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Summary: The aim of this study was to investigate the effects of microbial transglutaminase (MTGase) on the textural properties and meltability of vegan cheese formulated using cashew paste, tapioca starch, coconut oil, and pea protein. The parameters were evaluated at various MTGase concentrations (0.1, 0.25, 0.5, 0.75, and 1%) and different incubation times (30, 60, and 90 min). A MTGase concentration of 0.25% resulted in higher meltability Index of 3.02 ± 0.14 and acceptable hardness of 3479.44 ± 14.49 g at 30 min of incubation time. However, prolonged incubation times were found to adversely affect the texture and meltability of the cheese. A similar adverse effect on texture was observed at an MTGase concentration of 0.1% at 60 and 90 min of incubation. The results revealed that lower MTGase concentrations with longer incubation times produced similar effects as higher MTGase concentrations with shorter incubation times. However, at a lower enzyme concentration of 0.1%, meltability was significantly higher (3.24 ± 0.33, P < 0.05) at 60 min of incubation compared to 90 min. The findings suggest that vegan cheese with improved texture, meltability, and sensory properties can be developed by combining cashew, tapioca starch, coconut oil, and pea protein in a ratio of 50:25:20:10, with 1% each of carrageenan and agar, and 60% water, and incubating the mixture with 0.25% MTGase for 30 min at 40 °C. [ABSTRACT FROM AUTHOR]

ABSTRACT This study aimed to assess the effect of Persian gum (PG) and microbial transglutaminase enzyme (MTGase) on some physicochemical and microstructural characteristics of low‐fat ultrafiltrated Iranian white cheese during 60 days of cold storage. To manufacture the low‐fat cheese samples, PG (0%, 0.25%, and 0.5%) and MTGase (0, 0.5 and 1 U/g of protein) were used and cheeses (low‐ and full‐fat) without PG and MTGase were considered as controls. The obtained results revealed that the treated samples with PG had a higher titratable acidity, moisture content, and moisture‐to‐protein ratio (M:P), but a lower protein content as compared to the low‐fat control (p ≤ 0.05). Furthermore, the cheese samples treated with the higher level of MTGase (i.e., 1 U/g of protein) had a higher protein and fat content (p ≤ 0.05). Methyl palmitate (C16:0) and methyl oleate (C18:1) were found to be the most abundant fatty acids in the tested cheeses. Additionally, scanning electron micrographs revealed that addition of PG and MTGase improved the microstructure of the samples, so that the sample containing 0.5 U enzyme and 0.5% gum was more similar to its full‐fat counterpart and obtained the highest overall acceptance score among the cheeses. Therefore, our findings revealed that with incorporation of PG and MTGase, a low‐fat ultrafiltrated white cheese could be produced with an appropriate functional, physicochemical, and microstructural characteristics comparable to the full‐fat product. [ABSTRACT FROM AUTHOR]

ABSTRACT Bioactive peptides and protein hydrolysates have gained considerable attention in the food industry and functional food markets due to their diverse health effects, including antioxidant, antihypertensive, and antidiabetic properties. This study aimed to produce combined soy and corn protein hydrolysates using Alcalase (Al), modification of Al‐hydrolysates through sequential hydrolysis using Flavourzyme (Al‐FL), cross‐linking of Al‐hydrolysates using microbial transglutaminase (MTGase) (Al‐TG), and fractionation of Al‐hydrolysates by ultrafiltration (UF) with molecular weight (MW) cut‐off of 100 (Al‐F4), 30 (Al‐F3), 10 (Al‐F2), and 2 kDa (Al‐F1). Notably, the < 2 kDa fraction (Al‐F1) showcased exceptional biological activities, including antioxidant (81.54% DPPH, 98.02% ABTS), antihypertensive (95.45%), and antidiabetic effects (44.72% α‐glucosidase, 77.52% α‐amylase), linked to its high hydrophobic amino acid content and low molecular weights (111 and 263 Da). Conversely, the higher molecular weight fraction (Al‐TG) excelled in emulsion and foam stability, attributed to its balanced amino acid profile and larger peptides (1385–7057 Da). Our findings reveal that specific protein hydrolysate fractions, particularly Al‐F1 and Al‐TG, are promising for applications in food and pharmaceutical formulations due to their enhanced biological and functional properties. [ABSTRACT FROM AUTHOR]

During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the "mispairing problem." Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce "bsAb by external pairing and excision" (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells. [ABSTRACT FROM AUTHOR]

Summary: Background: In coeliac disease and environmental enteropathy, dietary gluten and enteric infections cause reversible inflammation and morphological changes to the small intestinal mucosa that can be detected in biopsy samples obtained by endoscopy. However, there is a clear need for non‐invasive biomarkers. Constant shedding of mucosal material into the bowel lumen and faeces, together with easy availability of stool, makes it an interesting sample matrix. Aims: To conduct a systematic literature search and summarize the existing evidence for host mucosa‐derived faecal biomarkers in evaluating small intestinal damage. Methods: We searched for studies on PubMed (MEDLINE) until 1 March 2024. Results: We identified 494 studies and included 35 original case–control and cohort studies. These assessed host mucosal transcripts and 14 other markers aiming specifically to reflect inflammation and cell‐mediated, innate and gluten‐induced immune responses. In coeliac disease, faecal calprotectin and anti‐gliadin, tissue transglutaminase, endomysium and deamidated gliadin peptide antibodies were the most studied but with inconsistent results. Single studies reported positive findings about microRNA transcripts, β‐defensin‐2, lipocalin‐2, zonulin‐related proteins and angiotensin‐converting enzyme. In environmental enteropathy, a non‐significant association was reported between calprotectin and urine lactulose/mannitol ratio; there were conflicting results for neopterin, myeloperoxidase and host transcripts. Single studies reported a positive association for lactoferrin, and a negative association for regenerating islet‐derived protein 1. Studies comparing faecal markers against small intestinal biopsy findings were not identified in environmental enteropathy. Conclusions: Further studies are needed to determine reliable faecal markers as a proxy for small intestinal mucosal damage. [ABSTRACT FROM AUTHOR]

