafficking to microglial morphology via the gastrosome.

Microglia play important roles in brain development and homeostasis by removing dying neurons through efferocytosis. Morphological changes in microglia are hallmarks of many neurodegenerative conditions, such as Niemann-Pick disease type C. Here, NPC1 loss causes microglia to shift from a branched to an ameboid form, though the cellular basis and functional impact of this change remain unclear. Using zebrafish, we show that NPC1 deficiency causes an efferocytosis-dependent expansion of the microglial gastrosome, a collection point for engulfed material. In vivo and in vitro experiments on microglia and mammalian macrophages demonstrate that NPC1 localizes to the gastrosome, and its absence leads to cholesterol accumulation in this compartment. NPC1 loss and neuronal cell death synergistically affect gastrosome size and cell shape, increasing the sensitivity of NPC1-deficient cells to neuronal cell death. Finally, we demonstrate conservation of cholesterol accumulation and gastrosome expansion in NPC patient-derived fibroblasts, offering an interesting target for further disease investigation.