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Background: Serum Cystatin S (CST4), a secretory protein that inhibits cellular matrix degradation, significantly influences the tumor microenvironment and tumor progression. However, the prognostic value of serum CST4 in gastric cancer (GC) remains unclear. This study aims to explore serum CST4's utility in GC prognostic assessment. Methods: A cohort of 334 patients with GC who underwent radical gastrectomy was assessed. Preoperative serum CST4 levels were measured alongside traditional tumor markers, correlating with clinical data and patient outcomes. The cohort was divided into training and test sets at a ratio of 3:1 for Cox regression analyses, which identified CST4 as an independent risk factor for overall survival (OS) and disease-free survival (DFS). A prognostic model was developed, validated with calibration curves, and its predictive value was evaluated using receiver operating characteristic (ROC) curves. In addition, CST4 expression was correlated with immune cell infiltration using data from The Cancer Genome Atlas (TCGA). Patients were stratified by median CST4 levels, and Kaplan-Meier curves for OS and DFS were plotted. Results: Cystatin S was confirmed as an independent risk factor for OS and DFS. Integrating CST4 with traditional markers and TNM pathological staging significantly enhanced the predictive value for prognosis. Cystatin S's impact on tumor progression is likely mediated through modulation of the immune microenvironment, including immune suppression and evasion. Conclusion: Cystatin S is an effective biomarker for GC prognostic assessment, assisting in the evaluation of prognosis and the selection of treatment strategies for patients with GC. [ABSTRACT FROM AUTHOR]

BackgroundCystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination.MethodsWildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3' single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios.ResultsJHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls.ConclusionsCystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.

Objective • To study the effects of acupuncture combined with the formula of Yi Qi Yang Yin and blood activating (A-YBF) on blood glucose levels and renal function in patients with early diabetic nephropathy. Methods • 96 patients with early diabetic nephropathy treated in our hospital from April 2021 to April 2022 were included in the study and divided into the control group (conventional medical treatment) and the study group (A-YBF), with 48 cases in each group. The efficacy and adverse effects were recorded by comparing the Chinese medicine symptom points, blood glucose level, renal function, and inflammatory factor level between the two groups before and after the treatment. Results • The clinical efficacy of the study group was significantly higher than that of the control group (P < .05). Before treatment, no difference was found between the primary and secondary symptom scores of the two groups (P > .05); after treatment, the primary and secondary symptom scores of the study group were lower than those of the control group (P < .05). Before treatment, there was no difference in fasting blood glucose (FPG) and 2h postprandial glucose (2hPG) levels; 24h urine protein quantification, cystatin C (Cys-C), urinary albumin excretion rate (UAER), and estimated glomerular filtration rate (eGFR) levels; and growth differentiation factor-15 (GDF-15), interleukin-1β (IL-1β), interleukin-17 (IL-17), and serum amyloid A (SAA) levels between the two groups (P > .05). After treatment, FPG and 2hPG levels; 24h urine protein quantification, Cys-C and UAER levels; and GDF15, IL-1β, IL-17, and SAA levels were lower in the study group than in the control group, while eGFR levels were higher than those in the control group (P < .05). Conclusion • A-YBF can effectively reduce blood glucose levels and improve renal function in patients with early diabetic nephropathy and can be promoted in clinical application. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a chronic autoimmune condition that causes synovitis and functional impairment. Currently, the most often utilized biomarkers for monitoring disease activity and severity are acute phase proteins, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). However, discrepancies have been found between the clinical inflammation and their levels. Therefore, there is a strong need for a novel biomarker to forecast how RA will proceed clinically and monitor the response to therapy. This study assessed the diagnostic value of serum cystatin D in RA patients and its potential as a biomarker for disease activity monitoring. Results: RA patients had considerably greater serum levels of cystatin D than the control group. These values showed a positive correlation with Disease Activity Score-28 (DAS28), ESR, CRP, grayscale synovitis, power Doppler synovitis, grayscale tenosynovitis (p < 0.001), and erosions, sum scores (p < 0.05). Nonetheless, no noteworthy association was observed between the serum cystatin D levels and sociodemographic data, rheumatoid factor (RF), and anti-cyclic citrullinated peptide (anti-CCP). Conclusion: Cystatin D serum levels are higher in RA patients compared to healthy subjects and are strongly correlated with the activity of RA. It can be a valuable biomarker for evaluating RA disease activity. [ABSTRACT FROM AUTHOR]