Transglutaminase enzymes catalyze Ca2+- and thiol-dependent posttranslational modifications of glutamine-residues that include esterification, hydrolysis and transamidation, which results in covalent protein–protein crosslinking. Among the eight transglutaminase family members in mammals, transglutaminase 1 (TG1) plays a crucial role in skin barrier formation via crosslinking and insolubilizing proteins in keratinocytes. Despite this established function in skin, novel functions have begun merging in normal tissue homeostasis as well as in pathologies. This review summarizes our current understanding of the structure, activation, expression and activity patterns of TG1 and discusses its putative novel role in other tissues, such as in vascular integrity, and in diseases, such as cancer and fibrosis. [ABSTRACT FROM AUTHOR]

Transglutaminases are enzymes that carry out post-translational modifications of proteins and participate in the regulation of their activities. Here, we show for the first time that the transglutaminase genes in the basal metazoan, the sea sponge Halisarca dujardinii, are organized in a cluster, similarly to mammalian transglutaminases. The regulatory regions of six transglutaminase genes and their differential expression in the course of the life cycle of H. dujardinii suggest independent regulation of these genes. The decrease in transglutaminase activities by cystamine facilitates restoration of the multicellular structures of this sponge after its mechanical dissociation. For the first time we observed that this decrease in transglutaminase activities was accompanied by generation of the reactive oxygen species in the cells of a basal metazoan. The study of transglutaminases in the basal metazoans and other sea-dwelling organisms might provide better understanding of the evolution and specific functions of these enzymes in higher animals. [ABSTRACT FROM AUTHOR]

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T50 (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T50 of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (kcat/Km) at 37.0 °C was increased from 3.3 × 102 M−1 s−1 for the wild type to 5.9 × 102 M−1 s−1. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency. Key points: • Improved heat resistance is essential to broaden the application of MTG. • The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability. • X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated. [ABSTRACT FROM AUTHOR]

ABSTRACT In this study, rainbow trout patties (Oncorhynchus mykiss Walbaum, 1792) were treated under a modified atmosphere (MA) (60/40:CO2/N2) packed with microbial transglutaminase enzyme (MTGase) at varying concentrations (0.5% and 1%) and kept for a full day at 2°C ± 2°C. Denser and more complex intermolecular cross‐linking formations between peptides and gel network structures were observed in the first and last scanning electron microscope images of MTGase‐containing groups. MTGase increased the springiness, hardness, shear force, and work‐of‐shear parameters during the storage period in uncooked and cooked patties compared to the control group. The protein, energy, moisture, and amino acid contents were maintained in the MTGase‐containing groups, but using MTGase at higher concentrations was ineffective. Microbiological analyses have shown that MTGase is partially effective on microbial fauna, and the most significant inhibitory effect was determined on total mesophilic aerobic bacteria (TMAB). In all groups, the total amounts of saturated, monosaturated, and polyunsaturated fatty acids remained unchanged on both production and expiration days (p > 0.05). After mixing the burger patty additives into the ground meat, the amounts of sodium, calcium, potassium, and magnesium increased in all groups (p < 0.05); however, a decrease in calcium was observed in the MTGase‐containing groups (p < 0.05) compared to the control group. The higher proportion of the MTGase‐contained group did not exhibit significant differences compared to the lower proportion of the MTGase‐contained group, except for textural characteristics and the TMAB inhibition effect. [ABSTRACT FROM AUTHOR]

Summary: Background: The European Society of Paediatric Gastroenterology, Hepatology and Nutrition established guidelines in 2012 for a no‐biopsy approach to diagnose coeliac disease in children. This guideline required symptoms suggestive of coeliac disease, positive human leukocyte antigen (HLA) DQ2/DQ8 haplotypes, tissue transglutaminase type‐2 immunoglobulin A antibody titre at levels greater than 10 times the upper limit of normal, and positive endomysial immune‐globulin A antibody test. An updated 2020 guideline excluded the need for symptoms and positive HLA. Aims: To assess the pooled positive predictive value (PPV) of the no‐biopsy approach with small bowel biopsy (SBB) data as the reference standard for comparison. Methods: Database searches (October 2023) provided data that we combined using a random‐effects meta‐analysis to provide a pooled PPV, representing the probability that a positive test result means that an individual truly has the condition. Results: We included 23 studies. Study sample sizes totalled 23,769 but only 3007 children had comparative SBB. The proportion of coeliac disease confirmed by the no‐biopsy approach and SBB ranged from 79.2% to 100%, with an overall pooled PPV of 97.4% (95% confidence interval 96.0, 98.6). Sensitivity analysis showed higher PPV for the criteria that included HLA (98.5% vs. 96.8%; p = 0.017). Conclusion: Both no‐biopsy criteria exhibit high PPV when compared to the reference standard. These results provide a consistent message of accuracy and feasibility to inform change and improve outcomes. [ABSTRACT FROM AUTHOR]

