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In this article, density functional theory computations at the PBE0‐D3/def2‐TZVP level are reported to unveil the type of bonding between β‐D‐glucopyranose—silver ion (1:1) complex ([Ag(C6H12O6)]+) and seven gas molecules, namely, H2, C2H2, C2H4, CO, N2, NO and O2. Moreover, the relative preference of trapping among these molecules within the sight of Ag metal ion in the complex is explored. The nature of interaction of these small molecules with the [Ag(C6H12O6)]+ ion is studied. Exergonic nature of binding is noted with the metal center for all the chosen small molecules except O2. Thermochemical data reveals the binding preference of C2H4 > C2H2 > CO > NO > N2 > H2. Natural bond orbital analysis, contour plot of the Laplacian of electron density, electron density descriptors, and gradient isosurface help in understanding the nature of interactions. Maximum bond formation is noted between the Ag‐complex and CO molecule. Assessed energy decomposition analysis discloses the nature of interaction as mainly orbital between the bound small gas molecules and the Ag‐complex. Frontier molecular orbital pictures further help in understanding the type of interaction as orbital. To disclose the kinetic stability of the gas molecule bound Ag complexes an ab initio molecular dynamics study is done at different temperatures up to 2 ps. These studies help in understanding the type of adsorption. Calculated conceptual density functional theory (CDFT) based reactivity descriptors corroborate well with results. β‐D‐glucopyranose—silver ion (1:1) complex may be used as small gas molecule scavenger. [ABSTRACT FROM AUTHOR]

Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-β1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis. [ABSTRACT FROM AUTHOR]

The study aimed to enhance Hatay cheese texture using microbial transglutaminase enzyme (mTG). Cheeses were manufactured using milk treated with mTG for different durations (30, 45 and 60 min) and assessed over 60 days. Longer treatment increased yield (18.79–20.81%) and hardness but reduced springiness. Composition, proteolysis and adhesiveness remained unaffected. Electrophoresis showed β‐casein and αs1‐casein declines during storage, with minor changes in αs1‐casein's breakdown product. Microbial transglutaminase enzyme created a denser protein network, visible in SEM, resulting in a compact structure. Sensory analysis favoured the 60‐min treatment. Overall, mTG improved Hatay cheese quality by modifying texture without major compositional changes. [ABSTRACT FROM AUTHOR]

Aims: To find clinical and immunological signatures of the SARS‐CoV‐2 and the COVID‐19 pandemic on children newly diagnosed with type 1 diabetes (T1D). Methods: A single‐centre, retrospective, observational study comparing the clinical and immunological characteristics of children diagnosed with T1D the year before and during the first 2 years of the COVID‐19 pandemic. Data extracted from the medical records included clinical and demographic parameters, COVID‐19 PCR results and the presence of anti‐islet, thyroid and celiac‐related antibodies. Also obtained from the medical records was a family history of T1D, celiac disease and autoimmune thyroid disease in a first‐degree family member. Results: A total of 376 children were diagnosed with T1D during the study period. A total of 132 in the pre‐COVID era and 246 in the first 2 years of the pandemic. At diagnosis, the pH in children with DKA was lower, and HbA1c tended to be higher in the COVID‐19 group compared to the pre‐COVID‐19 group (7.30 [7.18, 7.35] vs 7.33 [7.19, 7.36], p = 0.046) and (110.9 [86.9, 129.5] vs 100 [80.3, 129.5], p = 0.067]) respectively. Multiple islet antibodies (IA) were significantly more common among patients in the pre‐COVID‐19 group compared to the COVID‐19 group (72% vs 61%, p = 0.032). Tissue transglutaminase antibodies were more common among children diagnosed in the COVID‐19 compared to the pre‐COVID group (16.6% vs 7.9%, p = 0.022). Conclusions: Our findings suggest that SARS‐CoV‐2 and the environmental alterations caused by the pandemic affected the clinical characteristics and the immunological profile of children diagnosed with T1D. It is, therefore, plausible that the virus plays a role in the autoimmune process causing T1D. [ABSTRACT FROM AUTHOR]

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity. [ABSTRACT FROM AUTHOR]

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity. [ABSTRACT FROM AUTHOR]

In the cheese industry, whey, which is rich in lactose and proteins, is underutilized, causing adverse environmental impacts. The fractionation of its components, typically carried out through filtration membranes, faces operational challenges such as membrane fouling, significant protein loss during the process, and extended operating times. These challenges require attention and specific methods for optimization and to increase efficiency. A promising strategy to enhance industry efficiency and sustainability is the use of enzymatic pre‐treatment with the enzyme transglutaminase (TGase). This enzyme plays a crucial role in protein modification, catalyzing covalent cross‐links between lysine and glutamine residues, increasing the molecular weight of proteins, facilitating their retention on membranes, and contributing to the improvement of the quality of the final products. The aim of this study is to review the application of the enzyme TGase as a pretreatment in whey protein filtration. The scope involves assessing the enzyme's impact on whey protein properties and its relationship with process performance. It also aims to identify both the optimization of operational parameters and the enhancement of product characteristics. This study demonstrates that the application of TGase leads to improved performance in protein concentration, lactose permeation, and permeate flux rate during the filtration process. It also has the capacity to enhance protein solubility, viscosity, thermal stability, and protein gelation in whey. In this context, it is relevant for enhancing the characteristics of whey, thereby contributing to the production of higher quality final products in the food industry. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Summary: In this study, β‐conglycinin was treated with transglutaminase (TGase) and glucono‐δ‐lactone (GDL) to meticulously examine the resulting changes in both antigenicity and structural attributes. The findings elucidated a significant reduction (P < 0.05) in the antigenicity of β‐conglycinin to 72.1% and 77.5% after the modified treatment employing TGase alone or in conjunction with GDL, respectively, in contrast to the untreated samples. Electrophoretic analysis showed a decrease in the density of 7S α, 7S α′, and 7S β, indicating the formation of high molecular weight protein aggregates because of the catalytic effect of TGase. Additionally, the simultaneous application of TGase and GDL induced modifications in the structural configuration of β‐conglycinin, as evidenced by a decrease in intrinsic fluorescence intensity, an augmentation in surface hydrophobicity, and a transition from β‐sheet to β‐turn and random coil structures. These observed alterations suggest that the decline in antigenicity is intricately linked to conformational changes, potentially influencing the exposure of antigenic epitopes. Overall, this study holds significant theoretical and practical implications for the development of hypoallergenic soy products and the enhancement of food safety. [ABSTRACT FROM AUTHOR]

For autologous-disc-derived chondrocyte transplantation (ADCT) a transglutaminase crosslinked gelatine gel and an albumin hyaluronic acid gel, crosslinked with bis-thio-polyethylene glycol, were injected through a syringe into a degenerated intervertebral disc, where they solidified in situ. This biomechanical in vitro study with lumbar bovine motion segments evaluated disc height changes, motion characteristics in a quasi-static spine loading simulators, and the potential extrusion risk of these biomaterials in a complex dynamic multi-axial loading set-up with 100,000 loading cycles. After the injection and formation of the gel in the center of the nucleus, the disc height increase was about 0.3 mm. During cyclic testing, a gradual decrease in height could be detected due to viscoelastic effects and fluid loss. No gel extrusion could be observed for all specimens during the entire test procedure. A macroscopic inspection after dissections showed an accumulation of the solidified gel in the center of the nucleus. The results demonstrate that the injection of in situ solidifying gels through the intact annulus allows for the stable maintenance of the injected gel at the target location, with high potential for use as a suitable scaffold to anchor therapeutically applied cells for disc regeneration within the treated nucleus pulposus. [ABSTRACT FROM AUTHOR]

