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Tick-borne encephalitis virus (TBEV) is flavivirus transmitted to the host via tick saliva which contains various molecules with biological impacts. One of such molecules is Iristatin, a cysteine protease inhibitor from Ixodes ricinus that has been shown to have immunomodulatory properties. To characterize Iristatin in the relation to TBEV, we investigate whether this tick inhibitor has any capacity to influence TBEV infection. Mice were intradermally infected by TBEV with or without Iristatin and the viral multiplication was determined in skin and brain tissues by RT-PCR two and 5 days after infection. The viral RNA was detected in both intervals in skin and increased by time. The application of Iristatin caused a reduction in viral RNA in skin but not in the brain of infected mice 5 days post-infection. Moreover, anti-viral effect of Iristatin on skin was accompanied by a significant decline of interferon-stimulated gene 15 gene expression. The effect of Iristatin on TBEV replication was tested also in vitro in primary macrophages and dendritic cells; however, no changes were observed suggesting no direct interference of Iristatin with virus replication. Still, the Iristatin caused a suppression of Erk1/2 phosphorylation in TBEV-infected dendritic cells and had the anti-apoptotic effect. This is the first report showing that a tick cystatin decreases the viral RNA in the host skin, likely indirectly through creating skin environment that is less supportive for TBEV replication. Assuming, that viral RNA reflects the amount of infectious virus, decline of TBEV in host skin could influence the tick biology or virus transmission during cofeeding., Competing Interests: Declarations. Ethics approval: All animal experiments were performed in accordance with the Animal Protection Law of the Czech Republic No. 246/1992 Coll. and protocol approved by the Ministry of Education, Youth and Sports of the Czech Republic (protocol no. 14231/2019–2) and the responsible committee of the Faculty of Science, University of South Bohemia. Consent to participate and consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cystatin SN (CST1) appears to have pro-tumor effects in breast cancer (BC) and is involved in ferroptosis; however, there is no report on the regulation of ferroptosis by CST1 for BC development. The purpose of this study is to investigate the functions and mechanisms operated by CST1 in BC development and ferroptosis. Transcription Factor Activator Protein 2γ (TFAP2C) and CST1 levels in BC tissues and estrogen receptor (ER) + cells were quantified by RT-qPCR and western blotting. After knocking down TFAP2C and CST1 expression in MCF7 and T47D cells, the proliferation, colony formation ability, apoptosis, and cell cycle were assessed. Ferroptosis was verified by detecting glutathione peroxidase 4 (GPX4) and 4-hydroxy-2-nonenal (4HNE) levels. The kits were used to test Fe 2+ , reactive oxygen species, malondialdehyde, and glutathione levels, and ultrastructure of mitochondria was observed through transmission electron microscope. Dual-luciferase reporter assay and chromatin immunoprecipitation test were carried out to investigate the interaction of TFAP2C and CST1. A transplanted tumor model was established to explore the function of TFAP2C in tumorigenesis by quantifying TFAP2C, CST1, Ki67, and GPX4 levels through western blotting and immunochemistry after silencing TFAP2C. TFAP2C and CST1 were predominantly expressed in BC cells. Silencing of TFAP2C or CST1 expression suppressed ER + BC cell proliferation, promoted apoptosis and ferroptosis, and blocked cell cycle transition from G1 phase to S phase. TFAP2C knockdown in transplanted tumors inhibited tumor growth and GPX4 level. Upregulating CST1 nullified the anti-tumor effects of TFAP2C knockdown and TFAP2C promoted CST1 expression through transcription activation. TFAP2C activates CST1 transcription to facilitate BC development and block ferroptosis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

BACKGROUND Gastrointestinal diffuse large B-cell lymphoma (GI-DLBCL) is the most common histological subtype of extra-nodal DLBCL, but the risk factors, prognostic biomarkers, histopathological classifications, and treatment strategies have not had significant progress. Emerging evidence shows that cystatin SN (CST1) is involved in tumor progression in several cancer types, but its role in GI-DLBCL has not been revealed. MATERIAL AND METHODS We established a cohort consisting of 84 patients with GI-DLBCL who underwent surgical resection. The expression of CST1 in the cohort was investigated by immunohistochemistry, which divided the patients into subgroups with low or high expression of CST1. Moreover, the CST1 expression in GI-DLBCL tissues or adjacent GI tissues were compared with RT-qPCR. The correlation between CST1 expression and clinicopathological factors was analyzed with the chi-square test. The prognostic significance of CST1 was estimated by univariate and multivariate analysis, and statistical significance was analyzed with the log-rank test. RESULTS CST1 was aberrantly upregulated in GI-DLBCL tissues compared with in non-tumor GI tissues. High expression of CST1 indicated poor prognosis of GI-DLBCL (P=0.012), and CST1 can be regarded as an independent prognostic biomarker of GI-DLBCL (hazard ratio=3.07). In our study, serum lactate dehydrogenase (P=0.002), performance status (P=0.003), Lugano stage (P=0.002), and International Prognostic Index (P=0.001) were also prognostic factors of GI-DLBCL. CONCLUSIONS CST1 is an independent prognostic biomarker of GI-DLBCL, indicating unfavorable prognosis. Our results suggested that CST1 detection can be a promising method to stratify high-risk patients and guide individual treatment.

Purpose: Cystatin SA (CST2) belongs to the superfamily of cysteine protease inhibitors. Emerging research indicates that CST2 is often dysregulated across various cancers. Its role and molecular mechanisms in gastric cancer remain underexplored. This study aims to explore the expression and function of CST2 in gastric cancer., Methods: CST2 expression was analyzed and validated through Western blot. CST2 overexpression was induced by lentivirus in GC cells, and the correlation between CST2 expression levels and downstream signaling pathways was assessed. In addition, multiple assays, including cell proliferation, colony formation, wound-healing, and transwell migration/invasion, were considered to ascertain the influence of CST2 overexpression on gastric cancer. The cell cycle and apoptosis were detected by flow cytometry., Results: CST2 expression at the protein level was decreased to be reduced in both gastric cancer tissues and cell lines, and CST2 expression attenuate gastric cancer growth, an effect restricted to gastric cancer cells and absent in gastric epithelial GES-1 cells. Furthermore, CST2 was demonstrated to improve chemosensitivity to Oxaliplatin in gastric cancer cells through the PI3K/AKT signaling pathway., Conclusion: These findings indicate that CST2 is downregulated at the protein level in gastric cancer tissues and cell lines. Additionally, CST2 was found to attenuate the growth of gastric cancer cells and to enhance sensitivity to Oxaliplatin through the PI3K/AKT signaling pathway, specific to gastric cancer cell lines. CST2 may serve as a tumor suppressor gene increasing sensitivity to Oxaliplatin in gastric cancer., (© 2024. The Author(s).)

Meibomian gland dysfunction (MGD) is a highly prevalent condition and the most common cause of evaporative dry eye disease. Studying the proteome of MGD can result in important advances in the management of the condition. Here, we collected tear film samples from treatment naïve patients with MGD (n = 10) and age-matched controls (n = 11) with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography-tandem mass spectrometry. The proteins were considered differentially expressed if p < 0.05. A total of 88 proteins were significantly regulated. The largest change was observed in cystatin-SN, which was downregulated in MGD and correlated negatively with tear meniscus height. The downregulation of cystatin-SN was confirmed with targeted mass spectrometry by single reaction monitoring (SRM). Eighteen immunoglobulin components involved in B cell activation, phagocytosis, and complement activation were downregulated in MGD including Ig alpha-1 chain C region, immunoglobulin J chain, immunoglobulin heavy variable 3-15, and Ig mu chain C region. The changes in cystatin-SN and immunoglobulin chains are likely to result from the inflammatory changes related to tear film evaporation, and future studies may assess their association with the meibum quality.