The article in the Journal of Neurology discusses a family with a genetic mutation in the CST3 gene that leads to severe psychiatric-like symptoms, migraines, and progressive young-onset dementia. The mutation causes a range of symptoms, including hemineglect, behavioral issues, and cognitive decline. The study highlights the importance of genetic screening for CST3 mutations in patients with similar symptoms, emphasizing the need for further research to understand the impact of these variants. [Extracted from the article]

Hard ticks (family Ixodidae) are significant vectors of pathogens affecting humans and animals. This review explores the composition of tick saliva, focusing on proteases and protease inhibitors, their biological roles, and their potential in vaccines and therapies. Tick saliva contains various proteases, mostly metalloproteases, serpins, cystatins, and Kunitz-type inhibitors, which modulate host hemostatic, immune, and wound healing responses to facilitate blood feeding and pathogen transmission. Proteases inhibit blood clotting, degrade extracellular matrix components, and modulate immune responses. Serpins, cystatins, and Kunitz-type inhibitors further inhibit key proteases involved in coagulation and inflammation, making them promising candidates for anticoagulant, anti-inflammatory, and immunomodulatory therapies. Several tick proteases and protease inhibitors have shown potential as vaccine targets, reducing tick feeding success and pathogen transmission. Future research should focus on comprehensive proteomic and genomic analyses, detailed structural and functional studies, and vaccine trials. Advanced omics approaches and bioinformatics tools will be crucial in uncovering the complex interactions between ticks, hosts, and pathogens, improving tick control strategies and public health outcomes. [ABSTRACT FROM AUTHOR]

Abstract Background: Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately seven million individuals worldwide, with the highest number of cases in Latin America. CD has two phases, of which the chronic phase is characterized by reduced efficacy in drug therapies. This and other factors make developing new strategies that aim to identify molecules capable of becoming alternatives to or complement current chemotherapy vitally important. Methods: Cruzain and AsCystatin were obtained recombinantly through expression in E. coli. Bioinformatic assays were conducted with both molecules, followed by in vitro enzyme inhibition assays. Subsequently, in silico studies allowed for the design of peptides, which were then assessed for molecular interactions with cruzain. The designed peptides were synthesized, and their inhibitory potential on cruzain and their trypanocidal and cytotoxic effects in vitro were finally assessed. Results: AsCystatin, a potential inhibitor of cysteine proteases, was identified from previously published scientific literature. In silico assays suggested that AsCystatin interacts with key regions of cruzain, and was subsequently produced through heterologous expression, obtaining a protein with a high degree of purity. Next, the inhibition of AsCystatin on the activity of cruzain was assessed, observing that approximately 20 µM of cystatin could inhibit 50% of the catalytic activity of the recombinant enzyme. Based on the in-silico analysis performed previously, original, and modified peptides were designed and tested, which allowed for identifying four peptides with inhibitory capacity on the enzymatic activity of cruzain. Finally, three of these peptides showed trypanocidal activity on epimastigote forms of T. cruzi in in vitro models. Conclusion: It was possible to identify AsCystatin and four peptides derived from this protein with inhibitory activity on cruzain, highlighting the trypanocidal effect of these peptides observed in in vitro assays.

Aims Examining the systemic biological processes in the heterogeneous syndrome of heart failure with reduced ejection fraction (HFrEF), as reflected by circulating proteins, in relation to echocardiographic characteristics, may provide insights into heart failure pathophysiology. We investigated the link of 4210 repeatedly measured circulating proteins with repeatedly measured echocardiographic parameters as well as with elevated left atrial pressure (LAP), in patients with HFrEF, to provide insights into underlying mechanisms. Methods and results In 173 patients with HFrEF, we performed 6-monthly echocardiography and trimonthly blood sampling during a median follow-up of 2.7 (inter-quartile range: 2.5–2.8) years. We investigated circulating proteins in relation to echocardiographic parameters of left ventricular [left ventricular ejection fraction (LVEF), global longitudinal strain (GLS)] and left atrial function [left atrial reservoir strain (LASr)] and elevated LAP (E / e ʹ ratio >15) and used gene enrichment analyses to identify underlying pathophysiological processes. We found 723, 249, 792, and 427 repeatedly measured proteins, with significant associations with LVEF, GLS, LASr, and E / e ʹ ratio, respectively. Proteins associated with LASr reflected pathophysiological mechanisms mostly related to the extracellular matrix. Proteins associated with GLS reflected cardiovascular biological processes and diseases, whereas those associated with LVEF reflected processes involved in the sympathetic nervous system. Moreover, 49 proteins were associated with elevated LAP; after correction for LVEF, three proteins remained: cystatin-D, fibulin-5, and HSP40. Conclusion Circulating proteins show varying associations with different echocardiographic parameters in patients with HFrEF. These findings suggest that pathways involved in atrial and ventricular dysfunction, as reflected by the plasma proteome, are distinct. [ABSTRACT FROM AUTHOR]