Next to the choice of a material, the crosslinking characteristics play an important role in the development of hydrogels for applications in the biomedical field. By decreasing crosslinking time or concentration one can reduce the density of the network and hence the stiffness of the hydrogel. However, at the same time in many cases the stability of the matrix is significantly reduced, leading to a rapidly degrading hydrogel structure. In this study, we evaluate and compare two different crosslinking methods while maintaining the material chemistry. The hydrogel system evaluated consisted of oxidized hyaluronic acid and gelatin, and it was crosslinked with microbial transglutaminase. We found that by in situ crosslinking (CL) instead of post CL we can significantly increase the stability of the hydrogel while maintaining the matrix stiffness. Encapsulated embryonic mouse fibroblasts showed an increased viability in in situ crosslinked samples. These findings indicate that it is possible to change hydrogel parameters independently, even if they are otherwise interrelated. [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of proline- and glutamine-rich proteins, widely termed "gluten", in genetically susceptible individuals. CD induces an altered immune response that leads to chronic inflammation and duodenal mucosal damage. Currently, there are no specific tests for the accurate diagnosis of CD, and no drugs are available to treat this condition. The only available treatment strategy is lifelong adherence to a gluten-free diet. However, some studies have investigated the involvement of microRNAs (miRNAs) in CD pathogenesis. miRNAs are small noncoding ribonucleic acid molecules that regulate gene expression. Despite the growing number of studies on the role of miRNAs in autoimmune disorders, data on miRNAs and CD are scarce. Therefore, this study aimed to perform a literature review to summarize CD, miRNAs, and the potential interactions between miRNAs and CD in adults. This review shows that miRNA expression can suppress or stimulate pathways related to CD pathogenesis by regulating cell proliferation and differentiation, regulatory T-cell development, innate immune response, activation of the inflammatory cascade, focal adhesion, T-cell commitment, tissue transglutaminase synthesis, and cell cycle. Thus, identifying miRNAs and their related effects on CD could open new possibilities for diagnosis, prognosis, and follow-up of biomarkers. [ABSTRACT FROM AUTHOR]

In the previous work by the same authors, optimisation of sequential application of acid protease and transglutaminase was carried out to achieve Woolmark specification AW-1 for the machine washable care claim, that is, less than 3% total shrinkage. As a result of optimisation, the total warp shrinkage reduced to 2.64% with only 2.82% loss in warp tensile strength. In this work, simultaneous application of acid protease and transglutaminase was explored and optimised using Box–Behnken design of experiment. The optimisation was carried out using two criteria. In the first criterion, the model was used to predict treatment parameters for minimum total warp shrinkage with minimum loss in warp tensile strength. Taking these predicted treatment parameters, wool was treated with acid protease (1%, owf) and transglutaminase (2.8%, owf) at pH ∼5.3 and 50°C for 60 min. After treatment, the total warp shrinkage reduced to only 4.32% with 4.65% loss in warp tensile strength. This was not only higher than the optimised sequential application of both the enzymes but also failed to meet the Woolmark specification for machine washable care claim. So, the second criterion was used in which the prediction was taken for treatment parameters to achieve the minimum total warp shrinkage without any constraints on loss in warp strength. Again, taking these predicted treatment parameters, wool was treated with acid protease (1.3%, owf), transglutaminase (1.6%, owf), pH ∼4.7 at 55°C for 85 min. Under these treatment conditions, the total warp shrinkage reduced to 2.89% with 10.1% loss in warp strength. Furthermore, interaction between the enzymes also plays a role in the outcomes of the treatment. It was evident that there was a loss in transglutaminase activity as a result of acid protease digestion. However, there was no loss in the activity of acid protease, indicating that transglutaminase did not have any negative influence on the activity of acid protease. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fried foods are favored for their unique crispiness, golden color and flavor, but they also face great challenge because of their high oil content, high calories and the existence of compounds such as acrylamide and polycyclic aromatic hydrocarbons. Long‐term consumption of fried foods may adversely affect health. Therefore, it is necessary to explore fried foods with lower oil contents and a high quality to meet the demand. RESULTS: A method of enzyme treatment was explored to investigate the effects of maltogenic amylase (MA), transglutaminase (TG) and bromelain (BRO) on the physicochemical properties of the batter and the quality of fried spring roll wrapper (FSRW). The results showed that the MA‐, TG‐ or BRO‐treated batters had a significant shear‐thinning behavior, especially with an increase in viscosity upon increasing TG contents. FSRW enhanced its fracturability from 419.19 g (Control) to 616.50 g (MA‐6 U g−1), 623.49 g (TG‐0.75 U g−1) and 644.96 g (BRO‐10 U g−1). Meanwhile, in comparison with BRO and MA, TG‐0.5 U g−1 endowed batter with the highest density and thermal stability. MA‐15 U g−1 and TG‐0.5 U g−1 displayed FSRW with uniform and dense pores, and significantly reduced its oil content by 18.05% and 25.02%, respectively. Moreover, compared to MA and TG, BRO‐50 U g−1 improved the flavor of FSRW. CONCLUSION: MA, TG or BRO played a key role in affecting the physicochemical properties of the batter and the quality of FSRW. TG‐0.5 U g−1 remarkly reduced the oil content of FSRW with a great potential in practical application. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state. A structural study of the protein crosslinking enzyme tissue transglutaminase (TG2), combining time-resolved SAXS and cryo-EM approaches, details the mechanisms by which TG2 responds to activating calcium ions or to a small-molecule inhibitor. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma is a very lethal tumor entity with the late appearance of clinical signs and the absence of biomarkers for early diagnosis. Diabetes mellitus can increase the risk of pancreatic cancer, as well as the effect of treatment modalities, so patients with PDAC and DM represent a special study group. We validated miRNA1285-3p, TGM2, CDH-1, CD166, and S100A13 as potential meaningful biomarkers to characterize patients with PDAC + DM. The present study represents a pilot approach to address this question. The potential biomarkers presented in the current study could contribute to the search for biomarkers for the diagnosis of PDAC, enabling early detection of PDAC in DM patients in the future. Early detection of PDAC remains challenging due to the lack of early symptoms and the absence of reliable biomarkers. The aim of the present project was to identify miRNA and proteomics signatures discriminating PDAC patients with DM from nondiabetic PDAC patients. Proteomics analysis and miRNA array were used for protein and miRNA screening. We used Western blotting and Real-Time Quantitative Reverse Transcription polymerase chain reaction (qRT-PCR) for protein and miRNA validation. Comparisons between experimental groups with normal distributions were performed using one-way ANOVA followed by Tukey's post hoc test, and pairwise tests were performed using t-tests. p ≤ 0.05 was considered statistically significant. Protein clusters of differentiation 166 (CD166), glycoprotein CD63 (CD63), S100 calcium-binding protein A13 (S100A13), and tumor necrosis factor-β (TNF-β) were detected in the proteomics screening. The miRNA assay revealed a differential miRNA 1285 regulation. Previously described target proteins of miR-1285 cadherin-1 (CDH-1), cellular Jun (c-Jun), p53, mothers against decapentaplegic homolog 4 (Smad4), human transglutaminase 2 (TGM2) and yes-associated protein (YAP), were validated via Western blotting. miR-1285-3p was successfully validated as differentially regulated in PDAC + DM via qRT-PCR. Overall, our data suggest miRNA1285-3p, TGM2, CDH-1, CD166, and S100A13 as potential meaningful biomarker candidates to characterize patients with PDAC + DM. Data are available via ProteomeXchange with the identifier PXD053169. [ABSTRACT FROM AUTHOR]