Typically, airway remodeling caused by migration and proliferation of airway smooth muscle cells (ASMCs) plays a crucial role in the pathophysiological characteristics of asthma development. Cystatin 1 (CST1), a protein-coding gene referred to as Cystatin SN, is highly expressed in asthma patients. However, the role of CST1 and related molecular mechanisms in the development of asthma remains to be explored. This study aims to investigate the role of CST1 in asthma progression and present related molecular mechanisms. To explore these aspects, human ASMCs with platelet-derived growth factor BB (PDGF-BB) are initially stimulated and applied as a cellular model of asthma. Further, CST1 is knocked down with small interfering ribose nucleic acid (siRNA) overexpressed with plasmids. Then, 5-ethynyl-2'-deoxyuridine (EdU) and Cell Count Kit (CCK)-8 assays are applied to assess the cell proliferation rates. Further, Transwell and Western blot analyses for migration of cells and expression of MMP1 and MMP9 proteins are assessed, respectively. Under PDGF-BB stimulation, human ASMCs showed an increased CST1 expression, enhanced proliferation, and migration abilities, as well as up-regulated PI3K/AKT signaling pathway. Further, knockdown or overexpression of CST1 presented the declined or enhanced proliferation, migration, and up-regulation of the PI3K/AKT signaling pathway of human ASMCs. Inhibiting PI3K/AKT signaling pathway displayed the reduced migration and proliferation of human ASMCs. In summary, these findings indicated that CST1 played an essential role in the progression of asthma by activating the PI3K/AKT signaling pathway and promoting the migration and proliferation abilities of human ASMCs treated with PDGF-BB., (© 2022 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Metastasis is an important factor contributing to poor prognosis in patients with gastric cancer; yet, the molecular mechanism leading to this cell behavior is still not well understood. In this study, we explored the role of cysteine protease inhibitor SN (Cystatin SN, CST1) in promoting gastric cancer metastasis. We hypothesized that CST1 could regulate gastric cancer progression by regulating GPX4 and ferroptosis. Whole transcriptome sequencing suggested that the expression of CST1 was significantly increased in metastatic cancer, and high CST1 expression was correlated with a worse prognosis. Our data further confirmed that the overexpression of CST1 may significantly promote the migration and invasion of gastric cancer cells in vitro and enhance liver, lung, and peritoneal metastasis of gastric cancer in nude mice. Meanwhile, high expression of CST1 promoted the epithelial-mesenchymal transition (EMT) of gastric cancer cells. Mechanistically, a co-immunoprecipitation experiment combined with mass spectrometry analysis confirmed that CST1 could interact with GPX4, a key protein regulating ferroptosis. CST1 relieves GPX4 ubiquitination modification by recruiting OTUB1, improving GPX4 protein stability and reducing intracellular reactive oxygen species (ROS), thereby inhibiting ferroptosis and, in turn, promoting gastric cancer metastasis. Moreover, clinical data suggested that CST1 is significantly increased in peripheral blood and ascites of gastric cancer patients with metastasis; multivariate Cox regression model analysis showed that CST1 was an independent risk factor for the prognosis of gastric cancer patients. Overall, our results elucidated a critical pathway through which high CST1 expression protects gastric cancer cells from undergoing ferroptosis, thus promoting its progression and metastasis. CST1 may be used as a new oncological marker and potential therapeutic target for gastric cancer metastasis., (© 2022. The Author(s).)

Background: Cystatin SN (CST1) and cystatin SA (CST2) are cysteine protease inhibitors that protect against allergen, viral, and bacterial proteases. Cystatins are overexpressed in the setting of allergic rhinitis and chronic rhinosinusitis with nasal polyps (CRSwNP); however, their role in promoting type 2 inflammation remains poorly characterized., Objective: The purpose of this study was to use integrated poly-omics and a murine exposure model to explore the link between cystatin overexpression in CRSwNP and type 2 inflammation., Methods: In this institutional review board- and institutional animal care and use committee-approved study, we compared tissue, exosome, and mucus CST1 and CST2 between CRSwNP and controls (n = 10 per group) by using matched whole exome sequencing, transcriptomic, proteomic, posttranslational modification, histologic, functional, and bioinformatic analyses. C57/BL6 mice were dosed with 3.9 μg/mL of CST1 or PBS intranasally for 5 to 18 days in the presence or absence of epithelial ABCB1a knockdown. Inflammatory cytokines were quantified by using Quansys multiplex assays or ELISAs., Results: Of the 1305 proteins quantified, CST1 and CST2 were among the most overexpressed protease inhibitors in tissue, exosome, and mucus samples; they were localized to the epithelial layer. Multiple posttranslational modifications were identified in the polyp tissue. Exosomal CST1 and CST2 were strongly and significantly correlated with eosinophils and Lund-Mackay scores. Murine type 2 cytokine secretion and T H 2 cell infiltration increased in a time-dependent manner following CST1 exposure and was abrogated by epithelial knockdown of ABCB1a, a regulator of epithelial cytokine secretion., Conclusion: CST1 is a potent upstream initiator of epithelial-derived type 2 inflammation in CRSwNP. Therapeutic strategies targeting CST activity and its associated posttranslational modifications deserve further interrogation., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Cystatins are a class of proteins that can inhibit cysteine protease and are widely distributed in human bodily fluids and secretions. Cystatin SN (CST1), a member of the CST superfamily, is abnormally expressed in a variety of tumors. However, its effect on the occurrence and development of lung adenocarcinoma (LUAD) remains unclear., Methods: We obtained transcriptome analysis data of CST1 from The Cancer Genome Atlas (TCGA) and GSE31210 databases. The association of CST1 expression with prognosis, gene mutations and tumor immune microenvironment was analyzed using public databases. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed to investigate the potential mechanisms of CST1., Results: In this study, we found that CST1 was highly expressed in lung adenocarcinoma and was associated with prognosis and tumor immune microenvironment. Genetic mutations of CST1 were shown to be related to disease-free survival (DFS) by using the c-BioPortal tool. Potential proteins binding to CST1 were identified by constructing a protein-protein interaction (PPI) network. Gene set enrichment analysis (GSEA) of CST1 revealed that CST1 was notably enriched in epithelial-mesenchymal transition (EMT). Cell experiments confirmed that overexpression of CST1 promoted lung adenocarcinoma cells migration and invasion, while knockdown of CST1 significantly inhibited lung adenocarcinoma cells migration and invasion., Conclusions: Our comprehensive bioinformatics analyses revealed that CST1 may be a novel prognostic biomarker in LUAD. Experiments confirmed that CST1 promotes epithelial-mesenchymal transition in LUAD cells. These findings will help to better understand the distinct role of CST1 in LUAD., (© 2022. The Author(s).)

Background: Effective means for early diagnosis are imperative to reduce death rate of non-small cell lung cancer (NSCLC) patients. We aimed to find out high-performance serologic markers to distinguish early-stage NSCLC patients from benign pulmonary nodule patients and healthy controls (HC). Cystatin-SN (CST1) is an active cysteine protease inhibitor of the CST superfamily, involving in the processes of inflammation and tumorigenesis. This is the first exploration of the diagnostic and prognostic values of serum CST1 in NSCLC., Methods: We analyzed the transcriptome data from The Cancer Genome Atlas and the Gene Expression Omnibus database, screened biomarkers for NSCLC, and verified the candidate markers via the ONCOMINE database. Then, we performed ELISA, western blotting, and immunohistochemistry analysis to detect the expression levels of CST1 in NSCLC cell lines, tumor tissues, and serum samples of clinical cohorts., Results: We identified 3 up-regulated secreted protein-encoding genes, validated the expression levels of CST1 in NSCLC tumor tissues and cell lines, and found that serum CST1 levels of NSCLC (4289 ± 2405 pg/mL) were significantly higher than those of PBN patients (1558 ± 441 pg/mL, P < .0001) and healthy controls (1529 ± 416 pg/mL, P < .0001). The AUC of the combination of CST1, Cytokeratin 19 fragment (Cyfra21-1), and Carcinoembryonic antigen (CEA) for distinguishing early-stage NSCLC from PBN/HC was as high as .914/0.925. Furthermore, our results suggested that the NSCLC patient with low serum CST1 level had a better survival rate., Conclusions: Serum CST1 may serve as a novel diagnostic marker for differentiating early-stage NSCLC from PBN and HC, and could be used as a prognosis predictor in NSCLC patients.

