Search Results (36)

Transglutaminase 2 (TG2) is a multifunctional protein and plays a role in cancer progression. We previously identified TG2 as an early-recurrence biomarker in hepatocellular carcinoma (HCC). TG2-knockdown (shTG2) and control (shCtl) HCC cell lines were used for comparative analyses to clarify the molecular mechanisms underlying the contribution of this protein to HCC malignancy. The proliferation of shTG2 cells was slightly but significantly decreased compared with that of shCtl cells. Differential gene expression profiling based on GeneChip arrays revealed the enrichment of the PI3K-Akt signaling pathway and showed that the expression of Dickkopf-1 and -3 (DKK1 and DKK3, respectively), inhibitors and modulators of the Wnt/β-catenin signaling pathway, was increased in shTG2 cells. The expression of epithelial–mesenchymal transition (EMT)-related genes was similar in both shCtl and shTG2 cells before and after TGF-β1 treatment, even though TGF-β1 markedly upregulated TG2. Thus, TG2 may contribute to cancer malignancy via the stimulation of cell proliferation signaling, such as PI3K-Akt and Wnt/β-catenin signaling, but not EMT. This effect might be further enhanced by humoral factors such as TGF-β1 from the tumor microenvironment. Full article

Plant-based milk substitutes are becoming increasingly popular in the food industry. Among different plant proteins, chickpea proteins (CP) offer unique qualities as good functional and nutritional properties, followed by pleasant taste. This study examines the ability of the production of o/w emulsions resembling milk analogue (3% w/w chickpea protein, 3% w/w canola oil) by using chickpea protein isolate with/without the enzyme transglutaminase (TG) (50 U/g of protein). As a reference material, commercial soymilk was used. The emulsions were characterized by particle size distribution, zeta potential, viscosity, and microstructure. The TG-crosslinked chickpea protein milk analogue demonstrated improved stability, characterized by enhanced zeta potential (−24.7 mV) and extended shelf life compared to chickpea protein milk analogue without TG and soymilk. Stable particle size distribution (D[3,2] 0.11–0.17 µm) and shear-thinning behaviour (viscosity values of 2.16 mPas at 300 1/s) additionally contributed to their stability and desirable viscosity. Overall, chickpea protein milk analogue crosslinked by TG presents a promising alternative to traditional and plant-based milk products, offering clean-label, functional, and shelf-stable formulations. The additional optimization of protein concentration and processing conditions could enhance the overall functionality even further. Full article

With growing environmental and health concerns surrounding meat consumption, meat analogs have emerged as sustainable and health-conscious alternatives. A major challenge in developing these products is replicating the fibrous, elastic texture of meat, where microbial transglutaminase (MTG) has shown significant potential. MTG catalyzes protein cross-linking, enhancing the structural integrity of meat analogs. This study aimed to evaluate the effects of MTG concentrations (0%, 0.5%, and 1%) and incubation times (0, 1.5, and 3 h) on the quality and rheological properties of meat analogs. Analogs were tested for pH, protein content, dry matter, fat retention, and thermal loss. Textural properties, including hardness, cohesiveness, gumminess, springiness, and chewiness, were determined using texture profile analysis, while leakage parameters were evaluated through water and fat content tests. Results revealed that higher MTG concentrations and longer incubation times improved protein content (14.34% to 15.55%), dry matter (29.61% to 32.53%), and reduced total leakage (1.262% to 0.634%). Textural properties, including hardness (57.08 N to 83.14 N), gumminess (19.40 N to 30.00 N), and chewiness (17.60 N × mm to 29.58 N × mm), also significantly improved with increasing MTG levels. Thermal loss ranged from 98.37% to 100.9%, showing enhanced retention at higher MTG concentrations. These results support the role of MTG in creating meat analogs with improved meat-like textures, achieved through enhanced protein cross-linking and moisture retention. Full article

This study aimed to evaluate how adding whey protein and transglutaminase impacts the quality of fried instant noodles. This research focused on analyzing various quality characteristics of the noodles based on the type and quantity of additives used. In the study, the following samples were produced: a control sample without additives and samples with 1, 2, 3, 4 and 5% of whey protein added, and 1 and 2% of transglutaminase were applied to each sample with whey protein addition. The following features were determined: fat content, water content, hydration time, hardness, adhesiveness, firmness, colour, browning index and a sensory evaluation of the macarons. The addition of whey protein, either alone or in combination with transglutaminase, reduced the fat content and increased the water content. The lowest fat content was obtained for the sample containing 5% whey protein and 2% transglutaminase (15.13%). The water content was observed in the range 2.53–3.72%. The hydration time of the instant noodles obtained increased with the use of more additives, but did not exceed 5 min in any of the samples tested. The use of additives affected the colour parameters and improved the textural properties of the noodles. Full article