This study explored the changes in cross-linking of low-salt myosin mediated by microbial transglutaminase (MTGase) under high intensity ultrasound (HIU). HIU activated the fish endogenous transglutaminase by 14.11%. Meanwhile, HIU induced the conformational changes in low-salt myosin with decrease of α-helix and increase of β-sheet structures. At 0.1 mol/L salt, compared to solely treated MTGase/solely treated myosin, the most ε-(γ-Glu)-Lys bonds were formed in HIU-treated MTGase + myosin, evidenced by the lowest solubility. Correspondingly, the G' showed the highest increase rate. Consequently, the samples formed regular and continuous protein matrix structures. Specifically, the microstructures of HIU-treated MTGase + myosin samples at 0.1 mol/L salt were even more compact than those of control samples at 0.3 mol/L salt. Besides, HIU-treated MTGase + myosin improved the water holding capacity by 18.28%. In summary, HIU promoted the cross-linking between low-salt myosin mediated by MTGase through activating the enzyme, improving substrate dispersion, or both, with the largest improvement degree observed for HIU-treated MTGase + myosin group. [ABSTRACT FROM AUTHOR]

There is no effective and noninvasive solution for thrombolysis because the mechanism by which certain thrombi become tissue plasminogen activator (tPA)-resistant remains obscure. Endovascular thrombectomy is the last option for these tPA-resistant thrombi, thus a new noninvasive strategy is urgently needed. Through an examination of thrombi retrieved from stroke patients, we found that neutrophil extracellular traps (NETs), ε-(γ-glutamyl) lysine isopeptide bonds and fibrin scaffolds jointly comprise the key chain in tPA resistance. A theranostic platform is designed to combine sonodynamic and mechanical thrombolysis under the guidance of ultrasonic imaging. Breakdown of the key chain leads to a recanalization rate of more than 90% in male rat tPA-resistant occlusion model. Vascular reconstruction is observed one month after recanalization, during which there was no thrombosis recurrence. The system also demonstrates noninvasive theranostic capabilities in managing pigs' long thrombi (>8 mm) and in revascularizing thrombosis-susceptible tissue-engineered vascular grafts, indicating its potential for clinical application. Overall, this noninvasive theranostic platform provides a new strategy for treating tPA-resistant thrombi. There is no noninvasive solution for tPAresistant thrombi as the mechanism remains obscure. Here, the authors show a constructed ultrasound-responsive theranostic platform for real-time monitoring and efficient thrombolysis by breaking the keychain in tPA-resistant thrombi. [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]

Transglutaminase (TGase) is a key enzyme that mediates hemolymph coagulation and is thought to contribute to the elimination of pathogenic microorganisms in invertebrates. The objective of this study was to elucidate the involvement of TGase in insect immune responses via functional analysis of this enzyme in the oriental armyworm, Mythimna separata, using recombinant proteins and RNA interference technique. We identified two TGase genes, mystgase1 and mystgase2, in Mythimna separata and found that both genes are expressed in all surveyed tissues in M. separata larvae. Significant changes were induced in hemocytes following Escherichia coli injection. Injection of Gram‐positive bacteria (Micrococcus luteus) and Gram‐negative bacteria (Escherichia coli and Serratia marcescens) into larvae triggered a time‐specific induction of both mystgase1 and mystgase2 in hemocytes. Recombinant MysTGase1 and MysTGase2 proteins bound to both E. coli and M. luteus, localizing within bacterial clusters and resulting in agglutination in a Ca2+‐dependent manner. The hemocytes of larvae injected with recombinant MysTGase1 or MysTGase2 exhibited enhanced phagocytic ability against E. coli, improved in vivo bacterial clearance, and increased resistance to S. marcescens, decreasing larval mortality rate. Conversely, RNA interference targeting mystgase1 or mystgase2 significantly reduced hemocyte phagocytic capability, decreased bacterial clearance, and increased susceptibility to S. marcescens infection, thereby increasing larval mortality rate. The findings of this study are anticipated to expand our understanding of the function of TGases within insect immune responses and may contribute to developing new pest control strategies. [ABSTRACT FROM AUTHOR]

