Cathepsin V is a useful prognostic factor for colorectal cancer.

Molecular studies have identified various treatment-related prognostic molecules to enhance the effectiveness of colorectal cancer (CRC) treatment and improve survival rates. The expression of cathepsin V in gastrointestinal cancer cells prompted an investigation into its potential as a prognostic indicator for CRC. The evaluation of cathepsin V expression and its clinicopathological significance was conducted through immunohistochemistry in a tissue microarray, encompassing 142 CRC and normal colorectal tissues. Overall and disease-free survival rates, based on cathepsin V expression levels, were assessed using the Kaplan-Meier method and compared utilizing the log-rank test. Univariate and multivariate analyses, employing a Cox proportional hazards model, were performed to identify prognostic factors. Cathepsin V expression exhibited no correlation with age, sex, tumor location, tumor size, or histological grade. However, it was significantly correlated with depth of tumor invasion, regional lymph node (LN) metastasis, distant metastasis, and lymphovascular involvement (all p<0.001). Overall and disease-free survival rates were significantly better with low cathepsin V expression than with high expression (p<0.001). Univariate analysis identified several prognostic factors, including histological grade (low vs. high), tumor size (≤ vs. >5 cm), tumor depth (T1 vs. ≥T2), regional LN metastasis, distant metastasis, tumor-node-metastasis (TNM) stage (Stage I vs ≥II), lymphovascular involvement, and cathepsin V expression. Multivariate analysis revealed that tumor depth, distant metastasis, and cathepsin V expression are independent predictors of poor survival. Cathepsin V is frequently expressed in CRC, and its high expression is associated with poor prognosis. Therefore, cathepsin V is a useful prognostic marker for CRC.

Rapid identification of antibody impurities in size-based electrophoresis via CZE-MS generated spectral library.

Methods for the reliable and effective detection and identification of impurities are crucial to ensure the quality and safety of biopharmaceutical products. Technical limitations constrain the accurate identification of individual impurity peaks by size-based electrophoresis separations followed by mass spectrometry. This study presents a size-based electrophoretic method for detecting and identifying impurity peaks in antibody production. A hydrogen sulfide-accelerated degradation method was employed to generate known degradation products observed in bioreactors that forms the basis for size calibration. LabChip GXII channel electrophoresis enabled the rapid (< 1 min) detection of impurity peaks based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their accurate identification. We combine these techniques to examine impurities resulting from cell culture harvest conditions and forced degradation to assess antibody stability. To mimic cell culture harvest conditions and the impact of forced degradation, we subjected samples to cathepsin at different pH buffers or exposed them to high pH and temperature. Our method demonstrated the feasibility and broad applicability of using a CZE-MS generated spectral library to unambiguously assign peaks in high throughput size-based electrophoresis (i.e., LabChip GXII) with identifications or likely mass of the antibody impurity. Overall, this strategy combines the utility of CZE-MS as a high-resolution separation and detection method for impurities with size-based electrophoresis methods that are typically used to detect (not identify) impurities during the discovery and development of antibody therapeutics.

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type.

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture, Japan revealed by ELISA using recombinant cathepsin L1.

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.

Improved Production and Biophysical Analysis of Recombinant Silicatein-α.

Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon-oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His6-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed.

Laser capture microdissection in combination with mass spectrometry: Approach to characterization of tissue-specific proteomes of Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

Expression of elastolytic cathepsins in human skin and their involvement in age-dependent elastin degradation.

BACKGROUND: Skin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides. METHODS: Cathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry. RESULTS: Elastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies. CONCLUSION: Cathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.

Prognostic significance of cathepsin V (CTSV/CTSL2) in breast ductal carcinoma in situ.