The Trichinella spiralis novel cystatin (TsCstN) inhibits cathepsin L (CatL) activity and inflammation of macrophages during lipopolysaccharide (LPS) induction. To identify the protease inhibitory region, this study applied an in silico modeling approach to simulate truncation sites of TsCstN (Ts01), which created four truncated forms, including TsCstNΔ1–39 (Ts02), TsCstNΔ1–71 (Ts03), TsCstNΔ1–20, Δ73–117 (Ts04), and TsCstNΔ1–20, Δ42–117 (Ts05). The superimposition of these truncates modeled with AlphaFold Colab indicated that their structures were more akin to Ts01 than those modeled with I-TASSER. Moreover, Ts04 exhibited the closest resemblance to the structure of Ts01. The recombinant Ts01 (rTs01) and truncated proteins (rTs02, rTs03, and rTs04) were successfully expressed in a prokaryotic expression system while Ts05 was synthesized, with sizes of approximately 14, 12, 8, 10, and 2.5 kDa, respectively. When determining the inhibition of CatL activity, both rTs01 and rTs04 effectively reduced CatL activity in vitro. Thus, the combination of the α1 and L1 regions may be sufficient to inhibit CatL. This study provides comprehensive insights into TsCstN, particularly regarding its protein function and inhibitory domains against CatL. [ABSTRACT FROM AUTHOR]

Objectives. Cardiovascular disease (CVD) is the leading cause of death and disability among persons with diabetes. Early intervention on cardiovascular risk factors (CRFs) is important in reducing CVD burden. The SEARCH for Diabetes in Youth study assessed CRFs in incident cohorts of youth aged <20 years established from 2002 to 2016. Research Design and Methods. Regression models assessed trends over each incident year for lipids (total cholesterol (TC), HDL‐c, LDL‐c, triglycerides (TG), VLDL‐c, and non‐HDL‐c), kidney function (albumin/creatinine ratio (ACR) ≥30 and ≥300, cystatin C, serum creatinine and estimated glomerular filtration rate (eGFR)), systolic and diastolic blood pressure (BP) z‐scores, BMI z‐score, waist circumference (WC), and an inflammatory marker (C‐reactive protein (CRP)). Models were stratified by diabetes type (type 1 diabetes (T1D), N = 4,600; type 2 diabetes (T2D), N = 932) and adjusted for age at diagnosis, sex, race/ethnicity, and diabetes duration. An interaction analysis assessed differential time trends by type. Results. For youth with T1D, all CRFs significantly improved over time, with the exception of ACR > 300, cystatin C, serum creatinine, eGFR, and CRP. For youth with T2D, TC, LDL‐c, and non‐HDL‐c significantly improved, while eGFR, BMI z‐score, and CRP significantly worsened. Significant differences in trends over time by type were seen for TC, HDL‐c, BMI z‐score, BP z‐scores, WC, and CRP. Conclusions. Overall, improvements in CRFs were more often observed in youth with T1D. Youth with T2D had worsening trends over time in BMI z‐score, CRP, and kidney function. Further research is needed to better understand these trends and their implications for long‐term CVD risk. [ABSTRACT FROM AUTHOR]