Potential celiac disease (PCD) is a clinical condition characterised by the presence of a positive CD-specific serology and a normal intestinal architecture. Asymptomatic PCD patients are generally advised to continue on a gluten-containing diet (GCD), but long-term risks of this approach have never been explored. In the present study, we aimed to investigate nutritional and autoimmune complications possibly developing overtime in a cohort of asymptomatic PCD children on a GCD. We compared children’s parameters of growth, nutritional status, and autoimmunity between the time of diagnosis and on the occasion of their last medical check, after a long-term gluten-containing diet. Altogether, we collected data from 171 PCD children with a mean follow-up time of 3 years (range 0.35–15.3 years). During follow-up, although patients did not reduce their amount of daily gluten intake, their anti-tissue transglutaminase (anti-TG2) antibodies spontaneously and significantly decreased. Most parameters analysed had not changed during follow-up (height centile, ferritin, albumin, cholesterol, calcium, alkaline phosphatase, parathormone, and vitamin D) or even improved significantly (weight and BMI centile, haemoglobin, blood iron, HDL, glycaemia, and HbA1C, p < 0.05), always remaining within the limit of normality. Equally, autoantibodies for other concomitant autoimmune disorders did not increase overtime. Similar results were obtained excluding from analysis patients who had stopped producing anti-TG2 and those with a follow-up time < 3 years. Our pilot study has provided reassuring results regarding the maintenance of a gluten-containing diet in asymptomatic PCD children, even when long-term follow-up was considered. Full article

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the relentless deposition of extracellular matrix (ECM), causing lung distortions and dysfunction. Animal models of human IPF can provide great insight into the mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches. In this study, we describe the effect of bleomycin concentration on disease progression in the classical rat bleomycin model. In a dose–response study (1.5, 2, 2.5 U/kg i.t), we characterized lung fibrosis at day 14 after bleomycin challenge using endpoints including clinical signs, inflammatory cell infiltration, collagen content, and bronchoalveolar lavage fluid-soluble profibrotic mediators. Furthermore, we investigated fibrotic disease progression after 2 U/kg i.t. bleomycin administration at days 3, 7, and 14 by quantifying the expression of clinically relevant signaling molecules and pathways, epithelial mesenchymal transition (EMT) biomarkers, ECM components, and histopathology of the lung. A single bleomycin challenge resulted in a progressive fibrotic response in rat lung tissue over 14 days based on lung collagen content, histopathological changes, and modified Ashcroft score. The early fibrogenesis phase (days 3 to 7) is associated with an increase in profibrotic mediators including TGFβ1, IL6, TNFα, IL1β, CINC1, WISP1, VEGF, and TIMP1. In the mid and late fibrotic stages, the TGFβ/Smad and PDGF/AKT signaling pathways are involved, and clinically relevant proteins targeting galectin-3, LPA1, transglutaminase-2, and lysyl oxidase 2 are upregulated on days 7 and 14. Between days 7 and 14, the expressions of vimentin and α-SMA proteins increase, which is a sign of EMT activation. We confirmed ECM formation by increased expressions of procollagen-1Aα, procollagen-3Aα, fibronectin, and CTGF in the lung on days 7 and 14. Our data provide insights on a complex network of several soluble mediators, clinically relevant signaling pathways, and target proteins that contribute to drive the progressive fibrotic phenotype from the early to late phase (active) in the rat bleomycin model. The framework of endpoints of our study highlights the translational value for pharmacological interventions and mechanistic studies using this model. Full article

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro. Full article

Glycation by transglutaminase (TGase)-type could effectively improve the structure and functional properties of proteins. However, the influence on intestinal inflammation or the underlying mechanisms has not been investigated. The goal of this research was to compare the bioactivities between glycated casein generated from the TGase-catalyzed reaction and oligochitosan as well as casein using a mouse model of dextran sulfate sodium (DSS)-induced intestinal inflammation to examine the protective effects and the underlying mechanism of glycated casein on intestinal inflammation. Eight groups of C57BL/6 mice were randomly assigned in this study: Control group: standard diet for 35 days; Model group: standard diet for 28 days and then colitis induction; Pretreated groups: different levels (200, 400, 800 mg/kg BW) of casein or glycated casein for 28 days before colitis induction. The mice were drinking water containing a 3% DSS solution for seven days of mice to cause colitis. The results indicated that glycated casein and casein at 200–800 mg/kg BW all relieved DSS-induced weight loss, reduced disease activity index (DAI) score, alleviated colon length shortening, weakened the destruction of colonic mucosal structure, decreased serum LPS, and MPO, IL-1β, IL-6 and TNF-α levels in serum and colon, as well as regulated the expression of proteins involved in the TLR4/NF-κB signaling pathway in a concentration-dependent manner. Glycated caseinate showed a better protective effect against DSS-induced colitis than casein, highlighting that the TGase-type glycation of proteins as a potential functional food ingredient might be a helpful method for gut health. Full article

