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Background: Ionizing radiation (IR) is a double-edged sword for immunotherapy as it may have both immunosuppressive and immunostimulatory effects. The biological effects of IR on the tumor microenvironment (TME) are a key factor for this balance. Fibroblast activation protein (FAP) is expressed on the surface of cancer-associated fibroblasts (CAF) in many cancer types and its abundance is associated with the poor immune response to immune-checkpoint-blockade in patients. We hypothesized that IR increases FAP expression in CAFs, therefore the combination of IR with targeted immunomodulators such as an agonistic anti-FAP-4-1BBL fusion protein could enhance the immune-mediated antitumoral effects of these treatments., Methods: The murine transplantable TS/A tumor-cell-line co-engrafted with CAFs was used to investigate increases in FAP expression in tumors following irradiation using immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and multiplex tissue immunofluorescence. One lesion of bilateral tumor-bearing mice was only locally irradiated or combined with weekly injections of the bispecific muFAP-4-1BBL fusion protein (a mouse surrogate for RG7826). Tumor sizes were followed over time and TME was assessed by flow cytometry. Selective monoclonal antibody (mAb)-mediated depletions of immune cell populations, neutralizing interferon alpha/beta receptor 1 (IFNAR-I) IFNAR and interferon (IFN)-γ mAbs and gene-modified mice (4-1BB -/- ) were used to delineate the immune cell subsets and mechanisms required for efficacy. 67 Ga labeled muFAP-4-1BBL tracked by SPECT-CT was used to study biodistribution. In human colorectal carcinoma samples, the inducibility of FAP expression following radiotherapy was explored by multiplex immunofluorescence., Results: Irradiation of TS/A+CAF tumors in mice showed an increase in FAP levels after local irradiation. A suboptimal radiotherapy regimen in combination with muFAP-4-1BBL attained primary tumor control and measurable abscopal effects. Immune TME landscape analyses showed post-treatment increased infiltration of activated immune cells associated with the combined radioimmunotherapy treatment. Efficacy depended on CD8 + T cells, type I IFN, IFN-γ and ability to express 4-1BB. Biodistribution studies of muFAP-4-1BBL indicated enriched tumor targeting to irradiated tumors. Human colorectal cancer samples pre and post irradiation showed enhanced FAP expression after radiotherapy., Conclusion: Increased FAP expression in the TME as a result of radiotherapy can be exploited to target agonist 4-1BB immunotherapy to malignant tumor lesions using an FAP-4-1BBL antibody fusion protein., Competing Interests: Competing interests: EG-S, IS-M, LF-R, AR, CEDA, CB-A, CdPH, CLR, AT, JAS, MC, and PS-M declare no competing interests. MER-R reports receiving research funding from Roche and Highlight Therapeutics. She also has received speaker’s bureau honoraria from BMS and ROCHE. IM reports receiving commercial research grants from BMS, Highlight Therapeutics, Alligator, Pfizer Genmab and Roche; has received speaker’s bureau honoraria from MSD; and is consultant or advisory board member for BMS, Roche, AstraZeneca, Genmab, Pharmamar, F-Star, Bioncotech, Bayer, Numab, Pieris, Gossamer, Alligator and Merck Serono. CK, PU, CC, and TT declare employment, stock ownership and patents with Roche., (© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