Abstract The objective of this in vitro study was to assess the efficacy of CaneCPI-5, either alone or in combination with various concentrations of sodium trimetaphosphate (TMP) in protecting against initial enamel erosion. A total of 135 bovine enamel specimens were prepared and categorized into nine groups (n/group=15) according to the following treatments: Deionized water; Commercial solution (Elmex Erosion ProtectionTM); 0.1 mg/mL CaneCPI-5; 0.5% TMP; 1.0% TMP; 3.0% TMP; 0.1 mg/mL CaneCPI-5+0.5% TMP; 0.1 mg/mL CaneCPI-5+1.0%TMP; and 0.1 mg/mL CaneCPI-5+3.0%TMP. The specimens were treated with the respective solutions for 2 h, followed by acquired enamel pellicle formation for 2 h and exposure to 0.65% citric acid (CA) for 1 min. These procedures were repeated once a day for three consecutive days. Demineralization was assessed by the percentage change in surface hardness (%CSH) and calcium release into CA, analyzed by the Arsenazo III method. The data were evaluated using Kruskal-Wallis/Dunn's tests. Regarding %CSH, CaneCPI-5+3.0%TMP was the most effective treatment when compared to the CaneCPI-5 group alone. As for calcium release into CA, the CaneCPI-5+0.5% TMP and CaneCPI-5 groups (both with lower calcium release) did not significantly differ from the commercial solution. In conclusion, combination of CaneCPI-5 with TMP enhances the protective potential against initial enamel erosion in vitro.

The Acari Hypothesis posits that acarians, i.e., mites and ticks, are operative agents of allergy. It derived from observations that allergens are molecular elements of acarians or acarian foodstuffs. A corollary of The Hypothesis provides how acarian dietary elements are selected as allergens; namely, a pattern recognition receptor native to the acarian digestive tract complexes with dietary molecules problematic to the acarian. By virtue of its interspecies operability, the receptor then enables not only removal of the dietary elements by the acarian immune system, but also—should such a complex be inoculated into a human—production of an element-specific IgE. Because pattern recognition receptors bind to molecules problematic to the organism from which the receptors originate, it follows that molecules targeted by adaptive IgE, i.e., allergens, must be problematic to acarians. This claim is supported by evidence that host organisms, when infested by acarians, upregulate representative members of allergenic molecular families. Appreciation of the relationship between allergens and acarians provides insight well beyond allergy, shedding light also on the anti-acarian defenses of many living things, especially humans.

PURPOSE: Studies examining biomarkers associated with fatigue in breast cancer survivors treated with radiation are limited. Therefore, we examined the longitudinal association between serum biomarkers and post-breast cancer fatigue in survivors treated with radiation: [oxidative stress] 8-hydroxyguanosine, myeloperoxidase; [inflammation] interleukin-6 (IL-6), c-reactive protein, growth differentiation factor-15 (GDF-15), placental growth factor, transforming growth factor-beta, [cardiac damage] cystatin-C, troponin-I. METHODS: In a secondary analysis, we included participants from the Womens Health Initiative if they had: a previous breast cancer diagnosis (stages I-III), no prior cardiovascular diseases, pre-and post-breast cancer serum samples drawn approximately 3 years apart, and fatigue measured using the Short-Form 36 vitality subscale at both serum collections. Biomarkers were measured using ELISA or RT-qPCR and modeled as the log2 post-to pre-breast cancer ratio. RESULTS: Overall, 180 women with a mean (SD) age of 67.0 (5.5) years were included. The mean (SD) vitality scores were 66.2 (17.2) and 59.7 (19.7) pre- and post-breast cancer, respectively. Using multivariable weighted linear regression, higher biomarker ratios of cystatin-C, IL-6, and GDF-15 were associated with a lower vitality score (i.e., higher fatigue). For example, for each 2-fold difference in cystatin-C biomarker ratio, the vitality score was lower by 7.31 points (95% CI: -14.2, -0.45). CONCLUSION: Inflammatory and cardiac damage biomarkers are associated with fatigue in breast cancer survivors treated with radiation; however, these findings should be replicated in a larger sample. Biomarkers could be measured in clinical practice or assessed in risk prediction models to help identify patients at high risk for fatigue.

