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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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]