Background: The aim of the study was to determine the concentration of endogenous cystatin C and cystatin SN, as potential tumor biomarkers, in the serum and biological fluids of the eye in both healthy controls and patients with uveal melanoma., Patients and Methods: The concentration of both cystatins was determined in the intraocular fluid (IOF), tear fluid, and serum of patients with uveal melanoma and compared to baseline measurements in IOF, tears, serum, cerebral spinal fluid, saliva and urine of healthy controls., Results: The concentration of cystatin C in all the biological matrices obtained from healthy controls significantly exceeded the concentration of cystatin SN and was independent of gender. Cystatin C concentrations in the tear fluid of patients with uveal melanoma (both the eye with the malignancy, as well as the contralateral, non-affected eye), were significantly greater than cystatin C concentrations in the tear fluid of healthy controls and was independent of tumor size. The concentration of cystatin SN in IOF of patients with uveal melanoma was significantly less than the corresponding concentration of cystatin SN in healthy controls., Conclusions: The ratio of cystatins (CysC:CysSN) in both the serum and tear fluid, as well as the concentration of cystatin SN in IOF, would appear to strongly suggest the presence of uveal melanoma. It is further suggested that multiple diagnostic criteria be utilized if a patient is suspected of having uveal melanoma, such as determination of the cystatin C and cystatin SN concentrations in serum, tears, and IOF, ocular fundus and ultrasound imaging, and biopsy with histopathological evaluation., (© 2022 Maria A. Dikovskaya, Galina S. Russkikh, Konstantin V. Loktev, Thomas P. Johnston, Margarita M. Gevorgyan, Natalya P. Voronina, Valery V. Chernykh, Alexander N. Trunov, Tatiana A. Korolenko, published by Sciendo.)

The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1 + myofibroblasts with prognostic values and potential biological significance. CST1 + myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology., (© 2021. The Author(s).)

Psoralen (PSO) exerts anti‑inflammatory pharmacological effects and plays an important role in a variety of inflammatory diseases. However, the effects of PSO with allergic rhinitis (AR) are yet to be reported. In the present study, an in vitro AR model was generated by inducing JME/CF15 human nasal epithelial cells with IL‑13, after which MTT was used to assess the cytotoxicity of PSO. The expression levels of inflammatory cytokines (granulocyte‑macrophage colony‑stimulating factor and Eotaxin) were determined by ELISA. Furthermore, the expression of inflammatory IL‑6 and ‑8, as well as mucin 5AC, was assessed by reverse transcription‑quantitative PCR and western blotting, and cellular reactive oxygen species were detected using a 2',7'‑dichlorodihydrofluorescein diacetate fluorescent probe. Western blotting was also used to detect the expression and phosphorylation of c‑Fos and c‑Jun in the activator protein 1 (AP‑1) pathway, as well as the expression of cystatin‑SN (CST1). PSO inhibited the inflammatory response and mucus production in IL‑13‑induced JME/CF15 cells. Furthermore, the levels of c‑Fos and c‑Jun phosphorylation in the AP‑1 pathway were decreased in IL‑13‑induced JME/CF15 cells following PSO treatment. The expression of pathway proteins was activated by the addition of PMA, an AP‑1 pathway activator, which concurrently reversed the inhibitory effects of PSO on the inflammatory response and mucus formation. The addition of an AP‑1 inhibitor (SP600125) further inhibited pathway activity, and IL‑13‑induced inflammation and mucus formation was restored. In conclusion, PSO regulates the expression of CST1 by inhibiting the AP‑1 pathway, thus suppressing the IL‑13‑induced inflammatory response and mucus production in nasal mucosal epithelial cells.

Background: Integrated care pathways improve the management of patients with chronic rhinosinusitis with nasal polyps (CRSwNP). The application of integrated care pathways requires development of endotype-based biomarkers to stratify patients. The value of cytokines and markers induced by cytokines for the management of CRSwNP is largely unknown., Objectives: Our aim was to determine the prognostic and pharmacologic value of type 2, non-type 2 cytokines, and markers associated with type 2 inflammation, including CCL26, periostin, and cystatin SN, in nasal secretions for CRSwNP., Methods: This retrospective study assigned 151 patients with CRSwNP to the discovery and validation phases. Concentrations of cytokines, CCL26, periostin, and cystatin SN in nasal secretions were determined by using Luminex and ELISA. Predictive significance was assessed with receiver-operating characteristic curves. Survival analysis was performed by using Kaplan-Meier curves and Cox regression models., Results: Cystatin SN was an independent predictor of the uncontrolled status of CRSwNP over a 2-year follow-up after adjustment for other risk factors (hazard ratio = 1.168 and 1.132 in the discovery and validation phases, respectively; both P < .001). Patients with high cystatin SN concentrations presented with a faster onset and higher rate of uncontrolled status than did those with low levels (P < .001). Enhanced medical treatment for patients with high cystatin SN levels postponed the uncontrolled status in the discovery (P = .016) and validation (P = .002) phases but did not completely abolish it by the end of the follow-up., Conclusion: Cystatin SN levels in nasal secretions hold strong prognostic value and can facilitate medical instructions for managing CRSwNP., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Ovarian cancer (OC) is the most common gynecological malignant tumor with the highest mortality rate. However, identification of effective immune therapeutic targets and biomarkers are beset by many challenges. CIBERSORT was used to calculate the abundance of 22 immune cell types in 379 OC samples, and indicated that three immune cell types were associated with poor prognoses. Further analysis revealed that 17 hub genes were associated with these three cell types. We screened differentially expressed immune-related prognostic gene associated with clinicopathological factors, which was CST4. We used clinical specimens to detect the expression of CST4, and determined that CST4 was both highly expressed in OC patients and associated with poor prognoses. Our findings indicated that infiltration of immune cells affected the survival of patients with OC, provided therapeutic targets represented by CST4, deepened our understanding of the immune microenvironment of OC, and enhanced the theoretical basis of immunotherapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: This paper examines the tear concentration of cystatin S (CST4), calcyclin (S100A6), calgranulin A (S100A8), and matrix metalloproteinase 9 (MMP9), and the correlation between biomarker expression, clinical parameters, and disease severity in patients suffering from dry eye (DE). A comparison of the results is obtained via ELISA tests and customized antibody microarrays for protein quantification., Methods: This single-center, observational study recruited 59 participants (45 DE and 14 controls). Clinical evaluation included an Ocular Surface Disease Index (OSDI) questionnaire, a tear osmolarity (OSM) test, the Schirmer test (SCH), tear breakup time (TBUT), fluorescein (FLUO) and lissamine green (LG) corneal staining, and meibomian gland evaluation (MGE). Tear concentrations of CST4, S100A6, S100A8, and MMP9 were measured using standard individual ELISA assays. The levels of CST4, S100A6, and MMP9 were also measured using customized multiplexed antibody microarrays. Correlations between variables were evaluated, and a significance level was p value <0.05., Results: The quantification of tear protein biomarkers with ELISA showed that the concentration of CST4 was significantly (2.14-fold) reduced in tears of DE patients in comparison with control (CT) subjects (p < 0.001). S100A6 and S100A8 concentrations were significantly higher in the tears of DE patients (1.36- and 2.29-fold; p < 0.001 and 0.025, respectively) in comparison with CT. The MMP9 level was also higher in DE patients (5.83-fold), but not significantly (p = 0.22). The changes in CST4 and S100A6 concentrations were significantly correlated with dry eye disease (DED) severity. Quantification of CST4, S100A6, and MMP9, using antibody microarrays, confirmed the ELISA results. Similar trends were observed: 1.83-fold reduction for CST4 (p value 0.01), 8.63-fold increase for S100A6 (p value <0.001) and 9.67-fold increase for MMP9 (p value 0.94), but with higher sensitivity. The biomarker concentrations were significantly associated with the signs and symptoms related with DED., Conclusions: S100A6, S100A8, and CST4 diagnostic biomarkers strongly correlate with DED clinical parameters. S100A6 and CST4 are also useful for grading DE severity. The multiplexed antibody microarray technique, used here for tear multi-marker quantification, appears more sensitive than standard ELISA tests., (Copyright © 2021 Molecular Vision.)