The Reelin signaling pathway, particularly the RELN-APOER2-DAB1 complex, has emerged as a key contributor to the neuropathology of Alzheimer's disease (AD) and Schizophrenia (SZ). Despite being distinct clinical conditions, these disorders exhibit similar patterns of cognitive decline, including early disruptions in synaptic function and memory impairments. Notably, individuals with SZ have a 2-4 fold increased risk of developing AD or other dementias, highlighting potential shared molecular mechanisms, and positioning Reelin as a pivotal link between them. This systematic review explores the role of Reelin and its signaling components across these disorders. In AD, Reelin disruption correlates with hallmark features such as Tau hyperphosphorylation, amyloid-beta accumulation, and cognitive deficits. In SZ, alterations in Reelin signaling, including epigenetic modifications affecting RELN expression, are linked to disruptions in neuronal development and synaptic plasticity, particularly in the parietal and prefrontal cortices. Additionally, genomic studies reveal specific RELN variants and allelic imbalances that may influence disease severity and treatment response in SZ, suggesting RELN's role as a potential biomarker for therapeutic outcomes. Region-specific Reelin alterations in both AD and SZ suggest differing impacts yet underscore a potential common molecular origin. Our findings highlight the Reelin pathway as a molecular convergence point, warranting further investigation as a therapeutic and diagnostic target for AD, SZ, and potentially other neuropsychiatric disorders. The interplay between genetic and epigenetic regulation of RELN may provide novel insights into neurodegeneration, with implications for personalized intervention strategies in AD and SZ., Competing Interests: Declaration of Competing Interest No potential competing interest is reported by the authors., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The development of an effective antitumor response relies on the synergistic actions of various immune cells that recognize tumor cells via distinct receptors. Tumors, however, often manipulate receptor-ligand interactions to evade recognition by the immune system. Recently, we highlighted the role of neolacto-series glycosphingolipids (nsGSLs), produced by the enzyme β1,3-N-acetylglucosaminyltransferase 5 (B3GNT5), in tumor immune escape. We previously demonstrated that loss of signal peptide peptidase like 3 (SPPL3), an inhibitor of B3GNT5, results in elevated levels of nsGSLs and impairs CD8 T cell activation. The impact of loss of SPPL3 and an elevated nsGSL profile in tumor cells on innate immune recognition remains to be elucidated. This study investigates the antitumor efficacy of neutrophils, NK cells, and γδ T cells on tumor cells lacking SPPL3. Our findings demonstrate that SPPL3-deficient target cells are less susceptible to trogocytosis by neutrophils and killing by NK cells and γδ T cells. Mechanistically, SPPL3 influences trogocytosis and γδ T cell-instigated killing through modulation of nsGSL expression, whereas SPPL3-mediated reduced killing by NK cells is nsGSL-independent. The nsGSL-dependent SPPL3 sensitivity depends on the proximity of surface receptor domains to the cell membrane and the affinity of receptor-ligand interactions as shown with various sets of defined antibodies. Thus, SPPL3 expression by tumor cells alters crosstalk with immune cells through the receptor-ligand interactome thereby driving escape not only from adaptive but also from innate immunity. These data underline the importance of investigating a potential synergism of GSL synthesis inhibitors with current immune cell-activating immunotherapies., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

A critical aspect of the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the protease-mediated activation of the viral spike (S) protein. The type II transmembrane serine protease TMPRSS2 is crucial for SARS-CoV-2 infection in lung epithelial Calu-3 cells and murine airways. However, the importance of TMPRSS2 needs to be re-examined because the ability to utilize TMPRSS2 is significantly reduced in the Omicron variants that spread globally. For this purpose, replication profiles of SARS-CoV-2 were analyzed in human respiratory organoids. All tested viruses, including Omicron variants, replicated efficiently in these organoids. Notably, all SARS-CoV-2 strains retained replication ability in TMPRSS2-gene knockout (KO) respiratory organoids, suggesting that TMPRSS2 is not essential for SARS-CoV-2 infection in human respiratory tissues. However, TMPRSS2-gene knockout significantly reduces the inhibitory effect of nafamostat, indicating the advantage of TMPRSS2-utilizing ability for the SARS-CoV-2 infection in these organoids. Interestingly, Omicron variants regained the TMPRSS2-utilizing ability in recent subvariants. The basal infectivity would be supported mainly by cathepsins because the cathepsin inhibitor, EST, showed a significant inhibitory effect on infection with any SARS-CoV-2 strains, mainly when used with nafamostat. A supplementary contribution of other serine proteases was also suggested because the infection of the Delta variant was still inhibited partially by nafamostat in TMPRSS2 KO organoids. Thus, various proteases, including TMPRSS2, other serine proteases, and cathepsins, co-operatively contribute to SARS-CoV-2 infection significantly in the respiratory organoids. Thus, SARS-CoV-2 infection in the human respiratory tissues would be more complex than observed in cell lines or mice., Importance: We explored how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects human respiratory organoids, which are a cultured cell model made to mimic the physiological conditions of the human airways. We focused on understanding the role of different proteases of host cells in activating the virus spike proteins. Specifically, we looked at TMPRSS2, a transmembrane serine protease, and cathepsin L, a lysosomal enzyme, which helps the virus enter cells by cutting the viral spike protein. We discovered that while TMPRSS2 is crucial for the virus in certain cells and animal models, other proteases, including cathepsins and various serine proteases, also play significant roles in the SARS-CoV-2 infection of human respiratory organoids. We suggest that SARS-CoV-2 uses a more complex mechanism involving multiple proteases to infect human airways, differing from what we see in conventional cell lines or animal models. This complexity might help explain how different variants can spread and infect people effectively., Competing Interests: The authors declare no conflict of interest.