Liver diseases represent a considerable burden to patients and healthcare systems. Hydrogels play an important role in the engineering of soft tissues and may be useful for embedding hepatocytes for different therapeutic interventions or the development of in vitro models to study the pathogenesis of liver diseases or testing of drugs. Here, we developed two types of hydrogels by crosslinking hydrazide-functionalized gelatin with either oxidized dialdehyde hyaluronan or alginate through the formation of hydrazone bonds. Gel formulations were studied through texture analysis and rheometry, showing mechanical properties comparable to those of liver tissue while also demonstrating long-term stability. The biocompatibility of hydrogels and their ability to host hepatocytes was studied in vitro in comparison to pure gelatin hydrogels crosslinked by transglutaminase using the hepatocellular line HepG2. It was found that HepG2 cells could be successfully embedded in the hydrogels, showing no signs of gel toxicity and proliferating in a 3D environment comparable to pure transglutaminase cross-linked gelatin hydrogels used as control. Altogether, hydrazide gelatin in combination with oxidized polysaccharides makes stable in situ gelling systems for the incorporation of hepatocytes, which may pave the way for use in liver tissue engineering and drug testing. [ABSTRACT FROM AUTHOR]

Current international guidelines recommend duodenal biopsies to confirm the diagnosis of celiac disease in adult patients. However, growing evidence suggests that immunoglobulin A (IgA) anti-tissue transglutaminase (tTg) antibody levels ≥10 times the upper limit of normal (ULN) can accurately predict celiac disease, eliminating the need for biopsy. We performed a systematic review and meta-analysis to evaluate the accuracy of the no-biopsy approach to confirm the diagnosis of celiac disease in adults. We systematically searched MEDLINE, EMBASE, Cochrane Library, and Web of Science from January 1998 to October 2023 for studies reporting the sensitivity and specificity of IgA-tTG ≥10×ULN against duodenal biopsies (Marsh grade ≥2) in adults with suspected celiac disease. We used a bivariate random effects model to calculate the summary estimates of sensitivity, specificity, and positive and negative likelihood ratios. The positive and negative likelihood ratios were used to calculate the positive predictive value of the no-biopsy approach across different pretest probabilities of celiac disease. The methodological quality of the included studies was evaluated using the QUADAS-2 tool. This study was registered with PROSPERO, number CRD42023398812. A total of 18 studies comprising 12,103 participants from 15 countries were included. The pooled prevalence of biopsy-proven celiac disease in the included studies was 62% (95% confidence interval [CI], 40%–83%). The proportion of patients with IgA-tTG ≥10×ULN was 32% (95% CI, 24%–40%). The summary sensitivity of IgA-tTG ≥10×ULN was 51% (95% CI, 42%–60%), and the summary specificity was 100% (95% CI, 98%–100%). The area under the summary receiver operating characteristic curve was 0.83 (95% CI, 0.77 – 0.89). The positive predictive value of the no-biopsy approach to identify patients with celiac disease was 65%, 88%, 95%, and 99% if celiac disease prevalence was 1%, 4%, 10%, and 40%, respectively. Between-study heterogeneity was moderate (I 2 =30.3%), and additional sensitivity analyses did not significantly alter our findings. Only 1 study had a low risk of bias across all domains. The results of this meta-analysis suggest that selected adult patients with IgA-tTG ≥10×ULN and a moderate to high pretest probability of celiac disease could be diagnosed without undergoing invasive endoscopy and duodenal biopsy. [Display omitted] The diagnosis of celiac disease involves blood tests to detect elevated antibodies triggered by eating gluten, and endoscopy with biopsy from the small intestine to prove the damage to the intestinal lining. We found that when the levels of the antibodies are very high, damage to the intestinal lining is almost certain, and endoscopy may not be required in all cases. [ABSTRACT FROM AUTHOR]

Heat stress reduces the number of Sertoli cells, which is closely related to an imbalanced redox status. Glutamate functions to maintain the equilibrium of redox homeostasis. However, the role of glutamate in heat treated Sertoli cells remains unclear. Herein, Sertoli cells from 3-week-old piglets were treated at 44 °C for 30 min (heat stress). Glutamate levels increased significantly following heat stress treatment, followed by a gradual decrease during recovery, while glutathione (GSH) showed a gradual increase. The addition of exogenous glutamate (700 μM) to Sertoli cells before heat stress significantly reduced the heat stress-induced apoptosis rate, mediated by enhanced levels of antioxidant substances (superoxide dismutase (SOD), total antioxidant capacity (TAC), and GSH) and reduced levels of oxidative substances (reactive oxygen species (ROS) and malondialdehyde (MDA)). Glutamate addition to Sertoli cells before heat stress upregulated the levels of glutamate-cysteine ligase, modifier subunit (Gclm), glutathione synthetase (Gss), thioredoxin (Trx1) and B-cell leukemia/lymphoma 2 (Bcl-2), and the ratio of phosphorylated Akt (protein kinase B)/total Akt. However, it decreased the levels of Bcl2-associated X protein (Bax) and cleaved-caspase 3. Addition of the inhibitor of glutaminase (Gls1), Bptes (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, 30 μM)to Sertoli cells before heat stress reversed these effects. These results inferred that glutamate rescued heat stress-induced apoptosis in Sertoli cells by enhancing activity of antioxidant enzymes and activating the Trx1-Akt pathway. Thus, glutamate supplementation might represent a novel strategy to alleviate the negative effect of heat stress. • Heat treatment could cause apoptosis and oxidate stress of boar SCs. • Exogenous glutamate reduced apoptosis by enhancing the levels of antioxidant substances. • Glutamate improves oxidation resistance by regulating the expression of Gclm/Gss to increase GSH. • Protective effects of glutamate are mediated through the Trx1-Akt pathway. [ABSTRACT FROM AUTHOR]

Many of the biological processes of the cell, from its structure to signal transduction, involve protein–protein interactions. On this basis, our aim was to identify cellular proteins that interact with ERK5, a serine/threonine protein kinase with a key role in tumor genesis and progression and a promising therapeutic target in many tumor types. Using affinity chromatography, immunoprecipitation, and mass spectrometry techniques, we unveiled an interaction between ERK5 and the mitochondrial glutaminase GLS in pancreatic tumor cells. Subsequent co-immunoprecipitation and immunofluorescence studies supported this interaction in breast and lung tumor cells as well. Genetic approaches using RNA interference techniques and CRISPR/Cas9 technology demonstrated that the loss of ERK5 function led to increased protein levels of GLS isoforms (KGA/GAC) and a concomitant increase in their activity in tumor cells. It is well known that the tumor cell reprograms its intermediary metabolism to meet its increased metabolic needs. In this sense, mitochondrial GLS is involved in the first step of glutamine catabolism, one of the main energy sources in the context of cancer. Our data suggest that ERK5 contributes to the regulation of tumor cell energy metabolism via glutaminolysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tissue transglutaminase2 (TG2) has emerged as a key enigmatic protein in the development of various metabolic and age-related diseases. It catalyzes covalent cross-linking of countless proteins and provides strength to the extracellular matrix and resistance to proteolytic degradation via different pathways, including NF-kβ, TGF-β and PI3K/Akt as the major signaling pathways. The etiology of diabetes and associated diseases has been found to be linked to unbalanced TG2 activity that may not only result in impaired or delayed wound healing in diabetics but also worsen degenerative and metabolic disease conditions. TG2 is usually overexpressed in diabetes, fibrosis, cancer, and neurodegenerative disorders. These TG2-linked diseases are usually associated with prolonged activation of inflammatory pathways. Therefore, reducing the inflammatory mechanisms and improving tissue remodeling appear to be the main treatment strategies to exterminate TG2-linked diseases. The present review aims to deliver a detailed overview of the existing understanding of TG2 in diabetes and associated diseases' progression, as well as treatment strategies to regulate TG2 tightly and its potential clinical applications. Our research endorses the notion that TG2 can serve as an effective early-stage diagnostic biomarker for metabolic diseases and a therapeutic target for the development of potential drug. [ABSTRACT FROM AUTHOR]