Dietary polyphenol exposure is known to change protein saliva composition in rodents, but less is known in humans. The present study aimed to assess the relationship between saliva protein composition and adherence to Mediterranean Diet (MD) and polyphenol intake levels. Participants were assessed for their dietary habits, which were converted in Mediterranean adherence level, according to Mediterranean Diet Adherence Score (MEDAS) score. Total polyphenol and total flavanol intakes were extrapolated from dietary data, using Phenol explorer database. Whole saliva was collected, and proteins were separated by SDS-PAGE. Salivary S-type cystatins were highly expressed in the group with medium adherence to MD, being positively correlated with wine intake in overweight individuals. The association between salivary amylase and MD adherence also depended on Body Mass Index (BMI), with a positive association only in normal weight individuals. Polyphenol intake was positively associated with S-type cystatins levels, particularly when flavanols were considered separately. These results show that saliva relationship with MD adherence depend on BMI, suggesting that normal weight and overweight individuals may have different salivary responses to diet. Moreover, these results reinforce the link between saliva and dietary polyphenols (flavanols) levels, leading to the hypothesis that salivary proteome can have a role in polyphenol-rich foods acceptance.

Natural compounds that can stimulate salivary secretion are of interest in developing treatments for xerostomia, the perception of a dry mouth, that affects between 10 and 30% of the adult and elderly population. Chemesthetic transient receptor potential (TRP) channels are expressed in the surface of the oral mucosa. The TRPV1 agonists capsaicin and piperine have been shown to increase salivary flow when introduced into the oral cavity but the sialogogic properties of other TRP channel agonists have not been investigated. In this study we have determined the influence of different TRP channel agonists on the flow and protein composition of saliva. Mouth rinsing with the TRPV1 agonist nonivamide or menthol, a TRPM8 agonist, increased whole mouth saliva (WMS) flow and total protein secretion compared with unstimulated saliva, the vehicle control mouth rinse or cinnamaldehyde, a TRPA1 agonist. Nonivamide also increased the flow of labial minor gland saliva but parotid saliva flow rate was not increased. The influence of TRP channel agonists on the composition and function of the salivary proteome was investigated using a multi-batch quantitative MS method novel to salivary proteomics. Inter-personal and inter-mouth rinse variation was observed in the secreted proteomes and, using a novel bioinformatics method, inter-day variation was identified with some of the mouth rinses. Significant changes in specific salivary proteins were identified after all mouth rinses. In the case of nonivamide, these changes were attributed to functional shifts in the WMS secreted, primarily the over representation of salivary and nonsalivary cystatins which was confirmed by immunoassay. This study provides new evidence of the impact of TRP channel agonists on the salivary proteome and the stimulation of salivary secretion by a TRPM8 channel agonist, which suggests that TRP channel agonists are potential candidates for developing treatments for sufferers of xerostomia., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Houghton et al.)

Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Rhipicephalus microplus is a cattle ectoparasite found in tropical and subtropical regions around the world with great impact on livestock production. R. microplus can also harbor pathogens, such as Babesia sp. and Anaplasma sp. which further compromise cattle production. Blood meal acquisition and digestion are key steps for tick development. In ticks, digestion takes place inside midgut cells and is mediated by aspartic and cysteine peptidases and, therefore, regulated by their inhibitors. Cystatins are a family of cysteine peptidases inhibitors found in several organisms and have been associated in ticks with blood acquisition, blood digestion, modulation of host immune response and tick immunity. In this work, we characterized a novel R. microplus type 1 cystatin, named Rmcystatin-1b. The inhibitor transcripts were found to be highly expressed in the midgut of partially and fully engorged females and they appear to be modulated at different days post-detachment. Purified recombinant Rmcystatin-1b displayed inhibitory activity towards typical cysteine peptidases with high affinity. Moreover, rRmcystatin-1b was able to inhibit native R. microplus cysteine peptidases and RNAi-mediated knockdown of the cystatin transcripts resulted in increased proteolytic activity. Moreover, rRmcystatin-1b was able to interfere with B. bovis growth in vitro. Taken together our data strongly suggest that Rmcystatin-1b is a regulator of blood digestion in R. microplus midgut., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Purpose: Present study is designed to discover potential salivary biomarkers associated with predominantly antibody deficiencies, which include a large spectrum of disorders sharing failure of antibody production, and B cell defects resulting in recurrent infections, autoimmune and inflammatory manifestations, and tumor susceptibility. Understanding and clinical classification of these syndromes is still challenging., Methods: We carried out a study of human saliva based on liquid chromatography-mass spectrometry measurements of intact protein mass values. Salivary protein profiles of patients (n = 23) and healthy controls (n = 30) were compared., Results: Patients exhibited lower abundance of α-defensins 1-4, cystatins S1 and S2, and higher abundance of glutathionylated cystatin B and cystatin SN than controls. Patients could be clustered in two groups on the basis of different levels of cystatin SN, S1 and S2, suggesting that these proteins may play different roles in the disease., Conclusions: Quantitative variations of these pro-inflammatory and antimicrobial peptides/proteins may be related to immunodeficiency and infectious condition of the patients. The high incidence of tumors in the group with the highest level of cystatin SN, which is recognized as tumoral marker, appeared an intriguing result deserving of future investigations. Data are available via ProteomeXchange with identifier PXD012688.

Cystatin SN, a specific cysteine protease inhibitor, is thought to be involved in various malignant tumors. Therefore, we evaluated the role of cystatin SN in hepatocellular carcinoma (HCC). Notably, cystatin SN was elevated in tumorous samples and cells. Moreover, overexpression of cystatin SN was correlated with tumor diameter and TNM stage. Cox multivariate analysis displayed that cystatin SN was an independent prognosis indicator and that high cystatin SN level was associated with a dismal prognosis. Moreover, cystatin SN enhancement facilitated the proliferation, migratory, and invasive potential of Huh7 and HCCLM3 cells, whereas cystatin SN knockdown caused the opposite effect. Cystatin SN also modulated the epithelial-mesenchymal transition progression through the PI3K/AKT pathway. In vivo cystatin SN promoted HCCLM3 cell growth and metastasis in xenograft mice model. Thus, cystatin SN was involved in HCC progression and could be a latent target for HCC treatment., (© 2019 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins. An in vitro assay was developed based on a cell line from a human submandibular gland (HSG) that differentiated into acinus-like structures. Treatments with the β-adrenergic agonist isoproterenol caused a shift in the intracellular distribution of S-type cystatins and cystatin C, promoting their accumulation at the outer regions of the acinus prior to release and suggesting the activation of a directional transport involving co-migration of both molecules. In another treatment using non-differentiated HSG cells, it was evident that both expression and secretion of cystatin C increased upon addition of the β-adrenergic agonist, and these effects were essentially eliminated by the antagonist propranolol. The HSG cell line appears to have potential as a model for exploring the mechanism of cystatin secretion, particularly the S-type cystatins that originate primarily in the submandibular glands., Competing Interests: The authors have declared that no competing interests exist.