Aim: The serum creatinine/cystatin C ratio (CCR) or sarcopenia index is considered a useful marker of muscle mass. However, its usefulness in late‐stage older adults remains unclear. We aimed to determine the usefulness of CCR as an indicator of sarcopenia in community‐dwelling Japanese adults aged >75 years. Methods: Our study recruited participants aged 70, 80, and 90 ± 1 years during the baseline years, and included a 3‐year follow‐up in the Septuagenarians, Octogenarians, Nonagenarians, Investigation with Centenarians study. From 2015 to 2018, 955 participants were eligible: 367 in their 70s, 304 in their 80s, and 284 in their 90s. The diagnostic components of sarcopenia, including "low muscle mass, plus low muscle strength, and/or low physical performance," were evaluated using the bioelectrical impedance analysis‐measured skeletal muscle mass index (SMI), handgrip strength, and short physical performance battery (SPPB) score, respectively, in accordance with the Asia Working Group for Sarcopenia 2019 criteria. Separate analyses were performed between each component and CCR, adjusting for sex, body mass index, and other blood biomarkers in each group. Results: The relationship between CCR and sarcopenia components was significant for handgrip strength (β = 0.21, 0.13, 0.19, and P < 0.0001, =0.0088, <0.0001, for the 70s, 80s, and 90s age groups, respectively); however, it was limited for SMI (β = 0.14; P = 0.0022, only for the 90s) and not significant for the SPPB score. Conclusion: CCR is a limited indicator of sarcopenia in late‐stage older adults. Although its association with muscle strength was significant, its relationship with muscle mass and physical performance was less pronounced. Geriatr Gerontol Int 2024; 24: 529–536. [ABSTRACT FROM AUTHOR]

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In the present work, we have investigated a novel recombinant cystatin dgECP1 and its mutant form, dgECP1m1, focused on their impact on the adhesive behaviour of two breast cell lines: the cancerous, MDA-MB-231, and the normal, MCF-10A. DgECP1 cystatin is intriguing with its RGD motif, responsible for cell adhesion and typical for mammalian extracellular matrix proteins but uncommon for plant cystatins. The presence of the RGD sequence suggests the potential of the dgECP1 to influence the adhesion of cancer cells and, respectively, cancer metastasis. A mutant form of the dgECP1cystatin, dgECP1m1, where RGD is replaced with HGD tripeptide, was also investigated. We found that both phytocystatins exerted differential effects on the adhesion behaviour of normal and cancer cells. In the case of dgECP1 cystatins, the effect on cancer cell adhesion also depends on the mode of administration of the cystatin to cells. When dgECP1 is pre-adsorbed on a substrate, it improves the attachment of breast cancer cells and induces cell aggregation, which is more typical for normal breast cells, and oppositely suppressed adhesion of cancer cells when added to the medium. The mutant form, dgECP1m1, inhibited cancer cell adhesion independently on the way of administration. On the other hand, both plant cystatins only slightly reduced the adhesion of normal mammary cells pointing to the higher sensitivity of cancer cells to both cystatins. These preliminary results open the possibility of considering the plant cystatin dgECP1 for anti-cancer strategies. [ABSTRACT FROM AUTHOR]

Cystatins, a family of proteins known for their inhibitory role against cysteine proteases, have garnered significant attention in the field of neurodegeneration. Numerous genetic, experimental, and clinical studies concerning cystatin C suggest it plays an important role in the course of neurodegenerative diseases. Its beneficial effects are associated with cysteine protease inhibition, impact on β-amyloid aggregation, as well as regulation of cell proliferation, autophagy, and apoptosis. Cystatin isolated from chicken egg white, called ovocystatin, has been widely used in medical and pharmaceutical research due to its structural and biological similarities to human cystatin C. This article focuses on the potential use of cystatins, with special emphasis on easily obtained ovocystatin, in the treatment of neurodegenerative diseases, such as dementia. The current evidence on cystatin use has shed light on its mechanisms of action and therapeutic implications for neuroprotection and maintenance of cognitive functions. [ABSTRACT FROM AUTHOR]

Background Sarcopenia is an important prognostic factor, but its optimal screening methods remain challenging. Several new indices developed based on serum creatinine (Cr) and cystatin C (CysC) have been proposed to be diagnostic biomarkers for sarcopenia screening. Objective This review aimed to evaluate the diagnostic accuracy of serum Cr- and CysC-based indices for sarcopenia diagnosis. Methods We systematically searched MEDLINE, EMBASE, SCIE and SCOPUS from inception to 2 April 2023. Methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. A bivariate random-effects model was used to synthesise the pooled sensitivity, specificity and area under the curves of the summary receiver operating characteristic (SROC-AUC). Results We retrieved 936 publications and included 16 studies with 5,566 participants (mean age ranged: 51.0–78.4 years, 50.2% men). The prevalence of sarcopenia ranged from 7.8 to 69.5%. All included studies presented a moderate to high risk of bias. The serum Cr- and CysC-based indices showed moderate diagnostic accuracy for sarcopenia (pooled sensitivity: 0.67, 95% CI 0.57–0.75; pooled specificity: 076, 95% CI 0.67–0.83; pooled SROC-AUC: 0.78, 95% CI 0.74–0.81). The Cr/CysC ratio is the most widely studied index, followed by the Cr × eGFRcys index. Overall, both indicators had satisfactory and comparable performance in screening sarcopenia. Conclusion Serum Cr- and CysC-based indices showed moderate diagnostic accuracy for sarcopenia. The most studied indices—the Cr/CysC ratio and Cr × eGFRcys index—had comparable diagnostic accuracy for evaluating sarcopenia and may serve as surrogate markers for sarcopenia. However, further validation is required to verify these findings. [ABSTRACT FROM AUTHOR]