Enzymatic additives, particularly alkaline proteases, play a crucial role in enhancing detergent effectiveness against protein-based stains. Despite advancements in enzyme stabilization techniques, there is a need for innovative strategies to further improve protease stability in laundry detergents. However, research exploring the utilization of substrate imprinting technology to achieve this objective remains limited. Therefore, this study aims to enhance the stability of alkaline proteases in laundry detergents by employing casein as an imprinting substrate and utilizing transglutaminase-mediated (TGase) crosslinking to modify proteases 102 and 306. The optimal temperature, pH, and thermal stability of the modified alkaline proteases 102 and 306 showed no significant changes. However, these two modified alkaline proteases exhibited varying degrees of improvement in stability among the 14 detergent additives tested. Under 40 °C incubation for 24 h, the relative enzyme activity of modified alkaline protease 102 increased approximately 1.4–15-fold in AEO-9, BS-12, CMI, APG, FMEE, and SOE, while the relative enzyme activity of modified alkaline protease 306 increased approximately 1.2–3.7-fold across different additives (FMEE, AEO-9, BS-12, SOE, FAA, and AEC-9Na). These modified proteases demonstrated improved stability and wider applicability across commercial detergent formulations available. Integrated into standard laundry detergent at a 1:7 ratio before and after modification, they effectively removed protein stains from the cotton fabric after 24 h of 40 °C incubation. These findings provide insights into more effective stain-removal techniques. [ABSTRACT FROM AUTHOR]

Elafin, which is derived from trappin-2 or pre-elafin by proteolysis, is an endogenous serine protease inhibitor with a low molecular weight. Its inhibitory activity is dependent on anchoring to the extracellular matrix by forming covalent bonds with its distinctive N-terminal domain via tissue transglutaminases. In addition to inhibiting proteases, it also exhibits anti-inflammatory, antibiotic, antifungal, antiviral and immunomodulatory functions. Elafin plays an important role in inflammatory disease and is a promising candidate for the anti-inflammatory treatment of respiratory diseases. This review will discuss the therapeutic potential of elafin in airway inflammation and provide evidence and suggestions for the future treatment of airway inflammatory diseases. In addition, the therapeutic potential of elafin in lung cancer is also discussed. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases encompass a heterogeneous group of disorders that afflict millions of people worldwide. Characteristic protein aggregates are histopathological hallmark features of these disorders, including Amyloid β (Aβ)-containing plaques and tau-containing neurofibrillary tangles in Alzheimer's disease, α-Synuclein (α-Syn)-containing Lewy bodies and Lewy neurites in Parkinson's disease and dementia with Lewy bodies, and mutant huntingtin (mHTT) in nuclear inclusions in Huntington's disease. These various aggregates are found in specific brain regions that are impacted by neurodegeneration and associated with clinical manifestations. Transglutaminase (TG2) (also known as tissue transglutaminase) is the most ubiquitously expressed member of the transglutaminase family with protein crosslinking activity. To date, Aβ, tau, α-Syn, and mHTT have been determined to be substrates of TG2, leading to their aggregation and implicating the involvement of TG2 in several pathophysiological events in neurodegenerative disorders. In this review, we summarize the biochemistry and physiologic functions of TG2 and describe recent advances in the pathogenetic role of TG2 in these diseases. We also review TG2 inhibitors tested in clinical trials and discuss recent TG2-targeting approaches, which offer new perspectives for the design of future highly potent and selective drugs with improved brain delivery as a disease-modifying treatment for neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

A wound healing model was developed to elucidate the role of mesenchymal-matrix-associated transglutaminase 2 (TG2) in keratinocyte re-epithelialisation. TG2 drives keratinocyte migratory responses by activation of disintegrin and metalloproteinase 17 (ADAM17). We demonstrate that epidermal growth factor (EGF) receptor ligand shedding leads to EGFR-transactivation and subsequent rapid keratinocyte migration on TG2-positive ECM. In contrast, keratinocyte migration was impaired in TG2 null conditions. We show that keratinocytes express the adhesion G-protein-coupled receptor, ADGRG1 (GPR56), which has been proposed as a TG2 receptor. Using ADAM17 activation as a readout and luciferase reporter assays, we demonstrate that TG2 activates GPR56. GPR56 activation by TG2 reached the same level as observed with an agonistic N-GPR56 antibody. The N-terminal GPR56 domain is required for TG2-regulated signalling response, as the constitutively active C-GPR56 receptor was not activated by TG2. Signalling required the C-terminal TG2 β-barrel domains and involved RhoA-associated protein kinase (ROCK) and ADAM17 activation, which was blocked by specific inhibitors. Cell surface binding of TG2 to the N-terminal GPR56 domain is rapid and is associated with TG2 and GPR56 endocytosis. TG2 and GPR56 represent a ligand receptor pair causing RhoA and EGFR transactivation. Furthermore, we determined a binding constant for the interaction of human TG2 with N-GPR56 and show for the first time that only the calcium-enabled "open" TG2 conformation associates with N-GPR56. [ABSTRACT FROM AUTHOR]

Bacterial and mammalian cells are rich in putrescine, spermidine, and spermine. Polyamines are required for optimum fitness, but the biological function of these small aliphatic compounds has only been partially revealed. Known functions of polyamines include interaction with nucleic acids that alters gene expression and with proteins that modulate activity. Although polyamines can be incorporated into proteins, very few naturally occurring polyaminated proteins have been identified, which is due in part to the difficulty in detecting these adducts. In the current study, bovine albumin and the recombinant universal stress protein from Francisella tularensis were used as models for mass spectrometry analysis of polyaminated proteins. The proteins were covalently bound to putrescine, spermidine, or spermine by the action of carbodiimide or microbial transglutaminase. Tryptic peptides, subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS), were identified using Protein Prospector software. We describe the search parameters for identifying polyaminated peptides and show MS/MS spectra for adducts with putrescine, spermidine, and spermine. Manual evaluation led us to recognize signature ions for polyamine adducts on aspartate, glutamate, and glutamine, as well as neutral loss from putrescine, spermidine, and spermine during the fragmentation process. Mechanisms for the formation of signature ions and neutral loss are presented. Manual evaluation identified a false-positive adduct that had formed during trypsinolysis and resulted in peptide sequence rearrangement. Another false positive initially appeared to be a 71 kDa putrescine adduct on a cysteine residue. However, it was an acrylamide adduct on cysteine for a sample extracted from a polyacrylamide gel. The information presented in this report provides guidance and serves as a model for identifying naturally occurring polyaminated proteins. [ABSTRACT FROM AUTHOR]