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction. [ABSTRACT FROM AUTHOR]

Summary: Phytoproteins have been widely implemented in the food industry benefiting from their good functionalities, vast reserves and relatively low cost. However, phytoproteins frequently experience poor solubility, which limits their applications. In our study, the solubility of soy protein isolate (SPI) treated with pH‐shifting increased over 1.8 times; however, the transglutaminase‐induced gel properties were sacrificed. To solve this problem, walnut protein (WP) was used to co‐assemble with SPI during the treatment. The co‐assembly hybrid protein shows significantly better solubility, meanwhile, maintaining the transglutaminase‐induced gel properties and improving the cohesiveness and resilience properties compared with untreated SPI, which behaved an improved water holding capacity during storage. The detailed mechanisms were investigated with rheological analysis, SEM, textural analysis and Fourier transform infrared spectroscopy (FTIR). Overall, our study provided a promising strategy to develop SPI/WP hybrid composites with high solubility and improved gel properties to extend the application of SPI in the food industry. [ABSTRACT FROM AUTHOR]

Summary: Protein glycosylation modification is an effective way to enhance the functional properties of natural proteins. This study aims to explore the preparation of glycosylation products (PPCs) through the Maillard reaction or enzymatic glycosylation using transglutaminase. The study discusses the advantages and disadvantages of preparing PPCs using dry heat, wet heat, and enzymatic glycosylation, as well as factors influencing the glycosylation process. Compared to natural proteins, PPCs demonstrate superior functional properties, including solubility, viscosity, emulsifying ability, gelling ability, thermal stability, antioxidant activity, and antibacterial activity. With improved functional performance, PPCs have broad application prospects as raw materials in food processing. For example, PPCs can be used as antioxidants, emulsifiers, gelling agents, and carriers for various bioactive molecules in the food industry. In the pharmaceutical field, PPCs are expected to serve as contrast agents and dressings. Furthermore, the application of PPCs in food processing should also consider specific processing requirements to achieve the intended enhancement of glycosylated protein functionality. [ABSTRACT FROM AUTHOR]

Ubiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i. e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2 K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein. [ABSTRACT FROM AUTHOR]

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]

The microvascular network plays an important role in providing nutrients to the injured tissue and exchanging various metabolites. However, how to achieve efficient penetration of the injured tissue is an important bottleneck restricting the reconstruction of microvascular network. Herein, the hydrogel precursor solution can efficiently penetrate the damaged tissue area, and ultrasound triggers the release of thrombin from liposomes in the solution to hydrolyze fibrinogen, forming a fibrin solid hydrogel network in situ with calcium ions and transglutaminase as catalysts, effectively solving the penetration impedance bottleneck of damaged tissues and ultimately significantly promoting the formation of microvascular networks within tissues. First, the fibrinogen complex solution is effectively permeated into the injured tissue. Second, ultrasound triggered the release of calcium ions and thrombin, activates transglutaminase, and hydrolyzes fibrinogen. Third, fibrin monomers are catalyzed to form fibrin hydrogels in situ in the damaged tissue area. In vitro studies have shown that the fibrinogen complex solution effectively penetrated the artificial bone tissue within 15 s after ultrasonic triggering, and formed a hydrogel after continuous triggering for 30 s. Overall, this innovative strategy effectively solved the problem of penetration resistance of ultrasound‐triggered hydrogels in the injured tissues, and finally activates in situ microvascular networks regeneration. [ABSTRACT FROM AUTHOR]

