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Meibomian gland dysfunction is the most common cause of dry eye disease and leads to significantly reduced quality of life and social burdens. Because meibomian gland dysfunction results in impaired function of the tear film lipid layer, studying the expression of tear proteins might increase the understanding of the etiology of the condition. Machine learning is able to detect patterns in complex data. This study applied machine learning to classify levels of meibomian gland dysfunction from tear proteins. The aim was to investigate proteomic changes between groups with different severity levels of meibomian gland dysfunction, as opposed to only separating patients with and without this condition. An established feature importance method was used to identify the most important proteins for the resulting models. Moreover, a new method that can take the uncertainty of the models into account when creating explanations was proposed. By examining the identified proteins, potential biomarkers for meibomian gland dysfunction were discovered. The overall findings are largely confirmatory, indicating that the presented machine learning approaches are promising for detecting clinically relevant proteins. While this study provides valuable insights into proteomic changes associated with varying severity levels of meibomian gland dysfunction, it should be noted that it was conducted without a healthy control group. Future research could benefit from including such a comparison to further validate and extend the findings presented here., (© 2023. The Author(s).)

This study investigates the stability and cytotoxicity of biopolymer-coated liposomes for use in the oral cavity. Liposomes (3 mM and 6 mM) were prepared by the thin film method and hydrated with phosphate buffer (PB) or glycerol phosphate buffer (G-PB). For coating, liposomes were added to a biopolymer solution of opposite charge. Particle stability was evaluated by measuring the size, polydispersity index, and zeta potential for up to 60 weeks. In vitro interaction of fluorescent-labelled biopolymer-coated liposomes and dysplastic oral keratinocytes was analyzed by confocal microscopy. Potential cytotoxicity was assessed in dysplastic oral keratinocytes by cell proliferation and cell viability. All three biopolymers showed good coating abilities for both concentrations and hydration media. The alginate coated liposomes in PB, 3 mM chitosan-coated liposomes in PB, and chitosan-coated liposomes in G-PB were stable for up to 60 weeks. In vitro studies demonstrated low cytotoxicity for all coated liposomes and non-specific cellular uptake of biopolymer-coated liposomes, independent of biopolymer, surface charge, lipid concentration and hydration media. All three formulations demonstrated low cytotoxicity and were considered safe. Alginate- and chitosan-coated liposomes demonstrated good stability over time and may be promising agents for use in the oral cavity and should be investigated further., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs), are important for intercellular communication in both physiological and pathological processes. To explore the potential of cancer derived EVs as disease biomarkers for diagnosis, monitoring, and treatment decision, it is necessary to thoroughly characterize their biomolecular content. The aim of the study was to characterize and compare the protein content of EVs derived from three different cancer cell lines in search of a specific molecular signature, with emphasis on proteins related to the carcinogenic process. Oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC) and melanoma brain metastasis cell lines were cultured in CELLine AD1000 flasks. EVs were isolated by ultrafiltration and size-exclusion chromatography and characterized. Next, the isolated EVs underwent liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Functional enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different proteins were identified in EVs from each particular cell line. Here, 14%, 10%, and 24% of the identified proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell line, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the identified proteins were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as "biological adhesion", "cell motility", and "cellular component biogenesis". EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from the ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy., Competing Interests: The authors have declared that no competing interests exist.

Salivary and lacrimal gland involvement is a characteristic feature of primary Sjögren's syndrome (pSS), where tissue destruction is mediated by mononuclear cell infiltration, resulting in lacrimal and salivary gland impairment. We have previously shown distinct prevalence of adipose tissue replacement in the minor salivary gland tissue from pSS patients. The salivary gland microenvironment was further examined through microarray analysis, identifying signalling pathways that promoted adipose tissue development, inflammation, and lymphoma. As B cells may also contribute to disease progression, we now aimed to study the B cell pattern with regard to adipocyte development in pSS. Double immunohistochemical staining of paraffin-embedded salivary gland tissue from 22 pSS patients and 11 non-SS tissue controls was employed, using the characteristic pSS autoantigens Ro52 or Ro60, alongside CD27. Additional CD138/CD20 double staining was also performed to identify the plasma- and general B- cell pattern. Our results demonstrated CD27-positive Ro52 and Ro60 specific cells observed within and in close proximity to the adipose tissue. CD138-positive plasma cells were also seen in areas of adipose tissue replacement, while the CD20 + cells were located within focal infiltrates, forming distinct B cell zones. The quantification of CD138 + and CD20 + cells revealed elevated numbers of CD138 + cells in areas of fatty infiltration, and also interstitially, in the salivary glands of pSS patients when compared to non-SS controls. A significant increase ( p  < .01) in CD138 + cells close to areas of fatty infiltration, and interstitially, with increasing fatty infiltration and focus score was further observed in pSS patients. A correlation between the number of CD20 + B cell zones/mm 2 of salivary gland tissue and focus score values was also witnessed in the patients ( r 2 = 0.6047, p  < .001). In conclusion, autoantigen-specific B cells and plasma cells appear prominent in areas of fatty infiltration in salivary glands of pSS patients, where an increase in CD138 + plasma cells and CD20 + B cells, in relation to both fatty and focal infiltration, suggests their active involvement in promoting inflammation. Further studies are needed to assess whether these adipocytes are also a result of tissue repair.

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5-18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.

Rationale and Objectives: Most radiographic contrast media (CM) are hyperosmotic and pose an osmotic threat to cells they are in contact with. To study these effects at the cellular level, cell volume regulatory mechanisms were observed in proximal renal tubules following exposure to the CM iohexol, ioxaglate, and iodixanol., Materials and Methods: Isolated renal tubules from trout (Salmo trutta) were exposed to 5% vol/vol iohexol (326 mOsm), ioxaglate (314 mOsm), or iodixanol (300 mOsm) or mannitol (to achieve the same osmolalities), and cell volume changes were observed videometrically., Results: Iohexol and ioxaglate solutions induced a rapid shrinkage (12%-13%) not followed by cell volume regulation. Without CM (same osmolality), the cells shrank 11% but then showed a 77%-88% volume recovery. This reswelling was inhibited by 55% with the Na+, K+, Cl- symporter inhibitor bumetanide (50 micromol/L). Iodixanol did not significantly affect cell volume. Tubules preincubated with CM or mannitol were then stimulated with a hypoosmotic Ringer solution (160 mOsm) resulting in a 26%-36% cellular volume increase. Compared with results of experiments without mannitol and CM, preexposure to iohexol or ioxaglate almost completely inhibited the expected regulatory shrinkage phase, while previous exposure to hyperosmotic solutions with mannitol reduced the shrinkage response by 40%-53%., Conclusion: In this system, the hyperosmotic iohexol and ioxaglate cause cell shrinkage followed by an impaired cell volume regulatory response. Exposure to these two CM also inhibits cell volume regulation on hypoosmotic stimulation. The isosmotic iodixanol has no such effects. These changes appear to some extent to be a result of the CM's degree of hyperosmolality, but this property alone does not explain these findings.