Molecular histopathology of matrix proteins through autofluorescence super-resolution microscopy.

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training.

DPSCs regulate epithelial-T cell interactions in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion characterized by fibrous tissue deposition, the incidence of which correlates positively with the frequency of betel nut chewing. Prolonged betel nut chewing can damage the integrity of the oral mucosal epithelium, leading to chronic inflammation and local immunological derangement. However, currently, the underlying cellular events driving fibrogenesis and dysfunction are incompletely understood, such that OSF has few treatment options with limited therapeutic effectiveness. Dental pulp stem cells (DPSCs) have been recognized for their anti-inflammatory and anti-fibrosis capabilities, making them promising candidates to treat a range of immune, inflammatory, and fibrotic diseases. However, the application of DPSCs in OSF is inconclusive. Therefore, this study aimed to explore the pathogenic mechanism of OSF and, based on this, to explore new treatment options. METHODS: A human cell atlas of oral mucosal tissues was compiled using single-cell RNA sequencing to delve into the underlying mechanisms. Epithelial cells were reclustered to observe the heterogeneity of OSF epithelial cells and their communication with immune cells. The results were validated in vitro, in clinicopathological sections, and in animal models. In vivo, the therapeutic effect and mechanism of DPSCs were characterized by histological staining, immunohistochemical staining, scanning electron microscopy, and atomic force microscopy. RESULTS: A unique epithelial cell population, Epi1.2, with proinflammatory and profibrotic functions, was predominantly found in OSF. Epi1.2 cells also induced the fibrotic process in fibroblasts by interacting with T cells through receptor-ligand crosstalk between macrophage migration inhibitory factor (MIF)-CD74 and C-X-C motif chemokine receptor 4 (CXCR4). Furthermore, we developed OSF animal models and simulated the clinical local injection process in the rat buccal mucosa using DPSCs to assess their therapeutic impact and mechanism. In the OSF rat model, DPSCs demonstrated superior therapeutic effects compared with the positive control (glucocorticoids), including reducing collagen deposition and promoting blood vessel regeneration. DPSCs mediated immune homeostasis primarily by regulating the numbers of KRT19 + MIF + epithelial cells and via epithelial-stromal crosstalk. CONCLUSIONS: Given the current ambiguity surrounding the cause of OSF and the limited treatment options available, our study reveals that epithelial cells and their crosstalk with T cells play an important role in the mechanism of OSF and suggests the therapeutic promise of DPSCs.

Mucoadhesive polymers: Design of S-protected thiolated cyclodextrin-based hydrogels.

AIM: This study aims to design chemically crosslinked thiolated cyclodextrin-based hydrogels and to evaluate their mucoadhesive properties via mucosal residence time studies on porcine small intestinal mucosa and on porcine buccal mucosa. METHODS: Free thiol groups of heptakis(6-deoxy-6-thio)-ß-cyclodextrin (ß-CD-SH) were S-protected with 2-mercaptoethanesulfonic acid (MESNA) followed by crosslinking with citric acid. Cytotoxicity was assessed by hemolysis as well as resazurin assay. Hydrogels were characterized by their rheological and mucoadhesive properties. Ritonavir was employed as model drug for in vitro release studies from these hydrogels. RESULTS: The structure of S-protected ß-CD-SH was confirmed by IR and 1H NMR spectroscopy. Degree of thiolation was 390 ± 7 µmol/g. Hydrogels based on native ß-CD showed hemolysis of 12.5 ± 2.5 % and 13.6 ± 2.7 % within 1 and 3 h, whereas hemolysis of just 3.5 ± 2.8 % and 3.9 ± 3.0 % was observed for the S-protected thiolated CD hydrogels, respectively. Both native and S-protected thiolated hydrogels showed minor cytotoxicity on Caco-2 cells. Rheological investigations of S-protected thiolated ß-CD-based hydrogel (16.2 % m/v) showed an up to 13-fold increase in viscosity in contrast to the corresponding native ß-CD-based hydrogel. Mucosal residence time studies showed that thiolated ß-CD-based hydrogel is removed to a 16.6- and 2.4-fold lower extent from porcine small intestinal mucosa and porcine buccal mucosa in comparision to the native ß-CD-based hydrogel, respectively. Furthermore, a sustained release of ritonavir from S-protected thiolated ß-CD-based hydrogels was observed. CONCLUSION: Because of their comparatively high mucoadhesive and release-controlling properties, S-protected thiolated ß-CD-based hydrogels might be promising systems for mucosal drug delivery.