The objective of this in vitro study was to assess the efficacy of CaneCPI-5, either alone or in combination with various concentrations of sodium trimetaphosphate (TMP) in protecting against initial enamel erosion. A total of 135 bovine enamel specimens were prepared and categorized into nine groups (n/group=15) according to the following treatments: Deionized water; Commercial solution (Elmex Erosion ProtectionTM); 0.1 mg/mL CaneCPI-5; 0.5% TMP; 1.0% TMP; 3.0% TMP; 0.1 mg/mL CaneCPI-5+0.5% TMP; 0.1 mg/mL CaneCPI-5+1.0%TMP; and 0.1 mg/mL CaneCPI-5+3.0%TMP. The specimens were treated with the respective solutions for 2 h, followed by acquired enamel pellicle formation for 2 h and exposure to 0.65% citric acid (CA) for 1 min. These procedures were repeated once a day for three consecutive days. Demineralization was assessed by the percentage change in surface hardness (%CSH) and calcium release into CA, analyzed by the Arsenazo III method. The data were evaluated using Kruskal-Wallis/Dunn's tests. Regarding %CSH, CaneCPI-5+3.0%TMP was the most effective treatment when compared to the CaneCPI-5 group alone. As for calcium release into CA, the CaneCPI-5+0.5% TMP and CaneCPI-5 groups (both with lower calcium release) did not significantly differ from the commercial solution. In conclusion, combination of CaneCPI-5 with TMP enhances the protective potential against initial enamel erosion in vitro. [ABSTRACT FROM AUTHOR]

Background: Cystatin C (Cys C), deemed as a glomerular renal function biomarker with unique properties to be an independent predictor of cardiovascular disease (CVD), remains a matter of investigation among different categories of patients. The aim of the study is a determining the diagnostic and prognostic value of Cys C in the development of cardiovascular complications in patients with arterial hypertension (AH) and different comorbidities, such as type 2 diabetes mellitus (T2DM) and obesity, as a supplement to estimated glomerular filtration rate (eGFR). Material and methods: 111 patients with AH (men/women -- 50/61) and 20 control subjects were examined. All patients with AH at the age of 54.37 ± 1.18. During a thorough examination and follow-up of patients, they were classified into 4 groups depending on the comorbidities they had: patients with AH -- group 1 (22 people); patients with AH in combination with obesity -- group 2 (30 people); AH in combination with T2DM -- group 3 (31 people); patients with AH, T2DM -- group 4 (28 people). Cys C content and insulin levels in blood serum were measured by enzyme-linked immunosorbent assay on a Labline-90 analyzer (Austria) with commercial test systems manufactured by Elabscience (ELISA, China) and Monobind Inc. (ELISA, USA), according to the instructions included in the kits. Results: It has been proven that an increase in Cys C levels are associated with a decrease in eGFR in comorbid patients with T2DM (r = -0.676; p = 0.038) and without it (r = -0.589; p = 0.016). A significant increase in Cys C levels and cardiovascular accidents in comorbid patients was found. Conclusions: Cys C is a significant marker for predicting cardiovascular risk in comorbid patients with AH. [ABSTRACT FROM AUTHOR]