AIMS: Cathepsin V (CTSV/CTSL2) is a lysosomal cysteine proteinase and plays a role in extracellular matrix degradation. It is associated with poor prognosis in invasive breast cancer (IBC), but its role in breast ductal carcinoma in situ (DCIS) remains unclear. In this study, we aimed to evaluate the prognostic significance of CTSV in DCIS. METHODS: CTSV protein expression was immunohistochemically assessed in a well-characterised and annotated cohort of DCIS comprising pure DCIS (n=776) and DCIS coexisting with IBC (n=239). CTSV expression was analysed in tumour cells and surrounding stroma, including its association with clinicopathological parameters and outcome. RESULTS: In pure DCIS, high CTSV expression was observed in 29% of epithelial tumour cells and 20% of surrounding stroma. High expression in both components was associated with features of poor prognosis including higher nuclear grade, hormone receptor negativity and HER2 positivity. In addition, stromal CTSV expression was associated with larger DCIS size, comedo-type necrosis and high proliferation index. DCIS associated with IBC showed higher CTSV expression than pure DCIS either within the epithelial tumour cells or surrounding stroma (p<0.0001 and p=0.001, respectively). In DCIS/IBC, CTSV expression was higher in the invasive component than DCIS component either in tumour cells or surrounding stroma (both p<0.0001). CTSV stromal expression was associated with invasive recurrence independent of other prognostic factors in patients treated with breast conserving surgery (HR=3.0, p=0.005). CONCLUSION: High expression of CTSV is associated with poor outcome in DCIS and is a potential marker to predict DCIS progression to invasive disease.

Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous Treatment of Autoimmune Dry Eye Disease.

The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren's syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is "humanized", (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa is of further interest for systemic treatments for autoimmune diseases like SS.

Skeletal Muscle Lysosomal Function via Cathepsin Activity Measurement.

Muscle wasting or cachexia is commonly associated with aging and many diseases such as cancer, infection, autoimmune disorders, and trauma. Decrease in muscle mass, or muscle atrophy, is often caused by dysfunction of protein proteolytic systems, such as lysosomes, which regulate protein turnover and homeostasis. Lysosomes contain many hydrolases and proteases and, thus, represent the major organelle that control protein turnover. Recently, lysosomes have emerged as a signaling hub to integrate cellular functions of nutrient sensing and metabolism, autophagy, phagocytosis, and endocytosis, which are all related to tissue homeostasis. In this chapter, we describe the protocol used to measure lysosomal proteinase (cathepsins) activity in the skeletal muscle. A better understanding of lysosomal function in muscle homeostasis is critical in developing new therapeutic approaches to prevent muscle wasting.

Longitudinal analysis of tear cathepsin S activity levels in male non-obese diabetic mice suggests its potential as an early stage biomarker of Sjögren's Syndrome.

CONTEXT: Cathepsin S (CTSS) activity is elevated in Sjögren's Syndrome (SS) patient tears. OBJECTIVE: To evaluate longitudinal expression of tear and tissue CTSS activity relative to other disease indicators in Non-Obese Diabetic (NOD) mice. METHODS: CTSS activity was measured in tears and lacrimal glands (LG) from male 1-6 month (M) NOD and 1 and 6 M BALB/c mice. Lymphocytic infiltration was quantified by histopathology, while disease-related proteins (Rab3D, CTSS, collagen 1) were quantified using q-PCR and immunofluorescence. RESULTS: In NOD LG, lymphocytic infiltration was noted by 2 M and established by 3 M (p < 0.01). IFN-É£, TNF-α, and MHC II expression were increased by 2 M (p < 0.01). Tear CTSS activity was significantly elevated at 2 M (p < 0.001) to a maximum of 10.1-fold by 6 M (p < 0.001). CTSS activity in LG lysates was significantly elevated by 2 M (p < 0.001) to a maximum of 14-fold by 3 M (p < 0.001). CTSS and Rab3D immunofluorescence were significantly increased and decreased maximally in LG acini by 3 M and 2 M, respectively. Comparable changes were not detected between 1 and 6 M BALB/c mouse LG, although Collagen 1 was decreased by 6 M in LG of both strains. CONCLUSION: Tear CTSS activity is elevated with other early disease indicators, suggesting potential as an early stage biomarker for SS.

Cysteine cathepsins B, X and K expression in peri-arteriolar glioblastoma stem cell niches.

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.

Biomarkers of alveolar bone resorption in gingival crevicular fluid: A systematic review.