Expression of CLLD7 and CHC1L Proteins in Oral Epithelial Dysplasia in a Group of Thai Patients.

OBJECTIVES: Previous study showed aberrant CLLD7 and CHC1L protein expression in oral squamous cell carcinoma (OSCC) compared to normal oral mucosa (NOM). This study aimed to evaluate the expression of these proteins in oral epithelial dysplasia (OED). MATERIALS AND METHODS: Forty specimens of OED and 11 NOM were used. The expression of CLLD7 and CHC1L was determined by immunohistochemistry. In each case, at least 1000 cells were counted. Presence of nuclear, cytoplasmic, and/or membrane staining of CLLD7 and CHC1L were considered positive. Percentages of total positive cells and positive cells at different locations were recorded. SPSS version 18 was used to compare variation between groups with statistical significance at p<0.05. RESULTS: No significant differences in the percentages of total positive cells of CLLD7 and CHC1L were found between NOM and all grades of OED. Nevertheless, there were significant differences in subcellular staining of these two proteins. In CLLD7, the nuclear staining of the moderate and the severe OED groups was significantly lower than that of the NOM group (p<0.05). The percentages of membrane staining of CHC1L in moderate and severe OED were significantly higher than that of NOM (p<0.001). In addition, the nuclear staining of CHC1L in each grade of OED was significantly lower than that of NOM (p<0.05). CONCLUSION: The subcellular mislocalization of CLLD7 and CHC1L in OED suggests that the expression of these potential tumor suppressor proteins might be dysregulated during the dysplastic process. The distinct membrane staining of CHC1L observed in OED but not in NOM is a useful characteristic that can be used to separate OED from NOM. Thus, CHC1L may be a good marker to assist in the diagnosis of OED.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

BACKGROUND/AIM: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of ß-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. MATERIALS AND METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with ß-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. CONCLUSION: Oral epithelial cells express Dectin-1 and recognize ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

Evaluation of stromal myofibroblasts in oral submucous fibrosis and its malignant transformation: An immunohistochemical study.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion, with oral squamous cell carcinoma (OSCC) being the most prevalent malignancy affecting the oral mucosa. The malignant transformation of OSF into OSCC is estimated to occur in 7-13% of cases. Myofibroblasts (MFs) play pivotal roles in both physiological and pathological processes, such as wound healing and tumorigenesis, respectively. This study aimed to explore the involvement of MFs in the progression of OSF and its malignant transformation. MATERIALS AND METHODS: In total, 94 formalin-fixed paraffin-embedded tissue blocks were collected, including normal oral mucosa (NOM; n = 10), early-moderate OSF (EMOSF; n = 29), advanced OSF (AOSF; n = 29), paracancerous OSF (POSF; n = 21), and OSCC (n = 5) samples. Alpha-smooth muscle actin was used for the immunohistochemical identification of MFs. RESULTS: NOM exhibited infrequent expression of MFs. A higher staining index of MFs was found in AOSF, followed by EMOSF and NOM. Additionally, a significant increase in the staining index of MFs was found from EMOSF to POSF and OSCC. The staining index of MFs in NOM, EMOSF, AOSF, POSF, and OSCC was 0.14 ± 0.2, 1.69 ± 1.4, 2.47 ± 1.2, 3.57 ± 2.6, and 8.86 ± 1.4, respectively. All results were statistically significant (P < 0.05). CONCLUSIONS: The expression of MFs exhibited a gradual increase as the disease progressed from mild to malignant transformation, indicating the contributory role of MFs in the fibrogenesis and potential tumorigenesis associated with OSF.