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is treated using oral/topical steroids and surgery. Despite maximal medical therapy, some patients remain recalcitrant. Mucus cystatin 2, pappalysin-A, and periostin can predict the presence of CRSwNP and correlate with disease severity. This study was designed to determine whether prospective sampling of these mucus proteins could predict medical failure and the need for revision surgery., Methods: This investigation was an institutional review board-approved, prospective study of 66 patients with CRSwNP. All patients underwent surgery, administration of topical/oral steroids, and outpatient mucus sampling at 10 time-points over 2 years. Five proteins, including cystatin 2 (CST2), pappalysin-A (PAPP-A), and periostin (PST), were analyzed and correlated with subjective parameters (including scores on the 22-item Sino-Nasal Outcomes Test [SNOT-22]). Variables were then analyzed and compared between those requiring revision surgery within 2 years (n = 5) and those with stable disease (n = 61)., Results: All patients demonstrated a significant decline in CST2, PAPP-A, and periostin after their initial surgery. The recalcitrant group demonstrated escalations in all proteins despite steroids, with levels higher than those of the stable group at 1 year (CST2: 258.1 ± 205.2 pg/mL vs 235.3 ± 275.7 pg/mL, p = 0.86; PAPP-A: 170.3 ± 150.4 pg/mL vs 74.6 ± 106.7 pg/mL, p < 0.05; periostin: 188.8 ± 192.4 ng/mL vs 54.5 ± 47.6 ng/mL, p < 0.001). Escalation in all proteins correlated significantly with worsening SNOT-22 score at each time-point (domain 1: 8.2 ± 1.3 vs 5.5 ± 1.1; p < 0.05)., Conclusion: Early recurrences and medical recalcitrance in CRSwNP may be predicted noninvasively through the serial, prospective sampling of mucus CST2, PAPP-A, and periostin levels. These biosignatures may help to predict disease course and guide individualized therapy., (© 2019 The Authors International Forum of Allergy & Rhinology published by Wiley Periodicals, Inc. on behalf of American Academy of Otolaryngic Allergy and American Rhinologic Society.)

Background: Cystatins are epithelial protease inhibitors that participate in sinonasal immunity and inflammation. Nasal mucus-derived exosomes (NMDEs) are small vesicles secreted by epithelial cells that carry protein cargo reflective of their host cell. NMDEs have been used as a noninvasive biomarker source to study chronic rhinosinusitis with nasal polyps (CRSwNP) proteomics with superior sensitivity to whole mucus. The purpose of this study was to noninvasively quantify exosomal cystatins in a heterogenous population to determine their utility in predicting phenotype and disease severity., Methods: This was an Institutional Review Board-approved study in which NMDEs were purified from 105 patients undergoing sinonasal surgery by ultracentrifugation. Demographic and clinical variables were collected and phenotypes were assigned a priori. Linear discriminant analysis was executed based on normalized Cystatin values as phenotype predictor variables. Unsupervised cluster analysis was performed using Ward's linkage followed by Duda/Hart Je(2)/Je(1) index cluster stopping rules. Analysis of variance (ANOVA), Welch's test, and Fisher's exact tests were used for continuous and categorical variables., Results: NMDE Cystatin-2 expression segregated by phenotype (mean ± standard error [SEM]): control (23.4 ± 4.2 pg/µg, n = 32); CRS without NP (CRSsNP) (56.6 ± 8.3 pg/µg, n = 33); and CRSwNP (130.5 ± 16.7 pg/µg, n = 40) (p < 0.0001). Seven clusters were identified among patients where the highest NMDE Cystatin-2 levels clustered with asthma, tissue eosinophilia, and aspirin-exacerbated respiratory disease (AERD)., Conclusion: Cystatin levels in NMDEs predict CRS phenotype and disease severity. As a "liquid biopsy," noninvasive NMDE collection offers a promising opportunity to study disease pathophysiology, discriminate disease states, and potentially reveal novel therapeutic targets., (© 2019 ARS-AAOA, LLC.)

Background: Exosomes are secreted epithelial-derived vesicles that contain a conserved protein array representative of their parent cell. Exosomes may be reproducibly and noninvasively purified from nasal mucus. The exosomal proteome can be quantified using SOMAscan TM , a highly multiplexed, aptamer-based proteomic platform. The purpose of this study was to determine whether chronic rhinosinusitis with nasal polyps (CRSwNP) has a unique predictive exosomal proteomic biosignature., Methods: Exosomes were isolated from whole mucus sampled from control and CRSwNP patients (n = 20 per group) by differential ultracentrifugation. The SOMAscan TM platform was used to simultaneously quantify 1310 biologically relevant human proteins. Matched tissue and whole mucus proteomes were also analyzed. Differential protein expression and discriminatory power were calculated using the unweighted pair group method with arithmetic-mean and principal component analysis, respectively. Bioinformatic analysis was performed using Ingenuity Pathway, MetaCore, and GeneMANIA analyses., Results: The exosomal proteome demonstrated 123 significantly (p < 0.05) differentially regulated proteins in CRSwNP relative to control. Eighty of these proteins overlapped with the matched CRSwNP tissue proteome as compared with only 4 among matched whole mucus samples. Forty-three significantly dysregulated pathway networks overlapped between the exosomal and tissue proteome in CRSwNP as compared with only 3 among matched whole mucus samples. The best-performing protein set (cystatin-SN, peroxiredoxin-5, and glycoprotein VI) achieved an area under the curve (AUC) value of up to 99%., Conclusion: Our data contribute a significant advance in the development of a reproducible, noninvasive, serial, and quantitative "liquid biopsy" for rhinosinusitis. The exosomal proteomic approach has revealed a unique biosignature associated with CRSwNP, which outperforms whole mucus sampling, and thus provides a method of noninvasive disease detection and proposes new potential therapeutic targets., (© 2018 ARS-AAOA, LLC.)

Purpose: The purpose of this work was to analyze the expressions of matrix metalloproteinase 9 (MMP-9), calcyclin (S100A6), and cystatin S (CST4) in the tears of keratoconus (KC) patients. The correlations between the expressions of these proteins and the values of various ocular surface parameters were examined after accelerated corneal crosslinking (A-CXL) with pulsed ultraviolet light., Methods: This prospective, observational study enrolled patients with different grades of KC, scheduled to undergo the A-CXL procedure, as well as healthy subjects. Tear samples were analyzed by employing customized antibody microarray assays for MMP-9, S100A6, and CST4 proteins. The keratometry readings at the maximum keratometry (Kmax) and the simulated keratometry (SimK) values were obtained for examining the postoperative evolution of corneal topography. The state of the ocular surface was evaluated using the results of the Ocular Surface Disease Index (OSDI) questionnaire, tear osmolarity (OSM) test, Schirmer test (SCH), Tear Break Up Time (TBUT), tear clearance (CLR), and fluorescein (FLUO) and lissamine green (LG) corneal staining., Results: A total of 18 patients (22 eyes) and 10 healthy subjects were studied. The concentrations of MMP-9 and S100A6 decreased in tears, from 104.5 ± 78.98 ng/ml and 350.20 ± 478.08 ng/ml before the surgery to 48.7 ± 24.20 ng/ml and 55.70 ± 103.62 ng/ml, respectively, after 12 months of follow up. There were no changes in the CST4 concentration after 12 months of follow up (2202.75 ± 2863.70 versus 2139.68 ±2719.89 ng/ml). When the patients were divided into three groups according to the evolutive stage of KC, the trends for the three biomarkers in each group were the same as in the general group. Basal concentrations of MMP-9 and S100A6 from healthy subjects and KC patients were compared. The levels of MMP-9 and S100A6 in tears were (9.8 ± 5.11 and 104.55 ± 78.98 ng/ml, p <0.01; and 11.35 ± 3.18 and 350.26 ± 478.06 ng/ml, respectively, p <0.01). This was not the case for CST4, which did not exhibit statistically significant differences between the two groups (2261.94 ± 510.65 and 2176.73 ± 2916.27 ng/ml respectively, p=0.07)., Conclusions: A-CXL promoted a decrease in the concentrations of MMP-9 and S100A6 in the tear film. This effect may be related to the restoration of corneal homeostasis and the consequent repair of the tissue damage caused by KC. Moreover, the A-CXL treatment did not produce lasting alterations in the ocular surface, and the values of the evaluated clinical parameters did not change significantly.

