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β-galactosidase from Kluyveromyces lactis has been immobilized on chitosan-glutaraldehyde particles employing different enzyme loadings (in a range of 0.7–7 mg/g of support). The biocatalysts had been later utilized to produce lactulose by transglycosylation using milk whey supplemented with fructose. The amount of produced lactulose decreased when the enzyme loading increased, from around 18 mg/mL using the biocatalysts with only 0.7 mg/g to around 7 mg/mL using the biocatalyst with a loading of 7 mg/mL. The results showed in this paper shows the great effect of the substrates and product concentration gradients inside the biocatalyst particle, not only lactulose, but also glucose and galactose. That is, the use of a excessive enzyme loading can lead to a significant worsening of the β-galactosidase biocatalyst performance in kinetically controlled synthesis. [Display omitted] • β-galactosidase was immobilized using different biocatalysts enzyme loadings. • The biocatalysts were used to produce lactulose from lactose and fructose. • The higher the enzyme loading, the lower the lactulose production. • The substrate and products internal gradients explain the results. [ABSTRACT FROM AUTHOR]

Fabry disease, an X-linked lysosomal storage disorder caused by galactosidase α (GLA) gene mutations, exhibits diverse clinical manifestations, and poses significant diagnostic challenges. Early diagnosis and treatment are crucial for improved patient outcomes, pressing the need for reliable biomarkers. In this study, we aimed to identify miRNA candidates as potential biomarkers for Fabry disease using the KingFisher™ automated isolation method and NanoString nCounter® miRNA detection assay. Clinical serum samples were collected from both healthy subjects and Fabry disease patients. RNA extraction from the samples was performed using the KingFisher™ automated isolation method with the MagMAX mirVanaTM kit or manually using the Qiagen miRNeasy kit. The subsequent NanoString nCounter® miRNA detection assay showed consistent performance and no correlation between RNA input concentration and raw count, ensuring reliable and reproducible results. Interestingly, the detection range and highly differential miRNA between the control and disease groups were found to be distinct depending on the isolation method employed. Nevertheless, enrichment analysis of miRNA-targeting genes consistently revealed significant associations with angiogenesis pathways in both isolation methods. Additionally, our investigation into the impact of enzyme replacement therapy on miRNA expression indicated that some differential miRNAs may be sensitive to treatment. Our study provides valuable insights to identify miRNA biomarkers for Fabry disease. While different isolation methods yielded various detection ranges and highly differential miRNAs, the consistent association with angiogenesis pathways suggests their significance in disease progression. These findings lay the groundwork for further investigations and validation studies, ultimately leading to the development of non-invasive and reliable biomarkers to aid in early diagnosis and treatment monitoring for Fabry disease. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases have been implicated in a wide range of human conditions including lysosomal storage diseases. Consequently, many researchers have directed their efforts towards identifying new classes of glycoside hydrolase inhibitors, both synthetic and from natural sources. A large percentage of such inhibitors are reversible competitive inhibitors that bind in the active site often due to them possessing structural features, often a protonatable basic nitrogen atom, that mimic the enzymatic transition state. We report that mechanism-based small molecule galacto-like configured cyclohexenyl carbasugars form reversible covalent complexes with both α-galactosidase and β-galactosidase. In addition, we show that the β-galactosidase from Aspergillus oryzae reacts with three different carbasugar inhibitors, with three different second-order rate constants (kinact/Ki), to give the same enzyme–carbasugar covalent intermediate. The surprising observation that the α-galacto-configured inhibitor covalently labels the A. oryzae β-galactosidase highlights the catalytic versatility of glycoside hydrolases. We expect that cyclohexenyl covalent inhibitors will become an important class of compounds in the chemical biologist's tool box. [ABSTRACT FROM AUTHOR]