Rattus norvegicus Glutathione Transferase Omega 1 Localization in Oral Tissues and Interactions with Food Phytochemicals.

Glutathione transferases are xenobiotic-metabolizing enzymes with both glutathione-conjugation and ligandin roles. GSTs are present in chemosensory tissues and fluids of the nasal/oral cavities where they protect tissues from exogenous compounds, including food molecules. In the present study, we explored the presence of the omega-class glutathione transferase (GSTO1) in the rat oral cavity. Using immunohistochemistry, GSTO1 expression was found in taste bud cells of the tongue epithelium and buccal cells of the oral epithelium. Buccal and lingual extracts exhibited thiol-transferase activity (4.9 ± 0.1 and 1.8 ± 0.1 µM/s/mg, respectively). A slight reduction from 4.9 ± 0.1 to 4.2 ± 0.1 µM/s/mg (p < 0.05; Student's t test) was observed in the buccal extract with 100 µM GSTO1-IN-1, a specific inhibitor of GSTO1. RnGSTO1 exhibited the usual activities of omega GSTs, i.e., thiol-transferase (catalytic efficiency of 8.9 × 104 M-1·s-1), and phenacyl-glutathione reductase (catalytic efficiency of 8.9 × 105 M-1·s-1) activities, similar to human GSTO1. RnGSTO1 interacts with food phytochemicals, including bitter compounds such as luteolin (Ki = 3.3 ± 1.9 µM). Crystal structure analysis suggests that luteolin most probably binds to RnGSTO1 ligandin site. Our results suggest that GSTO1 could interact with food phytochemicals in the oral cavity.

Aberrantly downregulated FENDRR by arecoline elevates ROS and myofibroblast activation via mitigating the miR-214/MFN2 axis.

Long non-coding RNA FENDRR possesses both anti-fibrotic and anti-cancer properties, but its significance in the development of premalignant oral submucous fibrosis (OSF) remains unclear. Here, we showed that FENDRR was downregulated in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs), and overexpression of FENDRR mitigated various myofibroblasts hallmarks, and vice versa. In the course of investigating the mechanism underlying the implication of FENDRR in myofibroblast transdifferentiation, we found that FENDRR can directly bind to miR-214 and exhibit its suppressive effect on myofibroblast activation via titrating miR-214. Moreover, we showed that mitofusin 2 (MFN2), a protein that is crucial to the fusion of mitochondria, was a direct target of miR-214. Our data suggested that FENDRR was positively correlated with MFN2 and MFN2 was required for the inhibitory property of FENDRR pertaining to myofibroblast phenotypes. Additionally, our results showed that the FENDRR/miR-214 axis participated in the arecoline-induced reactive oxygen species (ROS) accumulation and myofibroblast transdifferentiation. Building on these results, we concluded that the aberrant downregulation of FENDRR in OSF may be associated with chronic exposure to arecoline, leading to upregulation of ROS and myofibroblast activation via the miR-214-mediated suppression of MFN2.

Multifunctional hyaluronic acid/gelatin methacryloyl core-shell microneedle for comprehensively treating oral mucosal ulcers.

Oral ulceration is the most common oral mucosal disease. Oral mucosal ulcers are extremely painful, may interfere with eating and speaking, and potentially complicate systemic symptoms in severe cases. The humid and highly dynamic environment of the oral cavity makes local drug administration for treating oral mucosal ulcers challenging. To overcome these challenges, we designed and prepared a novel dissolving microneedle (MN) patch containing multiple drugs in a core-shell to promote oral ulcer healing. The MNs contained a methacrylate gelatin shell layer of basic fibroblast growth factor (bFGF), a hyaluronic acid (HA) core loaded with dexamethasone (DXMS), and zeolite imidazoline framework-8 (ZIF-8) encapsulated in the HA-based backplane. Progressive degradation of gelatin methacryloyl (GelMA) from the tip of the MN patch in the oral mucosa resulted in sustained bFGF release at the lesion site, significantly promoting cell migration, proliferation, and angiogenesis. Moreover, the rapid release of HA and, subsequently, DXMS inhibited inflammation, and the remaining MN backing after the tip dissolved behaved as a dressing, releasing ZIF-8 for its antimicrobial effects. This novel, multifunctional, transmucosal core-shell MN patch exhibited excellent anti-inflammatory, antimicrobial, and pro-healing effects in vivo and in vitro, suggesting that it can promote oral ulcer healing.