Objective: Identification of molecular signatures having key roles in hypobaric hypoxia by analysing the salivary proteome. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being non-invasive., Methodology: We employed qualitative proteomics approach to develop discriminatory biomarker signatures from human saliva exposed to hypobaric hypoxia. Salivary proteins were analyzed and compared between age-matched healthy subjects exposed to high altitude (∼13700 ft) for seven days (HAD7) with control subjects at sea level (Normoxia) by using 2-Dimensional gel electrophoresis/Mass Spectrometry approach., Results: Several proteins with significant differential expression were found. The up-regulated proteins were apoptosis inducing factor-2, cystatin S, cystatin SN and carbonic anhydrase 6. The down regulated proteins were polymeric immunoglobulin receptor, alpha-enolase and prolactin-inducible protein. Further confirmation of the altered proteins such as alpha enolase, carbonic anhydrase 6, prolactin-inducible protein, apoptosis inducing factor 2, cystatin S and cystatin SN were performed using immunoblotting. The expression patterns of the selected proteins observed by immunoblot were in concurrence with 2-Dimesional gel electrophoresis results, therefore affirming the authenticity of the proteomic investigation., Conclusion: This study provides the proof of concept of salivary biomarkers for the non-invasive detection of hypobaric hypoxia induced effects. It is highly feasible to turn these biomarkers into an applicable clinical test after large scale validation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, we found Cystatin SN (CST1), a type 2 cystatin subfamily member, to be highly expressed in nasal polyps from patients with intractable chronic rhinosinusitis (CRS) with nasal polyps, using a whole-transcript analysis with next-generation sequencing. Eosinophilic CRS (ECRS) involves nasal polyps that are refractory and recur immediately after endoscopic sinus surgery. We hypothesized that CST1 may contribute to the pathogenesis of ECRS. We examined the expression of CST1 in nasal polyps from patients with ECRS by assessing mRNA expression levels using real-time PCR and immunohistochemistry. CST1 showed significantly greater expression in the epithelial cells of nasal polyps from patients with ECRS than in those from patients who did not have ECRS (non-ECRS). In particular, CST1 showed very strong expression in patients with severe ECRS. The expression of CST1 may be correlated with the recurrent and refractory nature of ECRS. We examined the function of CST1 using nasal epithelial cells and nasal fibroblasts. Stimulation by a combination of IL-4 plus double-stranded RNA plus CST1 significantly elevated mRNA expression levels and protein levels of TSLP in nasal epithelial cells. Stimulation by TSLP or IL-33 significantly elevated mRNA expression levels of CST1 in nasal epithelial cells. Stimulation of CST1 significantly elevated mRNA expression levels of CCL11 and POSTN in nasal fibroblasts. CST1 could amplify eosinophilic infiltration and T-helper cell type 2 inflammation by interacting with epithelial-derived cytokines and fibroblasts on nasal polyps. CST1 may be involved in the pathogenesis of ECRS, and may contribute to the severity and recurrence of CRS with nasal polyps after endoscopic sinus surgery.

Cystatin SN (cystatin 1, CST1) is a member of the cystatin superfamily which inhibits the proteolytic activity of cysteine proteases. CST1 is a tumor biomarker that provides useful information for the diagnosis of esophageal, gastric and colorectal carcinomas. MicroRNAs (miRNAs or miRs) play an important role in tumor cell proliferation. However, the exact role of let‑7d and CST1 in colon cancer remains unknown. The aim of this study was to assess whether let‑7d inhibits colorectal carcinogenesis through the CST1/p65 pathway, and determine whether it may be used as a potential target for clinical therapy. Microarray analysis of mRNAs extracted from colon cancer and normal tissues was performed. The results of gene expression microanalysis revealed that CST1 expression was upregulated in colon cancer compared with normal tissues. In addition, the upregulation of CST1 expression and the downregulation of let‑7d expression in patients with colon cancer and in several colorectal cancer cell lines were confirmed by reverse transcription-quantitative PCR (RT‑qPCR), immunohistochemistry and western blot analysis. In addition, siRNA targeting CST1 (CST1‑siRNA) and let‑7d-mimics were used in the HCT116 cells, and the results revealed that CST1 and let‑7d played a role in colorectal cancer cell proliferation. Let‑7d inhibited colorectal carcinogenesis through the CST1/p65 pathway. Thus, the findings of the present study indicate that CST1 may be a potential target for the future clinical therapy of colorectal cancer.

Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acid-soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated top-down and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, in particular, phosphorylation, N-terminal processing, and oxidation. This study increases the current knowledge of salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease. Data are available via ProteomeXchange with identifier PXD007170.

Background: Seasonal allergic rhinitis (SAR) caused by intermittent exposure to seasonal pollen causes itching, nasal congestion, and repeated sneezing, with profound effects on quality of life, work productivity, and school performance. Although both the genotype and environmental factors can contribute to the immunologic basis of allergic reactions, the molecular underpinnings associated with the pathogenesis of allergic rhinitis are not entirely clear., Methods: To address these questions, nasal epithelial brushings were collected from 29 patients with SAR and 31 control subjects during and after the pollen season. We then implemented an orbitrap-based, bottom-up, label-free quantitative proteomics approach, followed by multivariate analyses to identify differentially abundant (DA) proteins among the 4 sample groups., Results: We identified a total of 133 DA proteins for which the most significantly overrepresented functional category was found to be interferon 1 signaling. Two proteins, cystatin 1 and myeloblastin, the former of which protects against protease activity of allergens and the latter with a role in epithelial barrier function, were DA in patients with SAR and control subjects, irrespective of season. Moreover, interferon-inducible protein with tetratricopeptide repeats 1, cystatin 1, and interferon-inducible protein with tetratricopeptide repeats 3 were found to be differentially regulated between patients with SAR and control subjects, with inverse abundance dynamics during the transition from fall to spring., Conclusion: We identified type 1 interferon-regulated proteins as biomarkers in patients with SAR, potentially playing an important role in its pathogenesis. Moreover, when compared with patients with SAR, healthy subjects exhibit an antagonistic proteomic response across seasons, which might prove to be a therapeutic target for disease prevention., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Cystatin SN (CST1) belongs to the type 2 cystatin (CST) superfamily, which restricts the proteolytic activities of cysteine proteases. CST1 has been recently considered to be involved in the development of several human cancers. However, the prognostic significance and function of CST1 in breast cancer remains unknown. In the current study, we found that CST1 was generally upregulated in breast cancer at both mRNA and protein level. Furthermore, overall survival (OS) and disease-free survival (DFS) in the low CST1 expression subgroup were significantly superior to the high CST1 expression subgroup (OS, p < 0.001; DFS, p < 0.001), which indicated that CST1 expression level was closely correlated to the survival risk of these patients. Univariate and multivariate analyses demonstrated that CST1 expression was an independent prognostic factor, the same as ER status and nodal status. Next, CST1 overexpression promoted breast cancer cell proliferation, clonogenicity, migration, and invasion abilities. By contrast, knockdown of CST1 attenuated these malignant characteristics in breast cancer cells. Collectively, our study indicates that CST1 cannot only serve as a significant prognostic indicator but also as a potential therapeutic target for breast cancer., Key Messages: High CST1 expression is negatively correlated with survival of breast cancer patients. CST1 promotes cell proliferation, clone formation, and metastasis in breast cancer cells. CST1 is a novel potential prognostic biomarker and therapeutic target for breast cancer.