This study compared the effects of feeding a new strain of Saccharomyces cerevisiae HSA2020 with a commercial strain on in vitro rumen fermentation and production performance of dairy cows. Permeate was used as a substrate for the laboratory production of the new strain of S. cerevisiae after the hydrolysis by β-galactosidase (5000 µ/mL at 37°C). Two experiments were conducted: in Experiment 1, the effects of three levels (1, 2 and 3 g/kg dry matter) of S. cerevisiae on in vitro ruminal fermentation kinetics were evaluated. In Experiment 2, for 60 days, sixty multiparous Holstein cows (639±24.8 kg BW, 3±1 parity, 7±1 days in milk, with a previous milk production of 23±2.0 kg/d) during the previous lactation, were randomly assigned to 3 treatments in a completely randomized design. Cows were fed without any additives (control treatment) or supplemented with 2 g/kg feed daily of laboratory produced (PY) or commercial (CY) S. cerevisiae. In Experiment 1, inclusion of PY and CY increased (P<0.05) gas production, propionate, and nutrient disappearance, while decreased (P<0.05) methane production and protozoal count. Moreover, in Experiment 2, PY followed by CY increased (P<0.01) nutrient digestibility, and serum concentrations of total protein, albumin, and glucose (P<0.05). Higher daily milk yield, and milk energy output were observed with PY and CY without affecting concentrations of milk components or milk fatty acid profile. Compared to control, increased feed efficiency was observed with PY and CY. Compared to PY, CY increased serum concentrations of urea-N and decreased triglycerides, while PY decreased serum aspartate transaminase and increased concentration of conjugated linoleic acids in milk. In early lactating cow diets, both strains of S. cerevisiae improved production performance at 2 g/kg, and minimal differences between strains were found. [ABSTRACT FROM AUTHOR]

Bifidobacteria are well known as common and abundant colonizers of the human gut and are able to exert multiple beneficial effects on their host, although the cooperative and competitive relationships that may occur among bifidobacterial strains are still poorly investigated. Therefore, to dissect possible molecular interactions among bifidobacterial species that typically colonize the human gut, three previously identified bifidobacterial prototypes, i.e., B. bifidum PRL2010, B. breve PRL2012, and B. longum PRL2022 were cultivated individually as well as in bi- and tri-association in a human gut-simulating medium. Transcriptomic analyses of these co-associations revealed up-regulation of genes predicted to be involved in the production of extracellular structures including pili (i.e., flp pilus assembly TadE protein gene), exopolysaccharides (i.e., GtrA family protein gene) and teichoic acids (i.e., ABC transporter permease), along with carbohydrate, amino acid and vitamin metabolism-related genes (i.e., exoalpha- sialidase; beta-galactosidase and pyridoxamine kinase), suggesting that co-cultivation of bifidobacteria induces a response, in individual bifidobacterial strains, aimed at enhancing their proliferation and survival, as well as their ability to cooperate with their host to promote their persistence. Furthermore, exposure of the selected prototypes to human cell line monolayers unveiled the ability of the bifidobacterial tri-association to communicate with their host by increasing the expression of genes involved in adherence to/interaction with intestinal human cells. Lastly, bifidobacterial tri-association promoted the transcriptional upregulation of genes responsible for maintaining the integrity and homeostasis of the intestinal epithelial barrier. [ABSTRACT FROM AUTHOR]

Xyloglucan in plant cell walls has complex side-chain structures; Aspergillus oryzae produces various enzymes to degrade and assimilate xyloglucan. In this study, we identified and characterized α-1,2- l -fucosidase (AfcA) which is involved in xyloglucan degradation in A. oryzae. AfcA expression was induced in the presence of xyloglucan oligosaccharides. AfcA showed specific activity toward α-(1→2)-linked l -fucopyranosyl residues attached to the side chains of xyloglucan oligosaccharides and milk oligosaccharides, but not toward α-(1→3)-, α-(1→4)-, and α-(1→6)-linked l -fucopyranosyl residues. As fucopyranosyl residues in the side chains of xyloglucan oligosaccharides prevent the degradation of xyloglucan oligosaccharides by isoprimeverose-producing oligoxyloglucan hydrolase and β-galactosidase, the cooperative action of AfcA, isoprimeverose-producing oligoxyloglucan hydrolase, and β-galactosidase play a key role in degrading fucosylated xyloglucan in A. oryzae. [ABSTRACT FROM AUTHOR]