Conventional transnasal brain-targeted drug delivery strategies are limited by nasal cilia clearance and the nasal mucosal barrier. To address this challenge, we designed dissolving microneedles combined with nanocarriers for enhanced nose-to-brain drug delivery. To facilitate transnasal administration, a toothbrush-like microneedle patch was fabricated with hyaluronic acid-formed microneedles and tannic acid-crosslinked gelatin as the base, which completely dissolved in the nasal mucosa within seconds leaving only the base, thereby releasing the loaded cyclodextrin-based metal-organic frameworks (CD-MOFs) without affecting the nasal cilia and nasal microbial communities. As nanocarriers for high loading of huperzine A, these potassium-structured CD-MOFs, reinforced with stigmasterol and functionalized with lactoferrin, possessed improved physical stability and excellent biocompatibility, enabling efficient brain-targeted drug delivery. This delivery system substantially attenuated H 2 O 2 - and scopolamine-induced neurocyte damage. The efficacy of huperzine A on scopolamine- and D-galactose & AlCl 3 -induced memory deficits in rats was significantly improved, as evidenced by inhibiting acetylcholinesterase activity, alleviating oxidative stress damage in the brain, and improving learning function, meanwhile activating extracellular regulated protein kinases-cyclic AMP responsive element binding protein-brain derived neurotrophic factor pathway. Moreover, postsynaptic density protein PSD-95, which interacts with two important therapeutic targets Tau and β-amyloid in Alzheimer's disease, was upregulated. This fruitful treatment was further shown to significantly ameliorate Tau hyperphosphorylation and decrease β-amyloid by ways including modulating beta-site amyloid precursor protein cleaving enzyme 1 and a disintegrin and metalloproteinase 10. Collectively, such a newly developed strategy breaks the impasse for efficient drug delivery to the brain, and the potential therapeutic role of huperzine A for Alzheimer's disease is further illustrated. [Display omitted] • Lactoferrin-functionalized stigmasterol-reinforced CD-MOFs were developed for brain-targeting drug delivery. • Microneedles-mediated nose-to-brain drug delivery strategy were demonstrated. • Huperzine A improved memory deficits via regulating ERK-CREB-BDNF, PSD-95/Tau, and Aβ-BACE1-ADAM10 pathways. [ABSTRACT FROM AUTHOR]

Streptomyces mobaraensis transglutaminase (smTG) has been widely used in the food processing industry for protein crosslinking. However, its poor thermostability becomes a major obstacle to further applications. It is significant to develop a feasible strategy to improve the thermostability of smTG. Here, we developed a computational model based on the Siamese graph neural network framework to identify residues critical to the thermostability of smTG by predicting changes in folding free energy (ΔΔG) between the wild type and mutants. Four candidate residues were selected for mutation experiments, and the single mutant H44C exhibited 2.7 U mg−1 residual enzyme activity after 10 min incubation at 60 °C, twice as much as the wild type. Mutants Q74F and E87W also exhibited 3- and 2.3-times greater activity after 20 min. Molecular dynamics (MD) simulations revealed that smTG mutants (H44C, Q74F, and E87W) improved thermostability by enhancing hydrogen bond interactions, increasing additional residue interactions, and reducing loop flexibility. The MM-GBSA calculation demonstrated that mutants H44C and Q74F enhanced binding affinity with the substrate, and six residues crucial for substrate binding were identified. This study combines computational analysis with mutation assays for the rational design of smTG and offers a facile and efficient strategy to understand and improve the thermostability of proteins for industrial applications. [ABSTRACT FROM AUTHOR]

Background: Transglutaminase 2 (TGase 2) is a multifunctional protein and has a prominent role in various (patho)physiological processes. In particular, its transamidase activity, which is rather latent under physiological conditions, gains importance in malignant cells. Thus, there is a great need of theranostic probes for targeting tumor-associated TGase 2, and targeted covalent inhibitors appear to be particularly attractive as vector molecules. Such an inhibitor, equipped with a radionuclide suitable for noninvasive imaging, would be supportive for answering the general question on the possibility for functional characterization of tumor-associated TGase 2. For this purpose, the recently developed 18F-labeled Nε-acryloyllysine piperazide [18F]7b, which is a potent and selective irreversible inhibitor of TGase 2, was subject to a detailed radiopharmacological characterization herein. Results: An alternative radiosynthesis of [18F]7b is presented, which demands less than 300 µg of the respective trimethylammonio precursor per synthesis and provides [18F]7b in good radiochemical yields (17 ± 7%) and high (radio)chemical purities (≥ 99%). Ex vivo biodistribution studies in healthy mice at 5 and 60 min p.i. revealed no permanent enrichment of 18F-activity in tissues with the exception of the bone tissue. In vivo pretreatment with ketoconazole and in vitro murine liver microsome studies complemented by mass spectrometric analysis demonstrated that bone uptake originates from metabolically released [18F]fluoride. Further metabolic transformations of [18F]7b include mono-hydroxylation and glucuronidation. Based on blood sampling data and liver microsome experiments, pharmacokinetic parameters such as plasma and intrinsic clearance were derived, which substantiated the apparently rapid distribution of [18F]7b in and elimination from the organisms. A TGase 2-mediated uptake of [18F]7b in different tumor cell lines could not be proven. Moreover, evaluation of [18F]7b in melanoma tumor xenograft models based on A375-hS100A4 (TGase 2 +) and MeWo (TGase 2 −) cells by ex vivo biodistribution and PET imaging studies were not indicative for a specific targeting. Conclusion: [18F]7b is a valuable radiometric tool to study TGase 2 in vitro under various conditions. However, its suitability for targeting tumor-associated TGase 2 is strongly limited due its unfavorable pharmacokinetic properties as demonstrated in rodents. Consequently, from a radiochemical perspective [18F]7b requires appropriate structural modifications to overcome these limitations. [ABSTRACT FROM AUTHOR]

Background: Gluten sensitivity (GS) is one of the gluten-related disorders (GRDs). Patients with GS may have serum antibodies against tissue transglutaminase (tTG) (IgA and IgG) without any evidence of enteropathy. We aimed to evaluate both tTG-6 and tTG-2 antibodies to determine the prevalence of seropositive tTG-2 and tTG-6 antibodies in patients with multiple sclerosis (MS). Methods: In this cross-sectional study, we carried out serological tests (IgA & IgG anti-tTG-6 and tTG-2 antibodies) in patients with MS. Results: Seventy-two patients with MS were included in this study. Of them, seropositive patients for total (IgA + IgG) of tTG-6 and tTG-2 autoantibodies were 9.7% (95% CI, 4.2-18) and 5.6% (95% CI, 1.4-12.5), respectively. Conclusion: Anti-tTG-6 antibody testing may be necessary for early diagnosis of GS in patients with MS. More studies with larger sample sizes are warranted to confirm these data. [ABSTRACT FROM AUTHOR]

• Transglutaminase 2 binding to fibrillar FN is tuned by FN-fiber tension. • Transglutaminase 2 preferentially binds with higher affinity to relaxed than to stretched fibronectin fibers. • C-terminal β-barrel domains of transglutaminase 2 interact with regions outside of the canonical gelatin-binding domain of fibronectin, namely FNIII 14–15 and FNI 2. • Mechano-regulated binding of transglutaminase 2 to fibronectin is dependent on the spatial proximity between N-terminal and C-terminal domains of transglutaminase 2. • Collagen-mimicking peptide R1R2 competes in a dose-dependent manner with transglutaminase 2 for binding to FN-fibers under low as well as high tension. Transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal β-sandwich and C-terminal β-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2's C-terminal β-barrels that interacts with FN regions located outside of the canonical gelatin binding domain, specifically FNI 2 and FNIII 14–15. Combining XL-MS distance restraints with molecular docking revealed the mechano-regulated binding mechanism between TG2 and modules FNI 7–9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how forces regulate cell adhesion, spreading, migration, phenotype modulation, depending on the tensional state of ECM fibers. Data are available via ProteomeXchange with identifier PXD043976. [ABSTRACT FROM AUTHOR]