Entomopathogenic fungi play a critical role in regulating insect populations, and representative species from the Metarhizium and Beauveria genera have been developed as eco-friendly biocontrol agents for managing agricultural insect pests. Relative to the advances in understanding antifungal immune responses in Drosophila , knowledge of how fungi evade insect immune defenses remains limited. In this study, we report the identification and characterization of a virulence-required effector Fkp1 in Metarhizium robertsii . Library screening and protein pull-down analysis unveiled that Fkp1 targets the cathepsin protease CtsK1 to inhibit its cleavage maturation of the danger-sensing serine protease Persephone (Psh), thereby facilitating fungal evasion of the Drosophila immune defenses. The Fkp1 -like gene is also required in Beauveria bassiana for insect infection. Transgenic expression of Fkp1 in Drosophila suppressed hemolymph cysteine protease activity and down-regulated the expression of antifungal genes. Fkp1 can also mask the Psh cleavage site without interfering with its ability to bait fungal subtilisin proteases. Given the evident compensatory relationship, our data indicate that the protease cascade is more crucial than the molecular pattern pathway in defending flies against fungal infections. This work reveals that Metarhizium fungi have evolved distinct effectors to block the dual recognition pathways of flies for immune evasion and sheds lights on the effector mechanisms mediating microbe-animal interactions., Competing Interests: Competing interests statement:The authors declare no competing interest.

Positively charged residues are commonly located near the cytoplasm-membrane interface, which is known as the positive-inside rule in membrane topology. The mechanism underlying the function of these charged residues remains poorly understood. Herein, we studied the function of cytoplasmic residues in corin, a type II transmembrane serine protease in cardiovascular biology. We found that the positively charged residue at the cytoplasm-membrane interface of corin was not a primary determinant in membrane topology but probably served as a charge-repulsion mechanism in the endoplasmic reticulum (ER) to prevent interactions with proteins in the ER, including the signal peptidase. Substitution of the positively charged residue with a neutral or acidic residue resulted in corin secretion likely due to signal peptidase cleavage. In signal peptidase-deficient cells, the mutant corin proteins were not secreted but retained in the ER. Similar results were found in the low-density lipoprotein receptor and matriptase-2 that have positively charged residues at and near the cytoplasm-membrane interface. These results provide important insights into the role of the positively charged cytoplasmic residues in mammalian single-pass transmembrane proteins., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: The angiotensin-converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2) are central human molecules in the SARS-CoV-2 virus-host interaction. Evidence indicates that ACE1 may influence ACE2 expression. This study aims to determine whether ACE1, ACE2, and TMPRSS2 mRNA expression levels, along with the ACE1 Alu 287 bp polymorphism (rs4646994), contribute to the severity and mortality of COVID-19., Methods: Swabs were collected in two Brazilian cities in 2020: Belo Horizonte ( n  = 134) and Rio de Janeiro ( n  = 41). A swab of mild patients in Rio de Janeiro who were not hospitalized ( n  = 172) was also collected. All analyzed biological material was obtained from residual diagnostic samples in 2020, prior to the emergence of SARS-CoV-2 variants of concern. ACE1 , ACE2 , TMPRSS2, and B2M (reference gene) expression levels were evaluated in 40 cycles of quantitative PCR. ACE1 Alu 287 bp polymorphism was genotyped using the FastStart Universal SYBR Green Master kit., Results: The median age differed between clinical sites ( p  = 0.016), but no difference in median days of hospitalization was observed ( p  = 0.329). Age was associated with severity ( p  = 0.014) and mortality ( p  = 0.014) in the Belo Horizonte cohort. No alteration in ACE1 , ACE2 and TMPRSS2 expression was associated with severity or mortality. ACE1 polymorphism rs4646994 did not influence the likelihood of either outcome. A meta-analysis including available data from the literature showed significant effects: the D-allele conferred risk (OR = 1.39; 95% CI [1.12-1.72])., Competing Interests: Shana Priscila Coutinho Barroso is a researcher at BioVet, funded by the Foundation for Research Support of the State of Rio de Janeiro –FAPERJ. Renan Pedra de Souza is an Academic Editor for PeerJ., (©2025 de Araújo et al.)