The effects of transglutaminase (TGase), reductant, and thermal treatment on the cross-linking of white proteins in soft-shell turtle eggs were investigated. Egg white proteins were denatured by reductant (0.83% 2-mercaptoethanol, 2-ME) pretreatment and thermal pretreatment (95 °C and 5 min), and the denatured proteins were then catalyzed by TGase (1.0 unit/mL). SDS–PAGE showed that without any pretreatments, three major egg white proteins (210 kDa, 115 kDa, and 76 kDa proteins) were inferior substrates for TGase. Only portions of the 210 kDa protein (7.9%), 115 kDa protein (11.4%), and 76 kDa protein (42.9%) were polymerized by TGase into high-molecular-weight (MW) protein polymers (>180 kDa) after incubation for 3 h at 40 °C. However, the combined use of TGase with 0.83% 2-ME and thermal pretreatment led to a significant increase (p < 0.05) in the rate of white protein polymerization after 3 h: 210 kDa protein (90.8%), 115 kDa protein (69.5%), and 76 kDa protein (72.2%). Particle size analysis indicated that these cross-linked high-MW protein polymers were 2000–10,000 nm in size. Based on the experimental results, egg white proteins denatured by 2-ME and heat pretreatment are more prone to TGase-induced cross-linking. Full article

Screening for Type 1 Diabetes (T1D, incidence 1:300) with T1D autoantibodies (T1Ab) at ages 2 and 6, while sensitive, lacks a preventive strategy. Cholecalciferol 2000 IU daily since birth reduced T1D by 80% at 1 year. T1D-associated T1Ab negativized within 0.6 years with oral calcitriol in 12 children. To further investigate secondary prevention of T1D with calcitriol and its less calcemic analog, paricalcitol, we initiated a prospective interventional non-randomized clinical trial, the PRECAL study (ISRCTN17354692). In total, 50 high-risk children were included: 44 were positive for T1Ab, and 6 had predisposing for T1D HLA genotypes. Nine T1Ab+ patients had variable impaired glucose tolerance (IGT), four had pre-T1D (3 T1Ab+, 1 HLA+), nine had T1Ab+ new-onset T1D not requiring insulin at diagnosis. T1Ab, thyroid/anti-transglutaminase Abs, glucose/calcium metabolism were determined prior and q3–6 months on calcitriol, 0.05 mcg/Kg/day, or paricalcitol 1–4 mcg × 1–3 times/day p.o. while on cholecalciferol repletion. Available data on 42 (7 dropouts, 1 follow-up < 3 months) patients included: all 26 without pre-T1D/T1D followed for 3.06 (0.5–10) years negativized T1Ab (15 +IAA, 3 IA2, 4 ICA, 2 +GAD, 1 +IAA/+GAD, 1 +ICA/+GAD) within 0.57 (0.32–1.3) years or did not develop to T1D (5 +HLA, follow-up 3 (1–4) years). From four pre-T1D cases, one negativized T1Ab (follow-up 1 year), one +HLA did not progress to T1D (follow-up 3.3 years) and two +T1Ab patients developed T1D in 6 months/3 years. Three out of nine T1D cases progressed immediately to overt disease, six underwent complete remission for 1 year (1 month–2 years). Five +T1Ab patients relapsed and negativized again after resuming therapy. Four (aged <3 years) negativized anti-TPO/TG, and two anti-transglutaminase-IgA. Eight presented mild hypercalciuria/hypercalcemia, resolving with dose titration/discontinuation. Secondary prevention of T1D with calcitriol and paricalcitol seems possible and reasonably safe, if started soon enough after seroconversion. Full article

Fresh-cut fruit requires an edible and water-resistant coating to remain fresh. This article investigated the effects of transglutaminase (TGase) and sunflower oil on the water-resistant characteristics, mechanical properties, and microstructure of a whey protein-based film. The whey protein-based emulsion coating’s preservation effect on fresh-cut apples was confirmed. According to the findings, sunflower oil (added at 1.5% w/w) could interact with β-lactoglobulin, α-lactoglobulin dimer, and β-lactoglobulin dimer to form emulsion droplets that are evenly dispersed throughout the protein film. This effect, combined with the covalent cross-linking of TGase, significantly improves the films’ microstructure, mechanical properties, and water resistance. However, too much and unevenly distributed sunflower oil (add 3% w/w) partially prevented the covalent cross-linking of TGase, reducing the elongation at the break of the composite film. In the fresh-cut apple storage experiment, the whey protein-based emulsion coating treatment significantly reduced the weight loss rate and browning index of fresh-cut apples by 26.55% and 46.39%, respectively. This was accomplished by the coating treatment significantly inhibiting the respiration rate increase, PPO and CAT activity enhancement, H₂O₂ production, and MDA accumulation. This research provides practical, technical, and theoretical guidance for the preservation of fresh-cut fruit. Full article