Adenine diphosphate (ADP)‐ribosylation catalyzed by bacterial toxins is the addition of an ADP‐ribose moiety to specific amino acid residues in target proteins such as arginine, asparagine, glutamine, and histidine through 1,2‐cis(α)‐glycosidic bond formation. ADP‐ribosylation modifies host cell functions, altering normal cellular processes to facilitate their survival, and replication. Despite the development of click chemistry and solid‐phase peptide synthesis‐based approaches, the synthesis of structurally well‐defined naturally occurring N‐linked ADP‐ribosyl molecules remains challenging. Herein, we report a direct and α‐selective N‐ADP‐ribosylation using unprotected β‐ADP‐ribosyl azide and triphenylphosphine esters through traceless Staudinger ligation. The stereoselectivity of this protecting‐group‐free N‐ADP‐ribosylation was validated by enzymatic degradation experiment and high‐performance liquid chromatography, referencing synthetic standards. This method facilitated the facile and rapid synthesis of N‐ADP‐ribosyl acetamide, biotin, fluorescent dye, amino acids, and tripeptide with complete α‐selectivity and moderate yields (37–55 %). [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is an autoimmune disease of the small intestine triggered by the consumption of gluten-containing foods in individuals with a genetic predisposition. CD was a rare disease until 20 years ago, when the prevalence increased. Currently, there is no data on the prevalence of CD in high-risk adult populations in Indonesia, even though there is a trend of increasing gluten consumption. Therefore, basic research is needed to determine the magnitude of CD in high-risk adult patients in Indonesia while identifying clinical signs/symptoms, illness history, and lifestyle to determine factors associated with CD in Indonesia. This study is an observational study with a cross-sectional method.Two hundred eighty-three patients who fulfilled the selection and signed the informed consent were recruited from the gastroenterology clinic of Dr. Cipto Mangunkusumo General Hospital. Patients were asked to fill out a celiac disease-related questionnaire and then given anthropometry measurement and blood test for serologic examination with ELISA, consisting of IgA anti-tissue transglutaminase (anti-TTG) and IgG anti-deaminated gliadin peptide (anti-DGP). Statistical analysis was performed using Chi-square and Multivariate logistic regression tests with SPSS software ver. 26. Statistical test differences were considered significant if the p-values were < 0.05. Eight of 283 patients are serologically confirmed with CD (2,83%). On bivariate analysis, the significant variables are age (p < 0,05), constipation (p < 0,05) and history of autoimmune disease (p < 0,05). On multivariate analysis, the only significant variable is the history of autoimmune disease (p < 0,05). This study concluded that the prevalence of CD in high-risk patients with functional gastrointestinal disorder at Dr. Cipto Mangunkusumo Hospital is relatively high (2.83%). CD-associated factors are age, constipation, and history of autoimmune disease in patients. On simultaneous interaction between these factors, autoimmune is the only significant variable associated with CD. [ABSTRACT FROM AUTHOR]

Transglutaminase (EC 2.3.2.13, TGase), an enzyme that catalyzes the formation of covalent cross-links between protein or peptide molecules, plays a critical role in commercial food processing, medicine, and textiles. TGase from Streptomyces is the sole commercial enzyme preparation for cross-linking proteins. In this study, we revealed that the SOS response repressor protein LexA in Streptomyces mobaraensis not only triggers morphological development but also enhances TGase synthesis. The absence of lexA significantly diminished TGase production and sporulation. Although LexA does not bind directly to the promoter region of the TGase gene, it indirectly stimulates transcription of the tga gene, which encodes TGase. Furthermore, LexA directly enhances the expression of genes associated with protein synthesis and transcription factors, thus favorably influencing TGase synthesis at both the transcriptional and posttranscriptional levels. Moreover, LexA activates four crucial genes involved in morphological differentiation, promoting spore maturation. Overall, our findings suggest that LexA plays a dual role as a master regulator of the SOS response and a significant contributor to TGase regulation and certain aspects of secondary metabolism, offering insights into the cellular functions of LexA and facilitating the strategic engineering of TGase overproducers. [ABSTRACT FROM AUTHOR]

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders. [ABSTRACT FROM AUTHOR]

Mastitis-causing bacteria can establish persistent infections in the mammary glands of commercially important dairy animals despite the presence of strong specific humoral and cellular immune mechanisms. We investigated the effect of vitamin D3 in the diet at a set level, but in two different forms (i.e., unencapsulated and encapsulated by complex coacervation with sulfur-saturated bovine lactoferrin-alginate using microbial transglutaminase-catalyzed crosslinking) on the immune response in late-lactating dairy goats. Dairy goats (n = 18) were randomly assigned to three experimental groups (n = 6). Dairy goats were orally administered 0.35 mg of vitamin D3/day in the unencapsulated form and 0.35 mg of vitamin D3/day in the encapsulated powder form. Another group received the basal diet. The experimental period lasted 6 weeks. The blood serum concentrations of 25-hydroxyvitamin D3 [25-(OH)-D3], lactoferrin, immunoglobulin A (IgA), and interferon-gamma (INF-γ) were measured. There were major differences in these parameters between dietary groups. However, the delivery of vitamin D3 in the encapsulated powder form to dairy goats resulted in a marked increase in 25-(OH)-D3 concentration in serum, while the serum level of lactoferrin also increased. Alternatively, the serum levels of IgA and the immunomodulatory cytokine INF-γ were elevated following supplementation with the encapsulated vitamin D3. The observed effects suggest that the deliverable form of dietary vitamin D3 results in differences in the immune response in late-lactating dairy goats. [ABSTRACT FROM AUTHOR]

Background: Filaggrin (FLG) is an essential protein that plays a vital role in maintaining skin barrier function and moisture levels, allowing the skin to adapt to dry environments. However, the precise temporal dynamics of FLG metabolism in the human epidermis remain poorly understood, and suitable tools to study these time‐dependent effects are currently lacking. Objective: To investigate the molecular mechanisms and time course of FLG metabolism and skin barrier function under high‐ and low‐humidity conditions, utilizing a reconstructed epidermis model. Methods: EpiSkin specimens cultured under humid or dry conditions for varying durations (2–48 h) were compared by assessing FLG degradation and skin barrier formation using immunofluorescence staining and western blotting. Results: Under conditions of low humidity, the proteolysis of FLG in EpiSkin increased between 4 and 12 h and was accompanied by elevated levels of cysteine–aspartic protease (caspase)‐14. The expression of peptidyl arginine deiminase 1 and calpain 1 also increased at 4 h. However, after 24 h, the expression of these three FLG‐degrading proteins significantly decreased. Conversely, the levels of pyrrolidone‐5‐carboxylic acid and urocanic acid initially decreased at 2 h and then increased between 12 and 24 h. Additionally, the expression of skin barrier proteins, such as FLG, transglutaminase 5, loricrin and zonula occludens‐1, decreased starting from 12 h. Notably, epidermal cell viability and activity were also inhibited. Conclusion: We propose a reliable and ethical model to study the temporal dynamics of FLG metabolism and its role in skin barrier function. Using a commercially reconstructed epidermis to mimic dry skin formation obviates the need for animal and human testing. [ABSTRACT FROM AUTHOR]