A systematic literature review (SR) registered in PROSPERO (CRD42023412241) and compliant with EFSA guidelines and the PRISMA statement on systematic reviews, was performed for the retrieval of relevant studies on adverse outcome pathways for the capacity of proteins to trigger celiac disease (CD). Search queries, together with grey literature search, resulted in the identification of relevant documents that were used to collect original data by means of data extraction, categorisation, risk of bias appraisal and data synthesis of high‐level information. A summary table was used to propose AOPs for CD. The investigation revealed that gluten, non‐gluten proteins, bacterial peptides, viral peptides, and a 55 kDa autoantigen played a role in various molecular initiating events (MIEs), with gluten proteins being the most common initiators. Furthermore, several shared characteristics were identified in the resulting AOPs across various MIEs, with structural mimicry emerging as a prominent recurring pattern, i.e., when foreign proteins or peptides have a similar structure to gluten. The studies emphasized the resemblance between the TFIIA protein and gliadin, the ability of gluten‐reactive T cells to react to bacterial peptides, and the identification of viral proteins by anti‐transglutaminase IgA antibodies in individuals with CD. These observations emphasize that CD is an intricate interaction between genetic and environmental factors, where structural mimicry connects these components and contributes to the autoimmune response in individuals with CD. This work aims to facilitate the path towards the development of a consolidated AOP pertinent to proteins triggering CD consistently to the principles defined by the OECD, in particular for the assessment of the risk of bias and for the data reporting. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by the defective activity of acid β‐glucosidase (GCase) which results from mutations in GBA1. Neurological forms of GD (nGD) can be generated in mice by intra‐peritoneal injection of conduritol B‐epoxide (CBE) which irreversibly inhibits GCase. Using this approach, a number of pathological pathways have been identified in mouse brain by RNAseq. However, unlike transcriptomics, proteomics gives direct information about protein expression which is more likely to provide insight into which cellular pathways are impacted in disease. We now perform non‐targeted, mass spectrometry‐based quantitative proteomics on brains from mice injected with 50 mg/kg body weight CBE for 13 days. Of the 5038 detected proteins, 472 were differentially expressed between control and CBE‐injected mice of which 104 were selected for further analysis based on higher stringency criteria. We also compared these proteins with differentially expressed genes (DEGs) identified by RNAseq. Some lysosomal proteins were up‐regulated as was interferon signaling, whereas levels of ion channel related proteins and some proteins associated with neurotransmitter signaling were reduced, as was cholesterol metabolism. One protein, transglutaminase 1 (TGM1), which is elevated in a number of neurodegenerative diseases, was absent from the control group but was found at high levels in CBE‐injected mice, and located in the extracellular matrix (ECM) in layer V of the cortex and intracellularly in Purkinje cells in the cerebellum. Together, the proteomics data confirm previous RNAseq data and add additional mechanistic understanding about cellular pathways that may play a role in nGD pathology. [ABSTRACT FROM AUTHOR]

Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known downstream protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation. [ABSTRACT FROM AUTHOR]

Summary: The present study aimed to investigate the impact of TGase cross‐linking on the flavour characteristics, particularly focusing on its ability to reduce bitterness in enzymatically digested Alaska Pollock framework (APF) hydrolysates using Protease A 'Amano' 2SD (A2SD) and Protease A 'Amano' 6SD (A6SD). Electronic tongue results showed that the umami was enhanced, accompanied by a reduction in both bitterness and bitter aftertaste. The contents of glutamic acid (Glu) and nucleotides (AMP, IMP and GMP) exhibited significant differences among all treatment groups (P < 0.05). There was a notable increase in the types and relative concentrations of alcohols, ketones, alkanes and aldehydes in the cross‐linked APF enzymatic hydrolysate. The Fourier transform infrared spectroscopy, ultraviolet spectrum and fluorescence spectrum analyses indicate that TGase cross‐linking leads to a reduction in the exposure of hydrophobic amino acid residues. The surface hydrophobicity of HA2SD‐C and HA6SD‐C exhibited a significantly lower level compared with that of HA2SD and HA6SD. The present study presents an effective approach for mitigating the bitter taste of aquatic hydrolysate, thereby positively contributing to the advancement and utilisation of this valuable resource. [ABSTRACT FROM AUTHOR]

Pulse proteins, such as pea and chickpea proteins, have inferior functionality, specifically gelation, compared to soy protein, hindering their applications in different food products, such as meat analogs. To close the functionality gap, protein polymerization via targeted modification can be pursued. Accordingly, transglutaminase-induced polymerization was evaluated in pea protein isolate (PPI) and chickpea protein isolate (ChPI) to improve their functionality. The PPI and ChPI were produced following a scaled-up salt extraction coupled with ultrafiltration (SE-UF) process. Transglutaminase (TGase)-modified PPI and ChPI were evaluated in comparison to unmodified counterparts and to commercial protein ingredients. Protein denaturation and polymerization were observed in the TG PPI and TG ChPI. In addition, the TGase modification led to the formation of intermolecular β-sheet and β-turn structures that contributed to an increase in high-molecular-weight polymers, which, in turn, significantly improved the gel strength. The TG ChPI had a significantly higher gel strength but a lower emulsification capacity than the TG PPI. These results demonstrated the impact of the inherent differences in the protein fractions on the functional behavior among species. For the first time, the functional behavior of the PPI and ChPI, produced on a pilot scale under mild processing conditions, was comprehensively evaluated as impacted by the TGase-induced structural changes. [ABSTRACT FROM AUTHOR]

Certain CD8 T cell responses are particularly effective at controlling infection, as exemplified by elite control of HIV in individuals harboring HLA-B57. To understand the structural features that contribute to CD8 T cell elite control, we focused on a strongly protective CD8 T cell response directed against a parasite-derived peptide (HF10) presented by an atypical MHC-I molecule, H-2Ld. This response exhibits a focused TCR repertoire dominated by Vβ2, and a representative TCR (TG6) in complex with Ld-HF10 reveals an unusual structure in which both MHC and TCR contribute extensively to peptide specificity, along with a parallel footprint of TCR on its pMHC ligand. The parallel footprint is a common feature of Vβ2-containing TCRs and correlates with an unusual Vα-Vβ interface, CDR loop conformations, and Vβ2-specific germline contacts with peptides. Vβ2 and Ld may represent specialist components for antigen recognition that allows for particularly strong and focused T cell responses.

Using a system optimized for propagating human keratinocytes, culture of skin samples from white and green sturgeons generated epithelial cells capable of making cross-linked protein envelopes. Two distinct forms of TGM1-like mRNA were molecularly cloned from the cells of white sturgeon and detected in green sturgeon cells, accounting for their cellular envelope forming ability. The protein translated from each displayed a cluster of cysteine residues resembling the membrane anchorage region expressed in epidermal cells of teleosts and tetrapods. One of the two mRNA forms (called A) was present at considerably higher levels than the other (called B) in both species. Continuous lines of white sturgeon epidermal cells were established and characterized. Size measurements indicated that a substantial fraction of the cells became enlarged, appearing similar to squames in human epidermal keratinocyte cultures. The cultures also expressed CYP1A, a cytochrome P450 enzyme inducible by activation of aryl hydrocarbon receptor 2 in fish. The cells gradually improved in growth rate over a dozen passages while retaining envelope forming ability, TGM1 expression and CYP1A inducibility. These cell lines are thus potential models for studying evolution of fish epidermis leading to terrestrial adaptation and for testing sturgeon sensitivity to environmental stresses such as pollution.