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD. Full article

Transglutaminase (TGM) isoform catalyze the cross-linking reaction of identical or different substrate proteins. Eosinophil has been recognized in chronic rhinosinusitis with nasal polyps (CRSwNP) forming tissue eosinophil in nasal polyp (NP), and TGM isoforms are suggested to be associated with a critical role in asthma and other allergic conditions. The aim of this study was to reveal the association of specific TGM isoform with both the tissue eosinophil infiltration deeply concerning with the intractable severity of CRSwNP and the fibrin polymerization ability of TGM isoform associated with the tissue eosinophil infiltration, which lead to NP formation and/or maintenance in CRSwNP. NP tissues (CRSwNP group) and uncinate process (UP) (control group) were collected from patients with CRSwNP and control subjects. We examined: (1) the expression level of TGM isoforms by using a real-time polymerase chain reaction (PCR) and the comparison to the issue eosinophil count in the CRSwNP group, (2) the location of specific TGM isoform in the mucosal tissue using immunohistochemistry, (3) the inflammatory cell showing the colocalization of specific TGM isoform in Laser Scanning Confocal Microscopy (LSCM) imaging, and (4) the fibrin polymerase activity of specific TGM isoform using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A certain level of TGM 1, 2, 3, 5 expression was present in both the CRSwNP group and the control group. Only TGM 1 expression showed a positive significant correlation with the tissue eosinophil count in the CRSwNP group. The localization of TGM 1 in NP (CRSwNP) laid mainly in a submucosal layer as inflammatory cells and was at the cytoplasm in the tissue eosinophil. Fibrin polymerase activity of TGM 1 showed the same polymerase ability of factor XIIIA. TGM 1 might influence the NP formation and/or maintenance in CRSwNP related to the tissue eosinophil infiltration, which formed fibrin mesh composing NP stroma. Full article

(1) Background: Microbial transglutaminases (MTGase) catalyze protein crosslink. This is useful in the food industry to improve gelation, water holding capacity, and emulsifying capacity during foodstuff manufacturing. The production of MTGase in wild-type strains renders low yield and high costs of downstream purification, limiting its industrial applications. (2) Methods: In this work, MTGase from Bacillus amyloliquefaciens BH072 (BaMTGase) has been heterologously expressed in Lactococcus lactis, using the signal peptide Usp45 to direct the secretion of recombinant BaMTGase out of the cell for easier purification. (3) Results: In these conditions, MTGase was purified with a high yield (48.7 ± 0.2 mg/L) and high enzyme activity (28.6 ± 0.5 U/mg). Next, BaMTGase was tested for industrial applications. Recombinant BaMTGase and commercial MTGase were used for SPI solution crosslinking. BaMTGase formed a harder gel with higher water-holding capacity and a dense and smooth gel microstructure. (4) Conclusions: This work provides an attractive food-grade cell factory for the food industry and offers a suitable chassis for MTGase production. Full article

Passive smoking is extensively studied because of its harmfulness to human health. In this study, the effects of fermented green tea waste extract gels (GTEG) on oxidative damage in mice exposed to short-term cigarette smoke (CS) were investigated. The GTEG is prepared from green tea waste extract and microbial transglutaminase (MTGase). The lung injury model of mice was established through passive smoking for 5 days. The experimental results revealed the following findings. (1) The GTEG induced by MTGase has obvious gel properties; (2) GTEG has strong biological activity and antioxidant properties in vitro; (3) The passive smoking model was established successfully; specifically, the lung tissue of the model mice exhibited inflammatory symptoms, oxidative stress response appeared in their bodies, and their inflammatory indicators increased; (4) Compared with the passive smoking model group, the mice, which were exposed to CS and received GTEG treatment, exhibited increased food intake and body weight; increased total superoxide dismutase and glutathione peroxidase activity in serum; significant decreases (p < 0.05) in the content levels of the inflammatory factors malondialdehyde, interleukin (IL)-6, and tumor necrosis factor α (TNF-α); and inhibited expression of IL-6, IL-33, TNF-α, and IL-1β inflammatory genes. The results indicated that taking GTEG can relieve the oxidative stress injury of mice caused by short-term CS and has antioxidant properties. Full article