Gastric cancer (GC) mesenchymal stromal cells (GCMSCs) are the predominant components of the tumor microenvironment (TME) and play a role in the occurrence, development, and metastasis of tumors. However, GCMSCs exhibit phenotypic and functional heterogeneity. The key population of GCMSCs which are vital to tumor progression remains elusive. The expression of fibroblast activation protein (FAP) in gastric cancer was analyzed and verified using clinical pathology data and single-cell RNA sequencing database of gastric cancer patients. FAP positive GCMSCs (FAP + GCMSCs) were isolated via flow cytometry and characterized through transcriptomic sequencing. The impact of conditioned medium from FAP + GCMSCs on gastric cancer cell lines was assessed using Enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Additionally, immunohistochemistry (IHC) and Masson's trichrome staining were employed to explore the association between FAP + GCMSCs and extracellular matrix (ECM) deposition in gastric cancer tissues. Our study demonstrates that FAP is predominantly expressed in the mesenchymal stromal cells within the gastric cancer milieu. FAP + GCMSCs exhibited enhanced proliferation, migration, contraction, and tumor-promoting capabilities compared to their FAP - counterparts. These cells significantly increased proliferation and migration of gastric cancer cells through the paracrine secretion of Inhibin Subunit Beta A (INHBA) and activation of the SMAD2/3 signaling pathway. Moreover, FAP + GCMSCs also induced collagen deposition in ECM and then up-regulated invasion and stemness of GC cells. Mechanistically, this process was mediated by the interaction of collagen with Integrin Subunit Beta 1 (ITGB1), triggering the phosphorylation of Focal Adhesion Kinase (FAK) and Yes Associated Transcriptional Regulator (YAP). Our findings reveal that FAP + GCSMCs enhanced the GC progression via releasing cytokine INHBA and remodeling ECM providing a theoretical basis for further exploration of tumor stromal-targeting therapy of gastric cancer., 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 © 2024 Elsevier B.V. All rights reserved.)

TMPRSS2, a human transmembrane protease enzyme, plays a crucial role in the spread of certain viruses, including influenza and coronaviruses. This enzyme promotes viral infection by cleaving viral glycoproteins, which helps viruses like SARS-CoV-2 and influenza A enter cells more effectively. Genetic differences in TMPRSS2 may affect people's susceptibility to COVID-19, underscoring the need for studies that consider diverse populations. Beyond infectious diseases, TMPRSS2 has also been linked to some cancers, suggesting it could be a valuable target for drug development. This review provides a summary of TMPRSS2 inhibitors currently under study, with some already in clinical trials to test their effectiveness against viral infections. As we uncover more about TMPRSS2's role in pathogenesis, it could open new doors for therapies to combat future outbreaks.