Objectives: Salivary cystatin S is a defence protein mainly produced by submandibular glands and involved in innate oral immunity. This study aimed to verify whether cystatin S was diversely expressed in different disease subsets of primary Sjogren's syndrome (pSS) patients, defined on the basis of salivary flow [unstimulated salivary flow rate (USFR)], minor salivary gland (MSG) focus score and submandibular gland ultrasonography abnormalities. We also evaluated miR-126 and miR-335-5p expression in MSG biopsies to verify whether an aberrant regulation of cystatin S at the glandular level may influence its salivary expression., Methods: Forty pSS patients and 20 sex- and age-matched healthy volunteers were included. Salivary cystatin S levels were assessed by western blot analysis using a stain-free technology. The expression of miR-126, miR-335-5p and cystatin S was assessed by quantitative PCR in 15 MSG biopsies differing for USFR and MSG focus score., Results: We found that salivary cystatin S was significantly decreased in pSS patients vs healthy volunteers ( P = 0.000), especially in those with hyposalivation. A positive correlation was observed between cystatin S and USFR ( r = 0.75, P = 0.01). Salivary cystatin S was also significantly reduced in patients with a submandibular gland ultrasonography score ⩾2. The expression levels of miR-126 and miR-335-5P increased in inverse proportion with USFR. The mRNA of cystatin S did not change significantly, suggesting post-transcriptional regulation., Conclusion: Cystatin S emerged as a promising biomarker for pSS, strongly correlated with glandular dysfunction. An upregulation of miR-126 and miR-335-5P might be implicated in its expression., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated β-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3β phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.

Cystatin SN (CST1) is a specific inhibitor belonging to the cystatin superfamily that controls the proteolytic activities of cysteine proteases such as cathepsins. Our previous study showed that high CST1 expression enhances tumor metastasis and invasiveness in colorectal cancer. Recently, auranofin (AF), a gold(I)-containing thioredoxin reductase 1 (TrxR1) inhibitor, has been used clinically to treat rheumatoid arthritis. AF is a proteasome-associated deubiquitinase inhibitor and can act as an anti-tumor agent. In this study, we investigated whether CST1 expression induces autophagy and tumor cell survival. We also investigated the therapeutic effects of AF as an anti-tumor agent in colorectal cancer (CRC) cells. We found that CRC cells expressing high levels of CST1 undergo increased autophagy and exhibit chemotherapeutic resistance to AF-induced cell death, while those expressing low levels of CST1 are sensitive to AF. We also observed that knockdown of CST1 in high-CST1 CRC cells using CST1-specific small interfering RNAs attenuated autophagic activation and restored AF-induced cell mortality. Conversely, the overexpression of CST1 increased autophagy and viability in cells expressing low levels of CST1. Interestingly, high expression of CST1 attenuates AF-induced cell death by inhibiting intracellular reactive oxygen species (ROS) generation, as demonstrated by the fact that the blockage of ROS production reversed AF-induced cell death in CRC cells. In addition, upregulation of CST1 expression increased cellular glutathione reductase (GR) activity, reducing the cellular redox state and inducing autophagy in AF-treated CRC cells. These results suggest that high CST1 expression may be involved in autophagic induction and protects from AF-induced cell death by inhibition of ROS generation through the regulation of GR activity.

Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases., Competing Interests: The authors have declared that no competing interests exist.

Cystatin SN has been considered to be involved in human cancer, but its clinical significance in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to evaluate the clinical value of Cystatin SN expression in patients with surgically resected NSCLCs. A retrospective analysis of 174 patients with surgically resected NSCLCs from April 2002 to March 2005 was performed with immunohistochemistry and fluorescence in situ hybridization to analyze the protein expression and amplification of Cystatin SN. The associations between Cystatin SN expression and recurrence, metastasis, and survival were investigated. In recurrence and metastasis analysis, compared with low-Cystatin SN expression NSCLCs, high expression tumors were more likely to recur and metastasize (P < 0.001). Disease-free survival (DFS) and overall survival (OS) were significantly prolonged in the low-Cystatin SN expression subgroup compared with the high-Cystatin SN expression subgroup (DFS, P < 0.001; OS, P = 0.001). A multivariate analysis confirmed that high expression of Cystatin SN was associated with poor survival (DFS, P = 0.001; OS, P = 0.006) and was an independent prognostic indicator. The present study indicates that high expression of Cystatin SN is a significant prognostic indicator of a higher rate of recurrence, metastatic risk, and poor survival in patients with surgically resected NSCLCs.

SAP-1 is a 113 amino acid long single-chain protein which belongs to the type 2 cystatin gene family. In our previous study, we have purified SAP-1 from human seminal plasma and observed its cross-class inhibitory property. At this time, we report the interaction of SAP-1 with diverse proteases and its binding partners by CD-spectroscopic and molecular docking methods. The circular dichroism (CD) spectroscopic studies demonstrate that the conformation of SAP-1 is changed after its complexation with proteases, and the alterations in protein secondary structure are quantitatively calculated with increase of α-helices and reduction of β-strand content. To get insight into the interactions between SAP-1 and proteases, we make an effort to model the three-dimensional structure of SAP-1 by molecular modeling and verify its stability and viability through molecular dynamics simulations and finally complexed with different proteases using ClusPro 2.0 Server. A high degree of shape complementarity is examined within the complexes, stabilized by a number of hydrogen bonds (HBs) and hydrophobic interactions. Using HB analyses in different protein complexes, we have identified a series of key residues that may be involved in the interactions between SAP-1 and proteases. These findings will assist to understand the mechanism of inhibition of SAP-1 for different proteases and provide intimation for further research.

Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3.

Seasonal allergic rhinitis (SAR) to the Japanese cedar, Cryptomeria japonica (JC) pollen is an IgE-mediated type I allergy affecting nasal mucosa. However, the molecular events underlying its development remain unclear. We sought to identify SAR-associated altered gene expression in nasal epithelial cells during natural exposure to JC pollen. We recruited study participants in 2009 and 2010 and collected nasal epithelial cells between February and April, which is the period of natural pollen dispersion. Fifteen patients with SAR-JC and 13 control subjects were enrolled in 2009, and 17 SAR-JC patients, 13 sensitized asymptomatic subjects (Sensitized), and 15 control subjects were enrolled in 2010. Total RNA was extracted from nasal epithelial cells and 8 SAR-JC patients and 6 control subjects in 2009 were subjected to microarray analysis with the Illumina HumanRef-8 Expression BeadChip platform. Allergen-stimulated histamine release was examined in the peripheral blood basophils isolated from patients with SAR. We identified 32 genes with significantly altered expression during allergen exposure. One of these, CST1 encodes the cysteine protease inhibitor, cystatin SN. CST1 expression in nasal epithelial cells was significantly upregulated in both the 2009 and 2010 SAR-JC groups compared with the control groups. Immunohistochemical staining confirmed the increased expression of CST1 in the nasal epithelial cells of SAR patients. Addition of exogenous CST1 to basophils inhibited JC allergen-stimulated histamine release in vitro. We propose that CST1 may contribute to inactivation of protease allergens and help re-establish homeostasis of the nasal membranes.