Bleomycin exhibits effective chemotherapeutic activity against multiple types of tumors, and also induces various side effects, such as pulmonary fibrosis and neuronal defects, which limit the clinical application of this drug. Macroautophagy/autophagy has been recently reported to be involved in the functions of bleomycin, and yet the mechanisms of their crosstalk remain insufficiently understood. Here, we demonstrated that reactive oxygen species (ROS) produced during bleomycin activation hampered autophagy flux by inducing lysosomal membrane permeabilization (LMP) and obstructing lysosomal degradation. Exhaustion of ROS with N-acetylcysteine relieved LMP and autophagy defects. Notably, we observed that LMP and autophagy blockage preceded the emergence of cellular senescence during bleomycin treatment. In addition, promoting or inhibiting autophagy-lysosome degradation alleviated or exacerbated the phenotypes of senescence, respectively. This suggests the alternation of autophagy activity is more a regulatory mechanism than a consequence of bleomycin-induced cellular senescence. Taken together, we reveal a specific role of bleomycin-induced ROS in mediating defects of autophagic degradation and further regulating cellular senescence in vitro and in vivo. Our findings, conversely, indicate the autophagy-lysosome degradation pathway as a target for modulating the functions of bleomycin. These provide a new perspective for optimizing bleomycin as a clinically applicable chemotherapeutics devoid of severe side-effects. Abbreviations: AT2 cells: type II alveolar epithelial cells; ATG7: autophagy related 7; bEnd.3: mouse brain microvascular endothelial cells; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCL2: C-C motif chemokine ligand 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; FTH1: ferritin heavy polypeptide 1; γ-H2AX: phosphorylated H2A.X variant histone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cells; HT22: hippocampal neuronal cell lines; Il: interleukin; LAMP: lysosomal-associated membrane protein; LMP: lysosome membrane permeabilization; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NCOA4: nuclear receptor coactivator 4; PI3K: phosphoinositide 3-kinase; ROS: reactive oxygen species; RPS6KB/S6K: ribosomal protein S6 kinase; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SAHF: senescence-associated heterochromatic foci; SASP: senescence-associated secretory phenotype; SEC62: SEC62 homolog, preprotein translocation; SEP: superecliptic pHluorin; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB [ABSTRACT FROM AUTHOR]

BACKGROUND: The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non‐digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by β‐galactosidase (β‐gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450‐β‐gal) from lactose, lactulose and lactitol. RESULTS: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450‐β‐gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush‐border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose‐oligosaccharides were the most resistant to the physiological conditions of mice. In general β (1→6) linked products showed higher resistance compared to β (1→3) oligosaccharides. CONCLUSION: In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Senescent cells are characterized by multiple features such as increased expression of senescence-associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16. They accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin (Doxo) cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. This study shows that 15d-PGJ2 is also released by Doxo-induced senescent cells as an SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2 by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in C2C12 mouse skeletal myoblasts, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to Ras Binding Domain (RBD) of RAF Kinase (RAF-RBD), and activation of cellular Mitogen Activated Protein (MAP) kinase–Extracellular Signal Regulated Kinase (Erk) signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d-PGJ2 to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2 released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy. [ABSTRACT FROM AUTHOR]

The most extensively studied β‐d‐galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family β‐galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (DaβGal). Unlike fungal monomeric six‐domain β‐galactosidases, the DaβGal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for β‐d‐galactopyranosides, and its distinguishing feature is the ability to cleave pNP‐β‐d‐fucopyranoside. DaβGal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 °С, and retains activity at 95 °С with a half‐life time value equal to 73 min. These properties make archaeal DaβGal a more attractive candidate for biotechnology than the widely used fungal β‐galactosidases. [ABSTRACT FROM AUTHOR]

Cover IllustrationAllegorical representation of the dimer spatial structure of a novel hyperthermophilic beta‐galactosidase. Image provided by Georgy Rychkov and colleagues, authors of the Original Article included in this issue, pages 3686–3705.. [Extracted from the article]