Background: Pulmonary fibrosis features in damaged pulmonary structure or over-produced extracellular matrix and impaired lung function, leading to respiratory failure and eventually death. Fibrotic lungs are characterized by the secretion of pro-fibrotic factors, transformation of fibroblasts to myofibroblasts, and accumulation of matrix proteins. Hypothesis/purpose: Imperatorin shows anti-inflammatory effects on alveolar macrophages against acute lung injury. We attempt to evaluate the properties of imperatorin on the basis of fibroblasts. Methods: In in vitro, zymosan was introduced to provoke pro-fibrotic responses in NIH/3T3 or MRC-5 pulmonary fibroblasts. Imperatorin was given for examining its effects against fibrosis. The mice were stimulated by bleomycin, and imperatorin was administered to evaluate the prophylactic potential in vivo. Results: The upregulated expression of connective tissue growth factor (CTGF), a-smooth muscle actin (a-SMA), and collagen protein due to zymosan introduction was decreased by imperatorin in fibroblasts. Zymosan induced the activity of transglutaminase 2 (TGase2) and lysyl oxidase (LOX), which was also inhibited by the administration of imperatorin. Imperatorin alone enhanced sirtuin 1 (SIRT1) activity and growth differentiation factor 15 (GDF15) secretion in fibroblasts via LKB1/AMPK/CREB pathways. In addition, GDF15 exerted a beneficial effect by reducing the protein expression of CTGF, a-SMA, and collagen and the activities of TGase and LOX. Moreover, orally administered imperatorin showed prophylactic effects on bleomycin-induced pulmonary fibrosis in mice. Conclusion: Imperatorin reduces fibrotic marker expression in fibroblasts and also increases GDF15 secretion via the LKB1/AMPK/CREB pathway, attenuating profibrotic responses in vitro. Imperatorin also alleviates pulmonary fibrosis induced by bleomycin in vivo. [ABSTRACT FROM AUTHOR]

Introduction: Despite being one of the most frequent chronic digestive diseases worldwide, with a prevalence of 1%, celiac disease (CD) remains severely underdiagnosed. Among the instruments used to improve its diagnostic rate, hematologic parameters have been proposed as screening tests to select patients with an increased probability of having CD. Assessment of coagulation is included in routine check-ups, and CD has been reported to be associated with coagulopathy. We aimed to assess if subtle changes in coagulation tests could be used in clinical practice to prompt testing for CD. Methods: We retrospectively recruited all patients with clinical suspicion for CD during a study period of 7 years (between 2015 and 2022), who were tested using IgA tissue transglutaminase (tTG) serology and serum total IgA (IgG tTG in case of IgA deficiency) and who underwent upper gastrointestinal endoscopy with multiple biopsy sampling of the duodenal bulb and distal duodenum. We stratified patients into three groups: newly diagnosed CD, gluten-free diet-treated CD, and non-CD controls. Results: Altogether, there were 133 CD patients (71 newly diagnosed, 62 GFD-treated) and 57 non-CD controls. Mean age and gender distribution were similar among the three groups: 43.3 years for newly diagnosed CD, 41.6 years for non-CD controls, and 44 years for GFD-treated CD patients, with a male gender distribution of 21.1%, 28%, and 24.1%, respectively. Among the included newly diagnosed CD patients, 14% had a prolonged INR. The mean INR was slightly higher in newly diagnosed CD patients, compared to GFD-treated CD patients and non-CD controls: 1.12 ± 0.30, 1.02 ± 0.83, and 1.00 ± 0.08, respectively (p = 0.009). Consequently, prothrombin activity was slightly lower in newly diagnosed CD patients, compared to GFD-treated CD and non-CD controls: 94.9 ± 19.3%, 102.3 ± 12.8%, and 101.9 ± 15.15, respectively. Interestingly, after GFD, the mean INR and prothrombin activity of CD individuals reached a value similar to that of non-CD controls. Conclusions: Subtle changes in INR, defined as a value within the normal range, but closer to the upper limit, could be an indicator of probability for CD. [ABSTRACT FROM AUTHOR]

1. The aim of this study was to utilise low-value spent hen meat and investigate the possibility of using chia (Salvia hispanica L.) as a mucilage-based edible film to prevent loss of quality in meat products. For this purpose, spent hen meat was reconstructed with the addition of transglutaminase (TGase) at different concentrations (0.5, 1 and 1.5%) and used to produce nuggets. These were covered with chia mucilage-based film and kept at 4°C for 7 d. 2. The addition of transglutaminase and coating with film decreased meat pH (P < 0.01). Samples containing transglutaminase had higher thiobarbituric acid (TBA) value (P < 0.01), while the DPPH value of the samples was not affected by the transglutaminase and edible film (P > 0.05). Hence, mucilage-based edible film did not prevent or delay the oxidation of nuggets. 3. Transglutaminase improved the texture characteristics of the nuggets and the edible film reduced fat retention in the samples. The combined use of transglutaminase and film decreased cooking loss and increased water holding capacity (P < 0.01). In the sensory analysis, nuggets with transglutaminase and film had more overall acceptability scores, although the results were not statistically significant. [ABSTRACT FROM AUTHOR]

Styrax is used for prevention and treatment of cerebrovascular diseases. However, the underlying mechanism remains unclear. To elucidate styrax's anti-ischemic stroke protective effects and underlying mechanisms. An ischemic-stroke rat model was established based on middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were randomly assigned to the following groups (n = 10) and administered intragastrically once a day for 7 consecutive days: sham, model, nimodipine (24 mg/kg), styrax-L (0.1 g/kg), styrax-M (0.2 g/kg) and styrax-H (0.4 g/kg). Neurological function, biochemical assessment, and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS)-based serum metabonomics were used to elucidate styrax's cerebral protective effects and mechanisms. Pearson correlation and western blot analyses were performed to verify. The addition of 0.4 g/kg styrax significantly reduced cerebral infarct volume and neurobehavioral abnormality score. Different doses of styrax also decrease MDA, TNF-α, IL-6, and IL-1β, and increase SOD and GSH-Px in ischemic-stroke rats (p < 0.05; MDA, p < 0.05 only at 0.4 g/kg dose). Biochemical indicators and metabolic-profile analyses (PCA, PLS-DA, and OPLS-DA) also supported styrax's protective effects. Endogenous metabolites (22) were identified in ischemic-stroke rats, and these perturbations were reversible via styrax intervention, which is predominantly involved in energy metabolism, glutathione and glutamine metabolism, and other metabolic processes. Additionally, styrax significantly upregulated phosphorylated AMP-activated protein kinase and glutaminase brain-tissue expression. Styrax treatment could ameliorate ischemic-stroke rats by intervening with energy metabolism and glutamine metabolism. This can help us understand the mechanism of styrax, inspiring more clinical application and promotion. [ABSTRACT FROM AUTHOR]