Numerous studies of the human brain supported by experimental results from rodent and cell models point to a central role for intracellular amyloid beta (Aβ) in the onset of Alzheimer's disease (AD). In a rat model used to study AD, it was recently shown that in layer II neurons of the anteriolateral entorhinal cortex expressing high levels of the glycoprotein reelin (Re+alECLII neurons), reelin and Aβ engage in a direct protein-protein interaction. If reelin functions as a sink for intracellular Aβ and if the binding to reelin makes Aβ physiologically inert, it implies that reelin can prevent the neuron from being exposed to the harmful effects typically associated with increased levels of oligomeric Aβ. Considering that reelin expression is extraordinarily high in Re+alECLII neurons compared to most other cortical neurons, such a protective role appears to be very difficult to reconcile with the fact that this subset of ECLII neurons is clearly a major cradle for the onset of AD. Here, we show that this conundrum can be resolved if Re+alECLII neurons have a higher maximum production capacity of Aβ than neurons expressing low levels of reelin, and we provide a rationale for why this difference has evolved., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Kobro-Flatmoen, Omholt. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

FLG is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it colocalized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis using benzyloxycarbonyl-pro-prolinal induced the accumulation of FLG monomers. Downregulation of PREP expression in reconstructed human epidermis using RNA interference confirmed the impact of PREP on FLG metabolism and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, western blotting, and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification such as loricrin. Consequently, at the functional level, the transepidermal electric resistance was drastically reduced., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

SARS-CoV-2, the causative virus of COVID-19, increases the risk of pregnancy complications including hypertensive disorders and placental inflammation. The spike glycoprotein mediates viral cell entry by interacting with the angiotensin-converting enzyme (ACE)2 in conjunction with the transmembrane serine protease 2 (TMPRSS2). ACE1, ACE2 and renin are components of the renin-angiotensin system (RAS), which regulates blood pressure. As the placenta expresses all these proteins, it is a target for SARS-CoV-2 and a source of blood pressure modulators. Noteworthy, an ACE1/ACE2 ratio imbalance can lead to RAS dysregulation and a bad prognosis in COVID-19 patients. Calcitriol, the most active vitamin D metabolite, negatively regulates RAS, reduces inflammation, and enhances antiviral immunity, thereby protecting against COVID-19 severity. However, contrasting information exists on the regulatory role of calcitriol upon RAS components and SARS-CoV-2 receptors; while the impact of calcitriol on spike-induced inflammation in placental cells has not been explored. Thus, we studied the effects of calcitriol on these parameters using the trophoblast cell line HTR-8/SVneo and primary syncytiotrophoblasts. By RT-qPCR, ELISA, and immunocytochemistry, we found that the spike enhanced proinflammatory cytokines expression and secretion, while calcitriol significantly downregulated this effect. Calcitriol also diminished ACE1, ACE2, TMPRSS2, and renin gene expression, as well as ACE1/ACE2 mRNA ratio. CONCLUSIONS: In the human placenta, calcitriol reduced the gene expression of main RAS components and TMPRSS2, resulting in the inhibition of spike-induced inflammation. This outcome suggest that vitamin D participates in restricting SARS-CoV-2 placental infection by rendering trophoblasts less permissive to infection while helping to regulate maternal blood pressure and decreasing inflammation., Competing Interests: Declaration of Competing Interest All authors declare that there are no financial or personal relationships with other people or organizations that could inappropriately influence the work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SARS-CoV-2 infection is accompanied by elevated liver enzymes, and patients with pre-existing liver conditions experience more severe disease. While it was known that SARS-CoV-2 infects human hepatocytes, our study determines the mechanism of infection, demonstrates viral replication and spread, and highlights direct hepatocyte damage. Viral replication was readily detectable upon infection of primary human hepatocytes and hepatoma cells with the ancestral SARS-CoV-2, Delta, and Omicron variants. Hepatocytes express the SARS-CoV-2 receptor ACE2 and the host cell protease TMPRSS2, and knocking down ACE2 and TMPRSS2 impaired SARS-CoV-2 infection. Progeny viruses released from infected hepatocytes showed the typical coronavirus morphology by electron microscopy and proved infectious when transferred to fresh cells, indicating that hepatocytes can contribute to virus spread. Importantly, SARS-CoV-2 infection rapidly induced hepatocyte death in a replication-dependent fashion, with the Omicron variant showing faster onset but less extensive cell death. C57BL/6 wild-type mice infected with a mouse-adapted SARS-CoV-2 strain showed high levels of viral RNA in liver and lung tissues. ALT peaked when viral RNA was cleared from the liver. Liver histology revealed profound tissue damage and immune cell infiltration, indicating that direct cytopathic effects of SARS-CoV-2 and immune-mediated killing of infected hepatocytes contribute to liver pathology., (© 2025 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