Simple Summary: Type 2 cystatins are a group of small secreted protease inhibitors that regulate cysteine protease cathepsins and legumain. These enzymes regulate important cellular processes that are linked to the immune response and tumor progression, playing important roles in both autoimmune diseases and various types of cancers. This review aims to explore the roles of type 2 cystatins in immune regulation and cancer development, shedding light on their significance in maintaining health. Cystatins are a family of intracellular and extracellular protease inhibitors that inhibit cysteine cathepsins—a group of lysosomal cysteine proteases that participate in multiple biological processes, including protein degradation and post-translational cleavage. Cysteine cathepsins are associated with the development of autoimmune diseases, tumor progression, and metastasis. Cystatins are categorized into three subfamilies: type 1, type 2, and type 3. The type 2 cystatin subfamily is the largest, containing 10 members, and consists entirely of small secreted proteins. Although type 2 cystatins have many shared biological roles, each member differs in structure, post-translational modifications (e.g., glycosylation), and expression in different cell types. These distinctions allow the type 2 cystatins to have unique biological functions and properties. This review provides an overview of type 2 cystatins, including their biological similarities and differences, their regulatory effect on human immune responses, and their roles in tumor progression, immune evasion, and metastasis. [ABSTRACT FROM AUTHOR]

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity. [ABSTRACT FROM AUTHOR]

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins' gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5′-3′ untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes. [ABSTRACT FROM AUTHOR]

Acquired enamel pellicle plays an important role in the pathogenesis of early childhood caries (ECC), working as a protective interface between the tooth and the oral cavity. The aim of this cross‐sectional in vivo proteomic study was to compare the acquired enamel pellicle protein profile of 3–5‐year‐old children with ECC (n = 10) and caries‐free children (n = 10). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC‐ESI‐MS/MS). In total, 241 proteins were identified. Basic salivary proline‐rich protein 1 and 2, Cystatin‐B, and SA were found only in the caries free group. When comparing caries free and ECC groups, lower protein levels were found in the caries free group for hemoglobin subunit beta, delta, epsilon, gamma‐2, globin domain‐containing protein and gamma‐1, neutrophil defensin 3, serum albumin, protein S100‐A8, and S100‐A9. The proteins histatin‐1, statherin, salivary acidic proline‐rich phosphoprotein ½, proline‐rich protein 4, submaxillary gland androgen‐regulated protein 3B, alpha‐amylase 1 and 2B were found at higher levels in the caries free group. The exclusive and the proteins found at higher levels in the caries free group might have protective functions that play a role in the prevention of caries, besides providing important insights to be evaluated in future studies for the possible development of new therapeutic strategies for ECC. [ABSTRACT FROM AUTHOR]

Glomerular hyperfiltration (GHF) is an increase in single-nephron glomerular filtration rate (GFR) that occurs in both physiological states and pathological states. Whole-kidney GHF is often used as a surrogate for single-nephron hyperfiltration since determining single-nephron GFR is impossible in routine clinical care. A clear definition (read threshold) of GHF is lacking. The aim of the first part of this review was to find evidence for defining the threshold for GHF, based on literature review, including systematic reviews and meta-analysis data, with both measured and estimated GFR. The consensus pediatric threshold for GHF as obtained from reviews, measured and estimated GFR studies, can reliably be set to 135 mL/min/1.73 m2 for children aged > 2 years. Diagnosing GHF from SCr-based estimated GFR is not reliable in subjects with reduced muscle mass. In these cases, it could be of interest to confirm the state of GHF using cystatin C-based eGFR, or preferably, by measured GFR, using methods that are accurate in the high GFR-range. [ABSTRACT FROM AUTHOR]

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1–6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3–6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3–9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) treatment relies primarily on 70-year-old drugs, and prophylaxis suffers from the lack of an effective vaccine. Among the 10 million people exhibiting disease symptoms yearly, 450,000 have multidrug or extensively drug-resistant (MDR or XDR) TB. A greater understanding of host and pathogen interactions will lead to new therapeutic interventions for TB eradication. One of the strategies will be to target the host for better immune bactericidal responses against the TB causative agent Mycobacterium tuberculosis (Mtb). Cathepsins are promising targets due to their manipulation of Mtb with consequences such as decreased proteolytic activity and improved pathogen survival in macrophages. We recently demonstrated that we could overcome this enzymatic blockade by manipulating protease inhibitors such as cystatins. Here, we investigate the role of cystatin F, an inhibitor that we showed previously to be strongly upregulated during Mtb infection. Our results indicate that the silencing of cystatin F using siRNA increase the proteolytic activity of cathepsins S, L, and B, significantly impacting pathogen intracellular killing in macrophages. Taken together, these indicate the targeting of cystatin F as a potential adjuvant therapy for TB, including MDR and XDR-TB. [ABSTRACT FROM AUTHOR]