Background: Cystatin SN is a secreted protein and a cysteine proteinase inhibitor. It has been considered to be a tumor marker for gastrointestinal tract cancer in several functional researches. However, the clinicopathological and prognostic significance of Cystatin SN expression in esophageal squamous cell carcinoma (ESCC) has not been elucidated., Methods: In our study, the expression of Cystatin SN was detected in 209 surgically resected ESCC tissues and 170 peritumoral normal esophageal mucosae by immunohistochemistry. The prognostic significance of Cystatin SN expression was analysed with Kaplan-Meier plots and the Cox proportional hazards regression models., Results: The results showed that the immunostaining of Cystatin SN in ESCC tissues was less intense than that in the normal control tissue (P < 0.001). Compared with patients with low tumoral Cystatin SN expression, ESCC patients with tumors high-expression Cystatin SN exhibited increased disease-free survival (DFS) and overall survival (OS) (P < 0.001 and P < 0.001, respectively). Furthermore, the expression level of Cystatin SN could further stratify the ESCC patients by survival (DFS and OS) in the stage II subgroup (P < 0.001 and P < 0.001, respectively). Multivariate analyses showed that Cystatin SN expression, N status and differentiation were independent and significant predictors of survival., Conclusions: We concluded that ESCC patients whose tumors express high levels of Cystatin SN have favourable survival compared with those patients with low Cystatin SN expression. Tumoral Cystatin SN expression may be an independent predictor of survival for patients with resectable ESCCs.

Patients with the monogenic disease autoimmune polyendocrine syndrome type I (APSI) develop autoimmunity against multiple endocrine organs and suffer from chronic mucocutaneous candidiasis (CMC), a paradoxical complication with an unknown mechanism. We report here that saliva from APSI patients with CMC is defective in inhibiting growth of Candida albicans in vitro and show reduced levels of a salivary protein identified as cystatin SA1. In contrast, APSI patients without CMC express salivary cystatin SA1 and can inhibit C. albicans to the same extent as healthy controls. We evaluated the anti-fungal activity of cystatin SA1 and found that synthesized full length cystatin SA1 efficiently inhibits growth of C. albicans in vitro. Moreover, APSI patients exhibit salivary IgA autoantibodies recognizing myosin-9, a protein expressed in the salivary glands, thus linking autoimmunity to cystatin SA1 deficiency and CMC. This data suggests an autoimmune mechanism behind CMC in APSI and provides rationale for evaluating cystatin SA1 in antifungal therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain-cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.

Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter-GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein which is very likely due to the reduced activity of endogenous cysteine protease.

A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans., (© 2012 John Wiley & Sons A/S.)

Bone is the one of the most common sites of distant metastasis of solid tumors. Secreted proteins are known to influence pathological interactions between metastatic cancer cells and the bone stroma. To comprehensively profile secreted proteins associated with bone metastasis, we used quantitative and non-quantitative mass spectrometry to globally analyze the secretomes of nine cell lines of varying bone metastatic ability from multiple species and cancer types. By comparing the secretomes of parental cells and their bone metastatic derivatives, we identified the secreted proteins that were uniquely associated with bone metastasis in these cell lines. We then incorporated bioinformatic analyses of large clinical metastasis datasets to obtain a list of candidate novel bone metastasis proteins of several functional classes that were strongly associated with both clinical and experimental bone metastasis. Functional validation of selected proteins indicated that in vivo bone metastasis can be promoted by high expression of (1) the salivary cystatins CST1, CST2, and CST4; (2) the plasminogen activators PLAT and PLAU; or (3) the collagen functionality proteins PLOD2 and COL6A1. Overall, our study has uncovered several new secreted mediators of bone metastasis and therefore demonstrated that secretome analysis is a powerful method for identification of novel biomarkers and candidate therapeutic targets.

The extent of the change in salivary protein characteristics after repeated stimulations was shown to be correlated to differences in perceived astringency. Salivary characteristics of 77 subjects were compared after masticatory (S1) and taste/masticatory (S2) stimulations. The variations (S2 minus S1) of protein concentration and saliva haze-forming capacity (HFC) were used to define 3 subject groups: low responding (LR, n = 20), medium responding (MR, n = 37), and high responding (HR, n = 20). Salivary protein concentration did not change in LR subjects; decreased a little, but significantly, in MR subjects; and strongly decreased in HR subjects. After S2, HFC increased in LR subjects, slightly decreased in MR subjects, and strongly decreased in HR subjects. Salivary protein electrophoresis patterns for HR and LR subjects were analyzed. No significant modifications of glycosylated proline-rich proteins (PRPs), PRPs, and amylases and a slight decrease in cystatins and histatins were found when S2 and S1 samples were compared in LR subjects, whereas HR subjects showed a strong decrease in all the above proteins after S2. Significant modifications of mucins were not found. Tannic acid (TA, 3 g/L) astringency ratings after S1 from HR subjects were significantly higher than those from the other 2 groups, whereas no differences were found comparing LR and MR ratings. The "carryover" effect due to 4 sequential exposures to TA samples (1.4 g/L) was observed in both HR and MR groups, whereas no significant astringency rating variation was found in the LR group. The results support the inhibiting role of proteins with strong phenol-binding activity on astringency elicitation. Individual physiological variations of parotid gland functionality might account for differences in sensitivity to astringent phenolic stimuli.

Ticks feed exclusively on blood to obtain their nutrients, but the gene products that mediate blood-sucking processes in ticks are still unknown. We report here the molecular characterization and possible biological function of a cysteine protease inhibitor (HlSC-1) identified in the salivary gland of the ixodid tick Haemaphysalis longicornis. The HlSC-1 cDNA contains 423 bp that code for 140 amino acids with a predictable molecular weight of 12 kDa. The recombinant HlSC-1 expressed in Escherichia coli was shown to inhibit the activity of papain and cathepsin L, while cathepsin B activity was unaffected. Immunolocalization studies detected the endogenous enzyme in the salivary gland type II acini of an adult tick. Furthermore, quantitative RT-PCR analysis showed that the expression of HlSC-1 transcripts was associated with blood-feeding processes and was highly up-regulated in the early phase of feeding. Our results strongly suggest that HlSC-1 may play pivotal roles in the blood-feeding processes.

The goal of this study was to investigate Cystatin SN, a cysteine protease inhibitor, as a novel tumor marker for colorectal cancer (CRC). Gene expression profiles of mRNA from normal tissues and cancer cell lines were performed. Twenty-eight monoclonal antibodies for Cystatin SN were generated and serum Cystatin SN was quantified using ELISA in sera from 159 patients with CRC and 40 healthy controls. Cystatin SN was highly expressed in colon cancer cells. Employing a receiver-operating characteristic curve, we obtained an area under the curve of 0.708 for Cystatin SN, 0.819 for carcinoembryonic antigen (CEA) and 0.703 for carbohydrate antigen 19-9 (CA19-9). The combination assay of Cystatin SN, CEA and CA19-9 showed 62.9% sensitivity and 90.0% specificity. Especially, the sensitivity of the combination assay in stages I and II detection, in which stages curative operation would be possible, was improved over that of the assay testing only for CEA and CA19-9 (from 37.5 to 42.5% in stage I, from 49.0 to 60.8% in stage II). Furthermore, Western blot analysis revealed that Cystatin SN was increased in the urine from patients with CRC. Our results suggest the possibility of utilizing this novel tumor marker that can be tested in urine samples. These observations suggest that Cystatin SN in combination with CEA and CA19-9 is a useful tumor marker for detecting early stage CRC and that it is a unique urinary excretory protein, suggesting that Cystatin SN might be a novel candidate for use in mass screening for CRC.