Antifibrotic effect of silymarin on arecoline-induced fibrosis in primary human buccal fibroblasts: an in silico and in vitro analysis.

BACKGROUND: This study aimed to assess silymarin's anticancer and antifibrotic potential through in silico analysis and investigate its impact on in vitro arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS & RESULTS: The study utilized iGEMDOCK for molecular docking, evaluating nine bioflavonoids, and identified silymarin and baicalein as the top two compounds with the highest target affinity, followed by subsequent validation through a 100ns Molecular Dynamic Simulation demonstrating silymarin's stable behavior with Transforming Growth Factor Beta. HBF cell lines were developed from tissue samples obtained from patients undergoing third molar extraction. Arecoline, a known etiological factor in oral submucous fibrosis (OSMF), was employed to induce fibrogenesis in these HBFs. The inhibitory concentration (IC50) of arecoline was determined using the MTT assay, revealing dose-dependent cytotoxicity of HBFs to arecoline, with notable cytotoxicity observed at concentrations exceeding 50µM. Subsequently, the cytotoxicity of silymarin was assessed at 24 and 72 h, spanning concentrations from 5µM to 200µM, and an IC50 value of 143µM was determined. Real-time polymerase chain reaction (qPCR) was used to analyze the significant downregulation of key markers including collagen, epithelial-mesenchymal transition (EMT), stem cell, hypoxia, angiogenesis and stress markers in silymarin-treated arecoline-induced primary buccal fibroblast cells. CONCLUSION: Silymarin effectively inhibited fibroblast proliferation and downregulated genes associated with cancer progression and EMT pathway, both of which are implicated in malignant transformation. To our knowledge, this study represents the first exploration of silymarin's potential as a novel therapeutic agent in an in vitro model of OSMF.

RFX transcription factor in the human-associated yeast Candida albicans regulates adhesion to oral epithelium.

Adhesion to mucosal surfaces is a critical step in many bacterial and fungal infections. Here, using a mouse model of oral infection by the human fungal pathobiont Candida albicans, we report the identification of a novel regulator of C. albicans adhesion to the oral mucosa. The regulator is a member of the regulatory factor X (RFX) family of transcription factors, which control cellular processes ranging from genome integrity in model yeasts to tissue differentiation in vertebrates. Mice infected with the C. albicans rfx1 deletion mutant displayed increased fungal burden in tongues compared to animals infected with the reference strain. High-resolution imaging revealed RFX1 transcripts being expressed by C. albicans cells during infection. Concomitant with the increase in fungal burden, the rfx1 mutant elicited an enhanced innate immune response. Transcriptome analyses uncovered HWP1, a gene encoding an adhesion protein that mediates covalent attachment to buccal cells, as a major RFX1-regulated locus. Consistent with this result, we establish that C. albicans adhesion to oral cells is modulated by RFX1 in an HWP1-dependent manner. Our findings expand the repertoire of biological processes controlled by the RFX family and illustrate a mechanism whereby C. albicans can adjust adhesion to the oral epithelium.

Cell-penetrating peptides for transmucosal delivery of proteins.

Enabling non-invasive delivery of proteins across the mucosal barriers promises improved patient compliance and therapeutic efficacies. Cell-penetrating peptides (CPPs) are emerging as a promising and versatile tool to enhance protein and peptide permeation across various mucosal barriers. This review examines the structural and physicochemical attributes of the nasal, buccal, sublingual, and oral mucosa that hamper macromolecular delivery. Recent development of CPPs for overcoming those mucosal barriers for protein delivery is summarized and analyzed. Perspectives regarding current challenges and future research directions towards improving non-invasive transmucosal delivery of macromolecules for ultimate clinical translation are discussed.