The gut microbiota offers an extensive resource of enzymes, but many remain uncharacterized. To distinguish the activities of similar annotated proteins and mine the potentially applicable ones in the microbiome, we applied an effective Activity-Based Metaproteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for directly discovering active enzymes and their potential applications, not for exploring host-microbiome interactions. By using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), we successfully identified and characterized several gut microbiota enzymes possessing AGAL activities. Cryo-electron microscopy analysis of a newly characterized enzyme (AGLA5) revealed the covalent binding conformations between the AGAL5 active site and the cyclophellitol aziridine ABP, which could provide insights into the enzyme's catalytic mechanism. The four newly characterized AGALs have diverse potential activities, including raffinose family oligosaccharides (RFOs) hydrolysis and enzymatic blood group transformation. Collectively, we present a ABMP platform that facilitates gut microbiota AGALs discovery, biochemical activity annotations and potential industrial or biopharmaceutical applications. The gut microbiota offers an extensive resource of enzymes, however, many remain uncharacterized. Here, the authors apply an activity-based metaproteomics strategy using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), and identify and characterize several gut microbiota enzymes possessing AGAL activities. [ABSTRACT FROM AUTHOR]

β‐Galactosidases are crucial enzymes that hydrolyse oligosaccharides and polysaccharides with terminal β‐1,4‐glycosidic bonds. Though the traditional application of β‐Galactosidases has been to catalyse the breakdown of lactose in dairy products, its application extends beyond the production of lactose‐free products since variants capable of facilitating lactose condensation and exhibiting galactosyl transferase activity are extensively utilised for the synthesis of prebiotic galacto‐oligosaccharides. This review analyses β‐Galactosidase in multiple aspects, including sources, classification, characterisation, immobilisation, genetic engineering and applications in terms of whey treatment, biofuel production, production of lactose‐free dietary product, synthesis of galacto‐oligosaccharides and the early detection of cellular senescence and tumours. [ABSTRACT FROM AUTHOR]

We report on the synthesis of two fluorescent probes which can be activated by β-Galactosidase (β-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with β-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin–Halo fusion protein in live cells with overexpressed β-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to demonstrate that an adipocyte tissue‐derived conditioned medium (ACM) contains inflammatory molecules that induce senescence in B cells. Methods: We incubated blood‐derived B cells from lean donors with ACM obtained from the adipose tissue of adult female donors with obesity undergoing weight reduction surgery or with medium as control. After 24 h, cells were harvested, and the expression of transcripts for proinflammatory cytokines (TNF/IL‐6), chemokines (IL‐8), and for markers of the senescence‐associated secretory phenotype (SASP) was measured by quantitative polymerase chain reaction. B cells were also stained with the marker of immunosenescence β‐galactosidase, and their metabolic status was evaluated in Seahorse using a Mito Stress Test. Results: We show that the incubation of B cells from lean donors with ACM induces the expression of transcripts for inflammatory and SASP transcripts, increases the amount of β‐galactosidase staining, and induces a metabolic phenotype characterized by higher basal and maximal oxygen consumption, spare respiratory capacity (difference between maximal and basal respiration), nonmitochondrial oxygen consumption, ATP production, and proton leak. Conclusions: These results demonstrate that B cells from lean individuals, after incubation with ACM, become inflammatory and senescent, and this occurs through metabolic pathways needed to support their secretory phenotype. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis (PF) is a fatal interstitial lung disease associated with declining pulmonary function but currently with few effective drugs. Cellular senescence has been implicated in the pathogenesis of PF and could be a potential therapeutic target. Emerging evidence suggests wogonin, the bioactive compound isolated from Scutellaria baicalensis, owns the anti-senescence properties, however, the possible impact of wogonin on PF and the potential mechanisms remain unclear. In this study, a well-established mouse model of PF was utilized which mice were administrated with bleomycin (BLM). Strikingly, wogonin treatment significantly reduced fibrosis deposition in the lung induced by BLM. In vitro, wogonin also suppressed fibrotic markers of cultured epithelial cells stimulated by BLM or hydrogen peroxide. Mechanistic investigation revealed that wogonin attenuated the expressions of DNA damage marker y-H2AX and senescence-related markers including phosphorylated p53, p21, retinoblastoma protein (pRB), and senescence-associated β-galactosidase (SA-β-gal). Moreover, wogonin, as a direct and selective inhibitor of cyclin-dependent kinase 9 (CDK9), exhibited anti-fibrotic capacity by inhibiting CDK9 and p53/p21 signalling. In conclusion, wogonin protects against BLM-induced PF in mice through the inhibition of cell senescence via the regulation of CDK9/p53 and DNA damage pathway. This is the first study to demonstrate the beneficial effect of wogonin on PF, and its implication as a novel candidate for PF therapy. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer‐inverting α‐glucoside hydrolases (i.e., glucoamylase) and anomer‐retaining α‐galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an α‐glucoside hydrolase, targeting α‐(1→6)‐glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for α‐(1→6)‐glucoside linkages over α‐(1→4)‐linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for α‐(1→4)‐glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for α‐(1→4)‐ and α‐(1→6)‐glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer‐inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S‐I378F), A195S and A195S‐I378F exhibited increased activity toward α‐(1→4)‐glucoside bonds compared to α‐(1→6)‐glucoside bonds. This suggests that Ala195, located on the Gly184‐Thr203 loop (named loop‐N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for α‐(1→6)‐glucoside linkage. [ABSTRACT FROM AUTHOR]