BACKGROUND: Periodontitis is a prevalent oral disease with bone loss being it's hallmark. Clinical parameters used to measure periodontitis are retrospective and do not indicate active inflammation nor prognosis. GCF can be easily collected chairside and bone turnover biomarkers found in GCF can be evaluated to check for active inflammation and disease progression. This systematic review aims to evaluate the literature for association and predictive value of bone turnover biomarkers in GCF during periodontal disease. MATERIALS AND METHODS: This review was conducted and reported according to the PRISMA guidelines. The online databases Google Scholar and PubMed were used for data search. MeSH terms were used for PubMed search. All original studies from 1990 to 2017 conducted on human subjects in the English language were included in the review. Studies on non-human subjects, reviews and studies conducted in languages other than English were not considered. Reference lists of qualified articles were also searched. RESULTS: The search generated 2300 results whose titles were screened and 1571 articles were retreived. 23 articles were accepted in the review and full texts were accessed. These included 1 randomized controlled trial, 12 cross-sectional studies, five pre-post interventional studies, 4 longitudinal and 1 in-vitro in-vivo experimental study. The studies were conducted on patients of both genders ranging from 10 to 81 years in age. A total of 37 biomarkers were evalueted in the studies included in this review. Majority of the studies reported interleukin-1ß (IL-1ß) while receptor activated nuclear factor-kappa B ligand (RANKL) and matrix metalloproteinase-8 (MMP-8) were the other frequently reported biomarkers. Most of the studies evaluated more than two biomarkers. ELISA was the most commonly used biochemical test used for detection. CONCLUSION: A wide range of biomarkers have been established as indicators of alveolar bone resorption. Few of the biomarkers have also shown positive correlation with disease progression and outcome of periodontal therapies thus underscoring their predictive value in periodontal diagnosis and prognosis. Not one single biomarker has been reported to have a predictive advantage over another and a combination of two or more biomarkers along with clinical evaluation is recommended.

Cysteine Protease Zymography: Brief Review.

Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V.

Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost. This chapter will demonstrate the utility and interpretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S, and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with densitometry.

Expression of cathepsins V and S in thymic epithelial tumors.

Cathepsins are a group of proteolytic enzymes of the endosomal/lysosomal pathway involved in the thymic development of T cells restricted by major histocompatibility complex class II molecules. In the normal thymus, cathepsin V (CTV) and cathepsin S (CTS) are expressed in cortical and medullary epithelial cells, respectively. To investigate whether cathepsins could serve as a diagnostic marker, we performed immunohistochemical analysis for CTV and CTS in 77 cases of thymic epithelial tumors. Almost all cases (59/60) of thymoma expressed CTV, whereas 28 of 60 cases of thymoma expressed CTS. Notably, CTS was expressed in most cases of type A and type AB thymomas, but not in type B thymoma. The expression of cathepsins in type AB thymoma showed a clear correlation with histologic features; CTV was found predominantly in the type B component, and CTS was frequently expressed in the type A component. In thymic carcinoma, CTV was expressed in less than half cases (7/17), and the ratio of CTS-positive cases was equivalent to that of thymoma (8/17). Cases of CTV-negative thymic carcinoma tended to have a higher incidence of recurrence than did CTV-positive cases. Although further studies with a larger number of cases are required to confirm the utility of cathepsin immunostaining, CTV and CTS appear to serve as auxiliary diagnostic and/or prognostic markers in thymic epithelial tumors.

Preclinical Evaluation of Cathepsin-Based Fluorescent Imaging System for Cytoreductive Surgery.