Extracellular vesicles from oral mucosa stem cells promote lipid raft formation in human microglia through TLR4, P2X4R, and αVß3/αVß5 signaling pathways.

Targeting of disease-associated microglia represents a promising therapeutic approach that can be used for the prevention or slowing down neurodegeneration. In this regard, the use of extracellular vesicles (EVs) represents a promising therapeutic approach. However, the molecular mechanisms by which EVs regulate microglial responses remain poorly understood. In the present study, we used EVs derived from human oral mucosa stem cells (OMSCs) to investigate the effects on the lipid raft formation and the phagocytic response of human microglial cells. Lipid raft labeling with fluorescent cholera toxin subunit B conjugates revealed that both EVs and lipopolysaccharide (LPS) by more than two times increased lipid raft formation in human microglia. By contrast, combined treatment with LPS and EVs significantly decreased lipid raft formation indicating possible interference of EVs with the process of LPS-induced lipid raft formation. Specific inhibition of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody as well as inhibition of purinergic P2X4 receptor (P2X4R) with selective antagonist 5-BDBD inhibited EVs- and LPS-induced lipid raft formation. Selective blockage of αvß3/αvß5 integrins with cilengitide suppressed EV- and LPS-induced lipid raft formation in microglia. Furthermore, inhibition of TLR4 and P2X4R prevented EV-induced phagocytic activity of human microglial cells. We demonstrate that EVs induce lipid raft formation in human microglia through interaction with TLR4, P2X4R, and αVß3/αVß5 signaling pathways. Our results provide new insights about the molecular mechanisms regulating EV/microglia interactions and could be used for the development of new therapeutic strategies against neurological disorders.

The administration of oral mucosal mesenchymal-derived stem cells improves hepatic inflammation, oxidative stress, and histopathology following traumatic brain injury.

BACKGROUND: The inflammatory mediators produced after traumatic brain injury (TBI) are reaching peripheral organs causing organ and tissue damage, including the liver. Our study assessed the effect of intravenous (i.v.) infusion of oral mesenchymal stem cells (OMSCs) on TBI-induced liver damage by measuring liver inflammatory factors and liver oxidative stress. METHODS: Twenty-eight adult male Wistar rats were divided into four groups: 1) sham control; 2) TBI alone (TBI); 3) TBI vehicle (Veh)-control; and 4) TBI with OMSC treatment (SC). OMSCs were obtained from oral mucosa biopsies. OMSCs were administered and administered i.v. at 1 and 24 h after TBI. Within 48 h after TBI, multiple parameters were analyzed, including inflammation, oxidative stress, and histopathological changes. RESULTS: In comparison to sham controls, the TBI alone showed in liver significantly increased levels of interleukin-1ß (IL-1ß; P < 0.001), interleukin-6 (IL-6; P < 0.001), malondialdehyde (MDA; P < 0.001), and protein carbonyl (PC; P < 0.001). At the same time the TBI alone decreased the liver levels of superoxide dismutase (SOD; P < 0.001), total antioxidant capacity (TAC; P < 0.001), catalase (CAT; P < 0.001), and interleukin-10 (IL-10; P < 0.001). In comparison to the TBI alone group, the therapeutic group treated with i.v. infusion of OMSCs demonstrated significantly reduced changes of IL-1ß (P < 0.001), IL-6 (P < 0.01), MDA (P < 0.01), PC (P < 0.05), SOD (P < 0.001), TAC (P < 0.01), CAT (P < 0.01), and IL-10 (P < 0.01). Histopathological evaluation showed in TBI alone group that the total score of liver tissue injury included extensive hydropic degeneration, lobular necrosis, inflammation as well as central vein congestion with subendothelial hemorrhage increased compared the sham group (P < 0.001). Administration of OMSC showed significantly smaller increase in the injury score compared to the TBI alone group (P < 0.001). CONCLUSION: Therapy with i.v. OMSCs administration after TBI reduces liver injury, as measured by inflammation and oxidative stress. The use of OMSCs can be considered for treatment of liver injury caused by TBI.

Boosting systemic absorption of peptides with a bioinspired buccal-stretching patch.