The relationship between salivary proteome and dietary habits was studied in previous works, where a relationship between salivary proteins like cystatins and polyphenol/tannin levels in diet was observed. However, it remains to be elucidated if this association results from an effect of polyphenol-rich food ingestion on saliva composition. The aim of this work was to test the effects of apple intake on the saliva proteome, both in the short and medium term (after 4 days of continuous intake). By incubating saliva samples with apple phenolic-rich extract, protein bands containing α-amylase, S-type cystatins, and proline-rich proteins (PRPs) appeared in the fraction that precipitated, showing the potential of these (poly)phenols to precipitate salivary proteins. Among these, it was salivary cystatins that presented changes in their levels both in the saliva samples collected immediately after apple intake and in the ones collected after 4 days of intake of an extra amount of apple. These results support the thought that intake is reflected in the salivary proteome. The effect of a polyphenol-rich food, like the apple, on salivary cystatin levels is in line with results observed in animal models and, due to the involvement of these proteins in oral food perception, it would be interesting to explore in future studies the effect of these changes on sensory perception and acceptance of polyphenol-rich food. [ABSTRACT FROM AUTHOR]

Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys29-to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly29 to Cys29. The lack of exopeptidase activity may be due to other mutations, such as His110-to-Asn in the occluding loop and Asp224-to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases.

Abstract Background Saliva possesses antiviral activity, with submandibular-sublingual (SMSL) saliva having higher antiviral activity than parotid saliva. Various salivary proteins have inactivating effects on influenza A virus (IAV), but the detailed relationship between antiviral proteins and salivary anti-IAV activities in the parotid and SMSL glands is unknown. Here, to identify salivary proteins with anti-IAV activity, salivary proteins from parotid and SMSL glands were identified, quantified, and compared using liquid chromatography-mass spectrometry. Methods Twelve healthy male volunteers participated in the study. Parotid and SMSL saliva was collected by suction and collection devices. We assessed anti-IAV activities, protein concentrations, and protein-bound sialic acid concentrations in parotid and SMSL saliva. Results SMSL had significantly higher anti-IAV activity than parotid saliva. SMSL also had higher concentrations of glycoproteins, such as mucin 5B and mucin 7, protein-bound sialic acid, cystatins, and lysozyme C, compared with parotid saliva. Salivary mucin 5B and mucin 7 concentrations significantly positively correlated with the salivary protein-bound sialic acid concentration. Salivary anti-IAV activity significantly positively correlated with protein-bound sialic acid, mucin 5B, mucin 7, cystatin-C, -S, and -SN concentrations. Conclusion Salivary mucins, cystatins, and lysozyme C contribute to the high anti-IAV activity of SMSL saliva.

Purpose: Pemetrexed has shown efficacy as monotherapy or in combination with platinum salts in the treatment of non-small cell lung cancer and mesothelioma. However, severe hematological toxicities induced by pemetrexed-based chemotherapy have been observed. Some studies have suggested that drug interactions may be associated with pemetrexed toxicity. The objective of this study was to determine predictive factors, including drug interactions, associated with pemetrexed toxicity. Methods: This retrospective open monocentric study included patients consecutively treated with pemetrexed after a multidisciplinary risk assessment. Patients who experienced toxicity of grade 3 or 4 according to the Common Terminology Criteria for Adverse Events v5.0, or a grade 2 leading to a change in management, during the first four courses of pemetrexed, were assigned to the early limiting toxicities (ELT) group. Univariate and multivariable logistic regression models were used to test the association variables with the occurrence of ELT. Results: Seventy-four patients were included in this study (median age: 67 years, with non-small cell lung cancer adenocarcinoma (88%), mesothelioma (7%), or others (5%). Thirty-six patients (49%) were assigned to the ELT group (27 grades 3 and 4; 9 grade 2 with management modification). Three baseline factors were associated with pemetrexed ELT in univariate and multivariate analysis: cystatin clearance (p = 0.0135), albumin level (p = 0.0333), and proton pump inhibitors use (p = 0.035). Conclusion: To conclude, ELT induced by pemetrexed-based treatments occur frequently in cancer patients in a real-world setting. A pretherapeutic assessment before pemetrexed initiation should include three major checkpoints: use of proton pump inhibitors, sarcopenia, and denutrition evaluation. [ABSTRACT FROM AUTHOR]

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products. [ABSTRACT FROM AUTHOR]

The golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics. [ABSTRACT FROM AUTHOR]