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route to expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient manner. We constructed a host bacterial cell to link the genotype of REs to the phenotype of β-galactosidase expression based on the bacterial SOS response, and used a high-throughput microfluidic platform to isolate, detect and sort the REs in microfluidic drops at a frequency of ∼800 drops per second. We employed this strategy to screen for the XbaI gene from the constructed libraries of varied sizes. In a single round of sorting, a 90-fold target enrichment was achieved within 1 h. Compared to conventional RE-screening methods, the direct screening approach that we propose excels at efficient search of desirable REs in natural samples – especially unculturable samples – and can be tailored to high-throughput screening of a wide range of genotoxic targets. [ABSTRACT FROM AUTHOR]

BACKGROUND: Diabetic osteoporosis is gaining public attention. However, few studies have reported the effect of a high-glucose environment on the osteogenic differentiation of human umbilical cord mesenchymal stem cells and the corresponding therapeutic strategies. OBJECTIVE: To investigate whether vitamin D3 can restore the osteogenic differentiation potential of human umbilical cord mesenchymal stem cells in a high)glucose environment. METHODS: The viability of human umbilical cord mesenchymal stem cells was detected by CCK-8 assay to screen the appropriate vitamin D3 intervention concentration. Under the high-glucose environment, RT-qPCR, western blot assay, immunofluorescence, JC-1 mitochondrial membrane potential, alizarin red staining, and β-galactosidase staining were used to evaluate the osteogenic differentiation potential, intracellular reactive oxygen species accumulation, mitochondrial membrane potential alteration, and cell senescence of human umbilical cord mesenchymal stem cells after vitamin D3 intervention. The underlying mechanism was also discussed. RESULTS AND CONCLUSION: (1) Vitamin D3 significantly promoted the proliferation of human umbilical cord mesenchymal stem cells in the range of 0.1 μmol/L to 1 mmol/L. (2) High-glucose environment down-regulated the mRNA and protein level expressions of osteogenic-related genes α1-I collagen, alkaline phosphatase, Runt-associated transcription factor 2, and osteocalcin in human umbilical cord mesenchymal stem cells, which induced oxidative stress and cellular senescence. (3) Vitamin D3 at an intervention concentration of 10 μmol/L significantly restored the osteogenic phenotype of human umbilical cord mesenchymal stem cells under high-glucose conditions and attenuated intracellular oxidative stress and cellular senescence by activating the Nrf2/HO-1 signaling pathway. (4) These findings suggested that the osteogenic differentiation ability of human umbilical cord mesenchymal stem cells was reduced in the high-glucose environment, and vitamin D3 could partially improve their osteogenic differentiation ability and reduce cell damage. [ABSTRACT FROM AUTHOR]

The lactose intolerance—as a limiting factor for dairy milk consumption—has a high prevalence worldwide. Dairy milk and milk-derived products are major sources of multiple inorganic compounds and nutrients and thus are considered to be functional foods. β-galactosidases are able to hydrolyze lactose and are therefore widely applied for the production of lactose-free products. In addition, they are capable of the synthesis of galacto-oligosaccharides (GOSs); thus, the dairy industry has a special interest in applying them for the enrichment of dairy products with prebiotic GOSs. In this work, we studied two commercially available β-galactosidase products: Saphera 2600L and Nola Fit 5500. Both enzyme solutions contain a recombinant β-galactosidase of Bifidobacterium bifidum and have already been authorized for food industrial application, but the information about their hydrolytic and/or synthetic activities is only limited. After immobilization on chitosan beads, the enzymes were used for lactose hydrolysis and simultaneous synthesis of GOSs, by performing the reactions in pasteurized milk (skim milk). Both immobilized β-galactosidase exhibited elevated lactose hydrolysis (vmax increased from ~ 1 to ~ 4 mM/min) and GOS synthesis as compared to the free enzymes. The enzyme-coated beads were efficiently re-used at least 15 cycles; the residual lactose concentration was < 2 mg/ml after each cycle. After treatment, GOSs were present in ≤ 9% of the total sugar content, indicating that the prepared low-lactose milks were enriched in prebiotic GOSs. The application of immobilized Saphera 2600L and Nola Fit 5500 β-galactosidases may be implemented for the large-scale production of GOS-enriched low-lactose milk. [ABSTRACT FROM AUTHOR]