Biopharmaceuticals, including proteins and peptides, have revolutionized the treatment of a wide range of diseases, from diabetes and cardiovascular disorders to virus infections and cancer. Despite their efficacy, most of these macromolecular drugs require parenteral administration because of their high molecular weight and relative instability. Over the past 40 years, only a few oral peptide drugs have entered clinical trials, even when formulated with substantial amounts of permeation enhancers. To overcome the epithelial barrier, devices that inject drugs directly into the gastrointestinal mucosa have been proposed recently. However, the robustness and safety of those complex systems are yet to be assessed. In this study, we introduced an innovative technology to boost drug absorption by synergistically combining noninvasive stretching of the buccal mucosa with permeation enhancers. Inspired by the unique structural features of octopus suckers, a self-applicable suction patch was engineered, enabling strong adhesion to and effective mechanical deformation of the mucosal tissue. In dogs, this suction patch achieved bioavailability up to two orders of magnitude higher than those of the commercial tablet formulation of desmopressin, a peptide drug known for its poor oral absorption. Moreover, systemic exposure comparable to that of the approved oral semaglutide tablet was achieved without further optimization. Last, a first-in-human study involving 40 healthy participants confirmed the dosage form's acceptability, thereby supporting the clinical translatability of this simple yet effective platform technology.

Plasma Exosomal microRNA Profile Reveals miRNA 148a-3p Downregulation in the Mucosal-Dominant Variant of Pemphigus Vulgaris.

The mucosal-dominant variant of pemphigus vulgaris (MPV) is an autoimmune disease characterized by oral mucosal blistering and circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3), resulting in life-threatening bullae and erosion formation. Recently, microRNAs (miRNAs) have emerged as promising players in the diagnosis and prognosis of several pathological states. For the first time, we have identified a different expression profile of miRNAs isolated from plasma-derived exosomes (P-EVs) of MPV patients positive for antibodies against Dsg3 (Dsg3-positive) compared to healthy controls. Moreover, a dysregulated miRNA profile was confirmed in MPV tissue biopsies. In particular, a strong downregulation of the miR-148a-3p expression level in P-EVs of MPV patients compared to healthy controls was demonstrated. Bioinformatics prediction analysis identifies metalloproteinase-7 (MMP7) as a potential miR-148a-3p target. An in vitro acantholysis model revealed that the miR-148a-3p expression level was dramatically downregulated after treatment with Dsg3 autoantibodies, with a concomitant increase in MMP7 expression. The increased expression of MMP7 leads to the disruption of intercellular and/or extracellular matrix adhesion in an in vitro cellular model of MPV, with subsequent cell dissociation. Overexpression of miR-148a-3p prevented cell dissociation and regressed MMP7 upregulation. Our findings suggest a pivotal role of P-EV cargo in regulating molecular mechanisms involved in MPV pathogenesis and indicate them as potential MPV therapeutic targets.

Pattern Distribution of Connexins in the Ortho- and Parakeratinized Epithelium of the Lingual Mucosa in Birds.

Connexins are important proteins involved in cell-to-cell communication and cytodifferentiation during renewal and cornification of the multilayered epithelia. So far, there is a lack of reports on this subject in birds' structurally different ortho- and parakeratinized epithelium of the tongue. The study aims to describe the distribution and expression profiles of the α-connexins (Cx40 and 43) and ß-connexins (Cx26, 30, and 31) in those epithelia in duck, goose, and domestic turkey. Research revealed the presence of the mentioned connexins and the occurrence of interspecies differences. Connexins form gap junctions in the cell membrane or are in the cytoplasm of keratinocytes. Differences in connexin expression were noted between the basal and intermediate layers, which may determine the proliferation of keratinocytes. Cx40, 43, and Cx30 in the gap junction of the keratinocytes of the intermediate layer are related to the synchronization of the cornification process. Because of the exfoliation of cornified plaques, a lack of connexins was observed in the cornified layer of orthokeratinized epithelium. However, in parakeratinized epithelium, connexins were present in the cell membrane of keratinocytes and thus maintained cellular integrity in gradually desquamating cells. The current studies will be useful in further comparative analyses of normal and pathological epithelia of the oral cavity in birds.