Nε -lysine acetylation is recognized as a prevalent post-translational modification (PTM) that regulates proteins across all three domains of life. In Bacillus subtilis, the histone-like protein HBsu is acetylated at seven sites, which regulates DNA compaction and the process of sporulation. In Mycobacteria, DNA compaction is a survival strategy in response antibiotic exposure. Acetylation of the HBsu ortholog HupB decondenses the chromosome to escape this drug-induced, non-growing state, and in addition, regulates the formation of drug-tolerant subpopulations by altering gene expression. We hypothesized that the acetylation of HBsu plays similar regulatory roles. First, we measured nucleoid area by fluorescence microscopy and in agreement, we found that wild-type cells compacted their nucleoids upon kanamycin exposure, but not exposure to tetracycline. We analyzed a collection of HBsu mutants that contain lysine substitutions that mimic the acetylated (glutamine) or unacetylated (arginine) forms of the protein. Our findings indicate that some level of acetylation is required at K3 for a proper response and K75 must be deacetylated. Next, we performed time-kill assays of wild-type and mutant strains in the presence of different antibiotics and found that interfering with HBsu acetylation led to faster killing rates. Finally, we examined the persistent subpopulation and found that altering the acetylation status of HBsu led to an increase in persister cell formation. In addition, we found that most of the deacetylation-mimic mutants, which have compacted nucleoids, were delayed in resuming growth following removal of the antibiotic, suggesting that acetylation is required to escape the persistent state. Together, this data adds an additional regulatory role for HBsu acetylation and further supports the existence of a histone-like code in bacteria. [ABSTRACT FROM AUTHOR]

This study investigated the effects of high-pressure processing (HPP; 600 MPa/15 min) and microbial transglutaminase-catalyzed (MTG; 30 U·g of protein−1) crosslinking on the concentration of dissolved proteins (SOL), free sulfhydryl groups (SH), surface hydrophobicity (H0), and viscosity of pea protein isolates (PPI) at different concentrations (1–13%; w/v). The SOL increased by increasing protein concentration (max. 29%). MTG slightly affected SOL. HPP decreased SOL with increasing protein concentration, and the combination MTG + HPP resulted in a lower SOL than HPP alone. The concentration of SH in untreated PPI increased with increasing protein concentration, reaching a maximum of 8.3 μmol·mg prot−1. MTG increased SH at higher protein concentrations. HPP lowered SH, but its concentration increased by increasing protein concentration. HPP + MTG offset the effect of MTG, yielding lower SH. MTG did not affect H0 at 1% concentration but increased it for concentrations from 3–5%, and there was a decrease with 7–9%. HPP increased H0 up to 37% for intermediate protein concentrations but did not affect it at higher concentrations. MTG + HPP decreased H0 at all protein concentrations. The viscosity of the dispersions increased with protein concentration. HPP increased the viscosity of the dispersions for concentrations above 7%, while MTG only caused changes above 9%. Combined MTG + HPP resulted in viscosity increase. The results underscore the opportunity for innovative development of high-protein products with improved properties or textures for industrial application. [ABSTRACT FROM AUTHOR]

Transglutaminases (TGMs) cross-link proteins by introducing covalent bonds between glutamine and lysine residues. These cross-links are essential for epithelial cornification which enables tetrapods to live on land. Here, we investigated which evolutionary adaptations of vertebrates were associated with specific changes in the family of TGM genes. We determined the catalog of TGMs in the main clades of vertebrates, performed a comprehensive phylogenetic analysis of TGMs, and localized the distribution of selected TGMs in tissues. Our data suggest that TGM1 is the phylogenetically oldest epithelial TGM, with orthologs being expressed in the cornified teeth of the lamprey, a basal vertebrate. Gene duplications led to the origin of TGM10 in stem vertebrates, the origin of TGM2 in jawed vertebrates, and an increasing number of epithelium-associated TGM genes in the lineage leading to terrestrial vertebrates. TGM9 is expressed in the epithelial egg tooth, and its evolutionary origin in stem amniotes coincided with the evolution of embryonic development in eggs that are surrounded by a protective shell. Conversely, viviparous mammals have lost both the epithelial egg tooth and TGM9. TGM3 and TGM6 evolved as regulators of cornification in hair follicles and underwent pseudogenization upon the evolutionary loss of hair in cetaceans. Taken together, this study reveals the gain and loss of vertebrate TGM genes in association with the evolution of cornified skin appendages and suggests an important role of TGM9 in the evolution of amniotes. [ABSTRACT FROM AUTHOR]