BACKGROUND: Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) is a treatment option for peritoneal surface malignancies. The ability to detect microscopic foci of peritoneal metastasis intraoperatively may ensure the completeness of cytoreduction. In this study, we evaluated the suitability of a hand-held cathepsin-based fluorescent imaging system for intraoperative detection of appendiceal and colorectal peritoneal metastasis. METHODS: Peritoneal tumors and normal peritoneal tissues were collected from patients with appendiceal and colorectal peritoneal metastasis. Expression of different cathepsins (CTS-B, -D, -F, -G, -K, -L, -O, and -S) was determined by quantitative RT-PCR and immunohistochemistry. The hand-held cathepsin-based fluorescent imaging system was used to detect peritoneal xenografts derived from human colon cancer cells (HT29, LoVo and HCT116) in nu/nu mice. RESULTS: While the expression levels of CTS-B, -D, -L, and -S could be higher in peritoneal tumors than normal peritoneum with a median (range) of 6.1 (2.9-25.8), 2.0 (1.0-15.8), 1.4 (0.8-7.0), and 2.1 (1.6-13.9) folds by quantitative RT-PCR, respectively, CTS-B was consistently the major contributor of the overall cathepsin expression in appendiceal and colonic peritoneal tumors, including adenocarcinomas and low-grade appendiceal mucinous neoplasms. Using peritoneal xenograft mouse models, small barely visible colonic peritoneal tumors (<2.5 mm in maximum diameter) could be detected by the hand-held cathepsin-based fluorescent imaging system. CONCLUSIONS: Because cathepsin expression is higher in peritoneal tumors than underlying peritoneum, the hand-held cathepsin-based fluorescent imaging system could be useful for intraoperative detection of microscopic peritoneal metastasis during CRS-HIPEC and clinical trial is warranted.

Effect of technological factors on the activity and losses of cathepsins B, D and L during the marinating of Atlantic and Baltic herrings.

BACKGROUND: This study analyzes the effect of salt and acetic acid concentration, time, temperature and fish freezing on the activity and losses of cathepsins during the marinating of Atlantic and Baltic herrings. RESULTS: The highest contribution to meat general proteolytic activity was found for cathepsin D-like activity. This contribution decreased during the marinating process as a result of, among other things, cathepsin losses to brine. The methods of marinating had a significant impact on cathepsin activity losses. The average ratio of cathepsin D-like activity to L and B in brine accounted for 15:3.5:1.5, respectively. Depending on the method of calculation, cathepsin activity in brine was similar (per gram of tissue/milliliter of brine) or multiply higher (per gram protein in tissue/brine) than in the marinated herring meat. Statistical analysis demonstrated that the extent and structure of cathepsin losses were significantly correlated with the quantitative and qualitative composition of protein hydrolysis products in marinades. CONCLUSION: The presented results depict new phenomena of cathepsin losses and explain their impact on the process of fish marinating. Results allow better optimization of the process of meat ripening. The high activity of aspartyl and cysteine cathepsins in brine indicates the real feasibility of their application in the food industry for novel food design. © 2016 Society of Chemical Industry.

A potential role for glycated cross-links in abdominal aortic aneurysm disease.

BACKGROUND: Diabetes is a risk factor for atherosclerotic disease but negatively associated with the development and progression of abdominal aortic aneurysm (AAA). Advanced glycation end products (AGEs) are increased in diabetes and renders the vascular matrix more resistant to proteolysis. We assessed the concentration of AGEs in AAA biopsies obtained from diabetic and nondiabetic patients and hypothesized that (nonenzymatic) glycation of AAA tissue protects against proteolytic breakdown of collagen. METHODS: AAA biopsies were collected from 30 diabetic and 30 matched nondiabetic AAA patients at the time of open repair. Aortic control samples from 10 nondiabetic and 16 diabetic patients were collected, and concentrations of the AGE cross-link pentosidine was measured. Furthermore, noncross-linking AGEs (adducts), as well as proteolytic enzymes known to play a role in aneurysm development (matrix metalloproteinase [MMP]-2, MMP-9, cathepsin B and S) were quantified. Ex vivo, nondiabetic AAA biopsies were glycated and measured subsequently for collagen type I release. RESULTS: Pentosidine concentrations in AAA wall biopsies were increased in patients with diabetes compared with nondiabetics 9.4 (5.0-13.5) vs 6.0 (2.5-9.6) pmol/µmol lysine (P = .02). Increased pentosidine concentrations were also observed in nonaneurysmatic aortic wall biopsies from diabetic patients. In diabetic AAA vascular wall tissue, pentosidine concentration was negatively correlated with aortic diameter (r = -0.43; P = .02). Ex vivo glycated AAA biopsies were resistant against MMP-induced collagen type I degradation as compared with controls (7.0 vs 10.4 µg/L; P = .02). No differences were observed for AGEs that are not forming cross-links. CONCLUSIONS: These findings suggest that cross-linking AGEs like pentosidine play a protective role in AAA progression in diabetic patients.