3-Hydroxy coumarin demonstrates anti-biofilm and anti-hyphal efficacy against Candida albicans via inhibition of cell-adhesion, morphogenesis, and virulent genes regulation.

Candida albicans, a common fungus of human flora, can become an opportunistic pathogen and causes invasive candidiasis in immunocompromised individuals. Biofilm formation is the prime cause of antibiotic resistance during C. albicans infections and treating biofilm-forming cells is challenging due to their intractable and persistent nature. The study intends to explore the therapeutic potential of naturally produced compounds by competitive marine bacteria residing in marine biofilms against C. albicans biofilm. To this end, 3-hydroxy coumarin (3HC), a compound identified from the cell-free culture supernatant of the marine bacterium Brevundimonas abyssalis, was found to exhibit anti-biofilm and anti-hyphal activity against both reference and clinical isolates of C. albicans. The compound demonstrated significant inhibitory effects on biofilms and impaired the yeast-to-hyphal transition, wrinkle, and filament morphology at the minimal biofilm inhibitory concentration (MBIC) of 250 µg mL-1. Intriguingly, quantitative PCR analysis of 3HC-treated C. albicans biofilm revealed significant downregulation of virulence genes (hst7, ume6, efg1, cph1, ras1, als1) associated with adhesion and morphogenesis. Moreover, 3HC displayed non-fungicidal and non-toxic characteristics against human erythrocytes and buccal cells. In conclusion, this study showed that marine biofilms are a hidden source of diverse therapeutic drugs, and 3HC could be a potent drug to treat C. albicans infections.

Formulation and characterization of pramipexole containing buccal films for using in Parkinson's disease.

Parkinson's disease (PD) is neurodegenerative chronic illness which affects primarily the elderly over 45 years of age. The symptoms can be various, both non-motor and motor symptoms can appear. The biggest problem in the treatment of the disease is the difficulty in swallowing for the patients. However, buccal patches can solve this problem because the patients do not have to swallow the dosage form, and during application, the API can absorb from the area of the buccal mucosa quickly without causing a foreign body sensation. In our present study, we focused on the development of buccal polymer films with pramipexole dihydrochloride (PR). Films with different compositions were formulated and their mechanical properties and chemical interactions were investigated. The biocompatibility of the film compositions was examined on the TR146 buccal cell line. The permeation of PR was also monitored across the TR146 human cell line. It can be stated that the plasticizer can enhance the thickness and the breaking hardness of the films, while not decreasing their mucoadhesivity significantly. All formulations proved to have cell viability higher than 87%. Finally, we found the best composition (3% SA+1% GLY-PR-Sample1) which can be applied on the buccal mucosa in the treatment of PD.

Interleukin-13 contributes to the occurrence of oral submucosal fibrosis.

Oral submucous fibrosis (OSF) is a chronic progressive fibrosis disease that affects in oral mucosal tissues. Interleukin (IL)-13 has been implicated in the development of fibrosis in multiple organs. Indeed, it contributes to diseases such as pulmonary fibrosis, liver cirrhosis among others. Currently, its expression in OSF and the specific mechanisms are not well understood. The aim of this study was to investigate the role of IL-13 in OSF and further explore whether IL-13 regulates-polarization of M2-macrophages in OSF. Initially, in the tissues of patients with OSF, we observed a high expression of M2-macrophages and IL-13 protein. Additionally, we found a correlation between the expression of IL-13 and the stage of OSF. Arecoline inhibited the proliferation of fibroblasts (FBs) and promoted IL-13 production in vitro. Furthermore, our observations revealed that M2-macrophages increased upon co-culturing M0-macrophages with supernatants containing the IL-13 cytokine. In conclusion, our study demonstrated that arecoline stimulates FBs leading to increased secretion of IL-13, which in turn IL-13 leads to polarization of M2-macrophages and promotes the occurrence of OSF. This suggests that IL-13 may be a potential therapeutic target of OSF.