SARS-CoV-2 virus infects cells by engaging with ACE2 requiring protease TMPRSS2. ACE2 is highly expressed in kidneys. Predictors for severe disease are high age and male sex. We hypothesized that ACE2 and TMPRSS2 proteins are more abundant (1) in males and with increasing age in kidney and (2) in urine and extracellular vesicles (EVs) from male patients with COVID-19 and (3) SARS-CoV-2 is present in urine and EVs during infection. Kidney cortex samples from patients subjected to cancer nephrectomy (male/female; < 50 years/˃75 years, n = 24; ˃80 years, n = 15) were analyzed for ACE2 and TMPRSS2 protein levels. Urine from patients hospitalized with SARS-CoV-2 infection was analyzed for ACE2 and TMPRSS2. uEVs were used for immunoblotting and SARS-CoV-2 mRNA and antigen detection. Tissue ACE2 and TMPRSS2 protein levels did not change with age. ACE2 was not more abundant in male kidneys in any age group. ACE2 protein was associated with proximal tubule apical membranes in cortex. TMPRSS2 was observed predominantly in the medulla. ACE2 was elevated significantly in uEVs and urine from patients with COVID-19 with no sex difference compared with urine from controls w/wo albuminuria. TMPRSS2 was elevated in uEVs from males compared to female. ACE2 and TMPRSS2 did not co-localize in uEVs/apical membranes. SARS-CoV-2 nucleoprotein and mRNA were not detected in urine. Higher kidney ACE2 protein abundance is unlikely to explain higher susceptibility to SARS-CoV-2 infection in males. Kidney tubular cells appear not highly susceptible to SARS-CoV-2 infection. Loss of ACE2 into urine in COVID could impact susceptibility and angiotensin metabolism., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

SARS-CoV-2-related proteins, ACE2 and TMPRSS2, are determinants of SARS-CoV-2 infection. Although these proteins are expressed in oral-related tissues, their expression patterns and modulatory mechanisms in the salivary glands remain unknown. We herein showed that full-length ACE2, which has both a fully functional enzyme catalytic site and high-affinity SARS-CoV-2 spike S1-binding sites, was more highly expressed in salivary glands than in oral mucosal epithelial cells and the lungs. Regarding TMPRSS2, zymogen and the cleaved form were both expressed in the salivary glands, whereas only zymogen was expressed in murine lacrimal glands and the lungs. Metformin, an AMPK activator, increased stimulated saliva secretion and full-length ACE2 expression and decreased cleaved TMPRSS2 expression in the salivary glands, and exerted the same effects on soluble ACE2 (sACE2) and sTMPRSS2 in saliva. Moreover, metformin decreased the expression of beta-galactosidase, a senescence marker, and ADAM17, a sheddase of ACE2 to sACE2, in the salivary glands. In aged mice, the expression of ACE2 was decreased in the salivary glands, whereas that of sACE2 was increased in saliva, presumably by the up-regulated expression of ADAM17. The expression of TMPRSS2 in the salivary glands and sTMPRSS2 in saliva were both increased. Collectively, these results suggest that the protein expression patterns of ACE2 and TMPRSS2 in the salivary glands differ from those in other oral-related cells and tissues, and also that metformin and aging affect the salivary expression of ACE2 and TMPRSS2, which have the potential as targets for preventing the transmission of SARS-CoV-2., (© 2025 The Author(s). BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)