Gel microparticles are tiny, soft particles made from proteins or polysaccharides using particle size reduction. In creating texture-modified food and thickened liquids, gel microparticles are frequently employed to customise the rheological and textural features of foods or thin liquids. Gel microparticles aid by lowering flow behaviour, and modifying the taste perception of the texture-modified food. By cross-linking fish gelatine with the microbial transglutaminase enzyme (mTGA) using the homogenisation process, fish gelatine has tremendous potential to be used to create gel microparticles. The size measurements, texture profile, viscoelastic characteristics, and impact of sugar content on the texture profile of gel microparticles were all examined in the present work for the developed gel microparticles. The results of a particle size investigation revealed that gel microparticle sizes, which ranged from 18 to 1445 μm, increased with higher enzyme concentrations. With increasing mTGA concentration, the texture profile analysis (TPA) also revealed increasing values for hardness, adhesiveness, springiness, cohesiveness, gumminess, and chewiness parameters. The cross-linked gel microparticles had higher dominance on elastic behaviour than viscous behaviour from the rheological investigation, as evidenced by a higher G' compared to G". Additionally, larger TPA values were seen as the sugar concentration increased. These outcomes are anticipated because sugar will strengthen fish gelatine’s hydrogen bond. Based on all completed analyses, fish gelatine cross-linked with 0.7% mTGA to create gel microparticles has shown encouraging results, which can be used as a thickener in texture-modified food to aid those who have trouble swallowing. [ABSTRACT FROM AUTHOR]

Silver Carp (SC) is an under-utilized, invasive species in North American river systems. In this study, the synergistic effects of manufactured Microfiber (MMF), Transglutaminase (TG), and chicken skin collagen (CLG)) to enhance surimi gel quality from frozen SC were studied. The gel strength, textural properties, rheological properties, water-holding capacity (WHC), water mobility, microstructure, and protein composition of the gel samples were determined to assess the impact of the additives individually and synergistically. The results suggested that TG had the most pronounced effect on the surimi gel properties by promoting protein cross-linking. Synergistic effects between TG, MMF, and CLG can bring effective gel property enhancement larger than the individual effect of each additive alone. With the established response-surface models, the combination of CLG and MMF can be optimized to produce surimi gels with less TG but comparable in properties to that of the optimal result with high TG usage. The findings of this study provided a technical foundation for making high-quality surimi gel products out of frozen-stored SC with synergistic utilization of additives, which could serve as guidelines for the industrial development of new surimi products. [ABSTRACT FROM AUTHOR]

A pathway frequently altered in cancer is glutaminolysis, whereby glutaminase (GA) catalyzes the main step as follows: the deamidation of glutamine to form glutamate and ammonium. There are two types of GA isozymes, named GLS and GLS2, which differ considerably in their expression patterns and can even perform opposing roles in cancer. GLS correlates with tumor growth and proliferation, while GLS2 can function as a context-dependent tumor suppressor. However, both isoenzymes have been described as essential molecules handling oxidant stress because of their involvement in glutathione production. We reviewed the literature to highlight the critical roles of GLS and GLS2 in restraining ROS and regulating both cellular signaling and metabolic stress due to their function as indirect antioxidant enzymes, as well as by modulating both reductive carboxylation and ferroptosis. Blocking GA activity appears to be a potential strategy in the dual activation of ferroptosis and inhibition of cancer cell growth in a ROS-mediated mechanism. [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]

In recent years, protein nanoparticles have been well considered in the production of Pickering emulsions and low-calorie foods, but few of them have been introduced. Therefore, finding new sources of nanoparticles and modifying their physical and thermal properties is very important. In this paper, the effect of various levels of microbial transglutaminase (mTG) (0–30 μg/g of sample) and ultrasound time (0–20 min) on properties of nanoparticles prepared with Amaranth Protein Isolate (API) were investigated and optimal levels were achieved by Response surface methodology. Based on the data analysis, ultrasound had a significant effect on tested properties, so that with an increase in ultrasound time, the particle size became smaller. Excluding Zeta potential, all nanoparticles properties were influenced by the interaction of ultrasound and mTG. The enzyme had a great impact (P < 0.001) on Nanoparticles characteristics except for Glass Transition Temperature. However, depending on enzyme content, changeable behaviors in particle size, particle size distribution and denaturation temperature were detected which is generally associated with the changes in the structure and formation of covalent bonds with the function of mTG. Scanning electron microscopy of FE-SEM showed spherically synthesized nanoparticles with a size between 53 to 155 nm. This seems to facilitate the use of ultrasound reaction between nanoparticles and biopolymer matrix. Consequently, the optimized levels of 16.7 min for ultrasound and 9.47 micg/g for mTG resulted in generating a product with the considered characteristics. This novel and edible amaranth-based Nanoparticles could be suggested for Pickering emulsion preparation. [ABSTRACT FROM AUTHOR]

Antifreeze peptides have become effective antifreeze agents for frozen products, but their low quantity of active ingredients and high cost limit large-scale application. This study used the glycosylation of fish collagen peptides with glucosamine hydrochloride catalyzed by transglutaminase to obtain a transglutaminase-catalyzed glycosylation product (TGP) and investigate its antifreeze effect on tilapia. Compared with the blank group, the freshness (pH value of 6.31, TVB-N value of 21.7 mg/100 g, whiteness of 46.28), textural properties (especially hardness and elasticity), and rheological properties of the TGP groups were significantly improved. In addition, the protein structures of the samples were investigated using UV absorption and fluorescence spectroscopy. The results showed that the tertiary structure of the TGP groups changed to form a dense polymer. Therefore, this approach can reduce the denaturation and decomposition of muscle fibers and proteins in fish meat more effectively and has a better protective effect on muscle structure and protein aggregation, improving the stability of fish meat. This study reveals an innovative method for generating antifreeze peptides by enzymatic glycosylation, and glycosylated fish collagen peptide products can be used as new and effective green antifreeze agents in frozen foods. [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]

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]

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]

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]

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]