Acid phosphatases (EC 3.1.3.2) are the enzymes that catalyse transphosphorylation reactions and promotes the hydrolysis of numerous orthophosphate esters in acidic media, as a crucial element for the metabolism of phosphate in tissues. Inorganic phosphate (Pi) utilisation and scavenging, as well as the turnover of Pi-rich sources found in plant vacuoles, are major processes in which intracellular and secretory acid phosphatases function. Therefore, a thorough understanding of these enzymes' structural characteristics, specificity, and physiochemical properties is required to comprehend the function of acid phosphatases in plant energy metabolism. Furthermore, acid phosphatases are gaining increasing importance in industrial biotechnology due to their involvement in transphosphorylation processes and their ability to reduce phosphate levels in food products. Hence, this review aims to provide a comprehensive overview of the purification methods employed for isolating acid phosphatases from diverse plant sources, as well as their structural and functional properties. Additionally, the review explores the potential applications of these enzymes in various fields. [ABSTRACT FROM AUTHOR]

Background: Skin is one of the abundance side products of the striped catfish (Pangasius hypophthalmus) fillet industry in tropical countries. The previous histological study revealed that the skin of striped catfish contains high collagens which has the potential to supply the increasing halal gelatin demands. However, the texture of striped catfish skin gelatin (SCSG) at room temperature is mushy. This can be enhanced by the transglutaminase (TG) modification. SCSG modified with TG (SCSG_TG) needs to be tested for safety and consumer acceptance before being marketed. The research aims to decide the level of acute toxicity of SCSG_TG in mice (Mus musculus) and discover the organoleptic quality of SCSG_TG. Methods: Striped catfish skin gelatin modified with transglutaminase (SCSG_TG) was acquire by CH3COOH (acid) and NaOH (base) extraction at a temperature of 58°C. Toxicity tests using mice were worked by the Limit Test Procedure of the Organization for Economic Cooperation and Development (OECD) and then analyzed descriptively and qualitatively. While organoleptic tests were worked using hedonic assessment and the Friedman test. Results: The result shows SCSG_TG did not induce any toxic effects or death in mice. SCSG_TG 10 mg film was the most favored treatment group by the panelists (4.9-6.5) compared with SCSG_TG 0 mg (3.8-6.0) and SCSG_TG 50 mg film (4.1-6.3). Conclusion: Hence, the striped catfish skin gelatin film modified with transglutaminase (SCSG_TG) was declared safe, non-toxic, and accepted according to the organoleptic assessment that has met the standard ("dislike" criteria <25%). [ABSTRACT FROM AUTHOR]

Soluble starch (SS) could significantly accelerate the process of bovine serum albumin (BSA) cold-set gelation by glucono-δ-lactone (GDL) and microbial transglutaminase (MTGase) coupling inducers, and enhance the mechanical properties. Hardness, WHC, loss modulus (G″) and storage modulus (G′) of the gel increased significantly, along with the addition of SS, and gelation time was also shortened from 41 min (SS free) to 9 min (containing 4.0% SS); the microstructure also became more and more dense. The results from FTIR, fluorescence quenching and circular dichroism (CD) suggested that SS could bind to BSA to form their composites, and the hydrogen bond was probably the dominant force. Moreover, the ability of SS to bind the original free water in BSA gel was relatively strong, thereby indirectly increasing the concentration of BSA and improving the texture properties of the gel. The acceleration of gelling could also be attributed to the fact that SS reduced the negative charge of BSA aggregates and further promoted the rapid formation of the gel. The embedding efficiency (EE) of quercetin in BSA-SS cold-set gel increased from 68.3% (SS free) to 87.45% (containing 4.0% SS), and a controlled-released effect was detected by simulated gastrointestinal digestion tests. The work could put forward new insights into protein gelation accelerated by polysaccharide, and provide a candidate for the structural design of new products in the food and pharmaceutical fields. [ABSTRACT FROM AUTHOR]

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto‐inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin‐derived covalent activity‐based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP‐2373 (12) as a small molecule pan‐DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP‐2373 quantitatively engages more than 35 DUBs across a range of non‐toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP‐2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease‐relevant phenotypes. [ABSTRACT FROM AUTHOR]

Studies involving endophytic fungi aim to identify organisms inhabiting extreme and relatively unexplored environments, as these fungi possess unique characteristics and uncommon biochemical pathways that enable them to produce compounds with biotechnological potential. Among various enzymes, L -Asparaginase is employed in the treatment of Acute Lymphoblastic Leukemia. In this study, we identified endophytic fungi from Sanionia uncinata and Polytrichastrum alpinum collected on King George Island in Antarctica. The fungi were categorized into morphological groups based on their characteristics, molecularly identified, and assessed for L -Asparaginase (L -ASNase) enzyme production. Subsequently, production optimization was conducted. A total of 161 endophytes were isolated from 504 moss gametophytes, with 107 originating from P. alpinum and 54 from S. uncinata. These isolates were categorized into 31 morphotypes. Fungi exhibiting high enzyme production were identified molecularly. Among them, nine identified isolates belonged to the genera Aspergillus , Collariella , Diaporthe , Epicoccum , Peroneutypa , Xylaria , and Trametes. Three of these isolates were identified at the species level through multigene phylogeny, namely Epicoccum nigrum , Collariella virescens , and Peroneutypa scoparia. All 31 fungi were subjected to solid media testing for L -ASNase enzyme production, with 22 isolates demonstrating production capability, and 13 of them produced L -ASNase free from Urease and Glutaminase. The isolates displaying solid media production underwent further testing in liquid media, all of which exhibited enzyme production ranging from 0.75 to 1.29 U g-1. Notably, the three fungi identified at the species level were the highest producers of the enzyme (1.29, 1.17, and 1.13 U g-1). The production of these fungi was optimized using the Taguchi method, resulting in production values ranging from 0.687 to 2.461 U g-1. In conclusion, our findings indicate that Antarctic moss endophytic fungi exhibit significant potential for the production of the anti-leukemic enzyme L -ASNase. • Nine endophytic fungi isolated from two Antarctic mosses cultured in vitro for biotechnological screening. • Two of then shows a differential L -ASNase production free of glutaminase and urease. • Endophytic fungi from Antarctic moss have a high potential for the production of the antileukemic enzyme L -ASNase. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis (PF) is a serious progressive fibrotic disease that is characterized by excessive accumulation of extracellular matrix (ECM), thus resulting in stiff lung tissues. Lysyl oxidase (LOX) is an enzyme involved in fibrosis by catalyzing collagen cross-linking. Studies found that the ingredients in schisandra ameliorated bleomycin (BLM)-induced PF, but it is unknown whether the anti-PF of schisandra is related to LOX. In this study, we established models of PF including a mouse model stimulated by BLM and a HFL1 cell model induced by transforming growth factor (TGF)-β1 to evaluate the inhibition effects of Schisandrin C (Sch C) on PF. We observed that Sch C treatment decreased pulmonary indexes compared to control group. Treatment of Sch C showed a significant reduction in the accumulation of ECM as evidenced by decreased expressions of α-SMA, FN, MMP2, MMP9, TIMP1 and collagen proteins such as Col 1A1, and Col 3A1. In addition, the expression of LOX in the lung tissue of mice after Sch C treatment was effectively decreased compared with the MOD group. The inhibition effects in vitro were consistent with those in vivo. Mechanistic studies revealed that Sch C significantly inhibited TGF-β1/Smad2/3 and TNF-α/JNK signaling pathways. In conclusion, our data demonstrated that Sch C significantly ameliorated PF in vivo and vitro, which may play an important role by reducing ECM deposition and inhibiting the production of LOX. [ABSTRACT FROM AUTHOR]

Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation. Given the homology between HOIL-1 and ARIH1, an RBR E3 that functions with the large superfamily of cullin-RING E3 ligases (CRLs), a biochemical investigation was undertaken, showing ARIH1 catalyzes Ser ubiquitylation to CRL-bound substrates. However, the efficiency of ubiquitylation was exquisitely dependent on the location and chemical environment of the Ser residue within the primary structure of the substrate. Comprehensive mutagenesis of the ARIH1 Rcat domain identified residues whose mutation severely impacted both oxyester and isopeptide bond formation at the preferred site for Ser ubiquitylation while only modestly affecting Lys ubiquitylation at the physiological site. The results reveal dual isopeptide and oxyester protein ubiquitylation activities of ARIH1 and set the stage for physiological investigations into this function of emerging importance. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2), which mediates post‐translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used 1H‐NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF‐7 and MDA‐MB‐231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD‐MB‐231, inhibiting transamidation reactions and decreasing cell migration and invasiveness. This metabolomics study provides evidence of a major effect of AA9 on MDA‐MB‐231 cells, impacting glutamate and aspartate metabolism, rather than on MCF‐7 cells, characterised by choline and O‐phosphocholine decrease. Interestingly, AA9 treatment induces myo‐inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer. [ABSTRACT FROM AUTHOR]

Lysyl oxidase-like 4 (LOXL4), a member of lysyl oxidase family, is a copper and lysine tyrosylquinone-dependent amine oxidase that serves the role of catalyzing the cross-linking of elastin and collagen in the extracellular matrix. Numerous studies have shown a significant association between LOXL4 expression levels and tumor proliferation, migration, invasion and patients' prognosis and overall survival in different types of tumors. Here we review their relationship and the molecular pathogenesis behind them, aiming to explore the possibilities of LOXL4 as a prognostic marker for diverse carcinomas and provide some indications for further research in this field. [ABSTRACT FROM AUTHOR]

Streptomyces lividans is an efficient host for extracellular overproduction of recombinant proteins. To enhance the overexpression strength of S. lividans, we designed several kinds of expression plasmids with different positioning of repeat promoters. The effect of repeat promoters was evaluated by measuring the accumulated amounts of a stable transglutaminase or an unstable carboxypeptidase that was secreted into the medium. Successive tandem positions of repeat promoters upstream of the normal promoter did not enhance the expression of transglutaminase. Discrete positions of repeat promoters both upstream and downstream of the normal promoter enhanced the expression of transglutaminase to 2-fold, and the downstream ones also enhanced the expression of carboxypeptidase to 1.7-fold. On the other hand, there were still some constructs of plasmids with discrete repeat promoters that did not promote the expression of the target enzymes, indicating the complexity of the mechanisms of repeat promoters working on gene expression. Discrete repeat promoter either upstream or downstream of the normal promoter enhanced the expression of extracellular enzyme of transglutaminase in Streptomyces lividans. [ABSTRACT FROM AUTHOR]

The aim of this research was to determine the effect of transglutaminase (TGase; 0, 0.3 and 3.0 units/g of protein) crosslinked micellar casein concentrate (MCC) and milk protein concentrate (MPC) on the functionality of process cheese product (PCP) loaf‐type formulation. The results showed that TGase significantly (P ≤ 0.05) increased viscosity after manufacture. Higher TGase levels also significantly (P ≤ 0.05) affected post‐manufacture melt characteristics such as melt viscosity and melt area measured using a rapid visco analyser and Schreiber melt test respectively. The study shows that the TGase treatment alters meltability of PCP. [ABSTRACT FROM AUTHOR]

BACKGROUND: Quinoa protein is enriched with a wide range of amino acids, including all nine essential amino acids necessary for the human body, and in appropriate proportions. However, as the main ingredient of gluten‐free food, it is difficult for quinoa to form a certain network structure for lack of gluten protein. The aim of this work was to enhance the gel properties of quinoa protein. Therefore, the texture characteristics of quinoa protein treated with different ultrasound intensities coupled with transglutaminase (TGase) were investigated. RESULTS: The gel strength of quinoa protein gel increased markedly by 94.12% with 600 W ultrasonic treatment, and the water holding capacity increased from 56.6% to 68.33%. The gel solubility was reduced and free amino content increased the apparent viscosity and the consistency index. Changes in the free sulfhydryl group and hydrophobicity indicated that ultrasound stretched protein molecules and exposed active sites. The enhanced intrinsic fluorescence intensity at 600 W demonstrated that ultrasonic treatment affected the conformation of quinoa protein. New bands emerged in sodium dodecylsulfate–polyacrylamide gel electrophoresis indicating that high‐molecular‐weight polymers were generated through TGase‐mediated isopeptide bonds. Furthermore, scanning electron microscopy showed that the gel network structure of TGase‐catalyzed quinoa protein was more uniform and denser, thereby improving the gel quality of quinoa protein. CONCLUSION: The results suggested that high‐intensity ultrasound combined with TGase would be an effective way to develop higher‐quality quinoa protein gel. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The emulsions prepared by three non-meat proteins, sodium caseinate (SC), soy protein isolate (SPI) and egg white protein (EPI), were individually added to the continuous phase of myofibrillar protein (MP) sol to form MP composite gels to simulate meat products. The research aimed to investigate the effects of Transglutaminase (TGase) on the physicochemical properties, microstructure and water phase distribution of non-meat protein emulsion MP composite gels. The results of this study revealed that TGase played a crucial role in forming a tight gel network structure in the composite gels. This enhanced their ability to retain water and improved their overall gel strength. Additionally, TGase increased the gel formation temperature of myofibrillar proteins. Electrophoresis analysis showed that when catalyzed by TGase, there was a lighter band compared to those not catalyzed by TGase. This indicated that the addition of TGase facilitated cross-linking interactions between meat proteins and non-meat proteins in the composite gels. Furthermore, microscopy observations demonstrated that composite gels treated with TGase exhibited a more uniform microstructure. This could be attributed to an acceleration in relaxation time T2. The uniform network structure restricted the movement of water molecules in the gel matrix, thereby improving its water-holding capacity. Overall, these findings highlight how incorporating non-meat proteins into myofibrillar systems can be effectively achieved through enzymatic treatment with TGase. Such modifications not only enhanced important functional properties but also contributed towards developing alternative meat products with improved texture and moisture retention abilities. [ABSTRACT FROM AUTHOR]

Microbial transglutaminase (mTG) is a bacterial survival factor, frequently used as a food additive to glue processed nutrients. As a result, new immunogenic epitopes are generated that might drive autoimmunity. Presently, its contribution to autoimmunity through epitope similarity and cross-reactivity was investigated. Emboss Matcher was used to perform sequence alignment between mTG and various antigens implicated in many autoimmune diseases. Monoclonal and polyclonal antibodies made specifically against mTG were applied to 77 different human tissue antigens using ELISA. Six antigens were detected to share significant homology with mTG immunogenic sequences, representing major targets of common autoimmune conditions. Polyclonal antibody to mTG reacted significantly with 17 out of 77 tissue antigens. This reaction was most pronounced with mitochondrial M2, ANA, and extractable nuclear antigens. The results indicate that sequence similarity and cross-reactivity between mTG and various tissue antigens are possible, supporting the relationship between mTG and the development of autoimmune disorders 150W. [ABSTRACT FROM AUTHOR]