A new β-galactosidase (EC 3.2.1.23) discovered in Kluyvera intermedia (β-gal-Kli) was recombinantly produced in Escherichia coli BL21 (DE3). The bioreactor cultivation resulted in a maximum β-gal-Kli activity of 124.6 ± 25.5 μkatoNPGal,37 °C/Lculture. The β-gal-Kli was purified 4.5fold to a specific activity of 172.3 μkatoNPGal,37 °C/gprotein. The β-gal-Kli showed maximum activity at pH 6.5 and 50°C and was sufficiently active and stable at an industrially relevant temperature of 8°C (half-life of 63 days). The β-gal-Kli was compared to a commercially available β-galactosidase (opti-lactase LX2) for lactose hydrolysis in milk (45.3 ± 0.9 glactose/L) at 8°C. With the activity of 2.7 nkatlactose/mLmilk, "lactose-free" (lactose <0.1 g/L) was realized by β-gal-Kli after 72 h, while 192 h was needed for opti-lactase LX2. Additionally, the galactooligosaccharide synthesis was observed with both these two enzymes. In conclusion, the β-gal-Kli showed its potential for the production of "lactose-free" dairy products. [ABSTRACT FROM AUTHOR]

Anderson–Fabry disease (AFD) is an X-linked multisystemic disorder with a heterogeneous phenotype, resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) and leading to globotriaosylceramide systemic accumulation. Lysosomal storage is not the unique player in organ failure and different mechanisms could drive tissue damage, including endoplasmic reticulum (ER) stress and its related signaling pathway's activation. We identified a new missense variant in the signal peptide of α-GLA gene, c.13 A/G, in a 55-year-old woman affected by chronic kidney disease, acroparesthesia, hypohidrosis, and deafness and exhibiting normal values of lysoGb3 and αGLA activity. The functional study of the new variant performed by its overexpression in HEK293T cells showed an increased protein expression of a key ER stress marker, GRP78, the pro-apoptotic BAX, the negative regulator of cell cycle p21, the pro-inflammatory cytokine, IL1β, together with pNFkB, and the pro-fibrotic marker, N-cadherin. Transmission electron microscopy showed signs of ER injury and intra-lysosomal inclusions. The proband's PBMC exhibited higher expression of TGFβ 1 and pNFkB compared to control. Our findings suggest that the new variant, although it did not affect enzymatic activity, could cause cellular damage by affecting ER homeostasis and promoting apoptosis, inflammation, and fibrosis. Further studies are needed to demonstrate the variant's contribution to cellular and tissue damage. [ABSTRACT FROM AUTHOR]

Background: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. Results: In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. Conclusion: For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes. [ABSTRACT FROM AUTHOR]

Delivery of active protein especially enzyme is one of the major therapeutic challenge. Replacing or substituted invalid/improper acting protein offer fast and effective treatment of disease. Herein, we describe the synthesis and properties of biotinylated peptidomimetics consisting of oxoacid—modified 2,3, l-diaminopropionic acid residues with guanidine groups on its side chains. Electrophoretic analysis showed that the obtained compounds interact with FITC-labeled streptavidin or a streptavidin-β-galactosidase hybrid in an efficient manner. Complexes formed by the abovementioned molecules are able to cross the cell membranes of cancer or healthy cells and show promising compatibility with live cells. Analysis of β-galactosidase activity inside the cells revealed surprisingly high levels of active enzyme in complex-treated cells compared to controls. This observation was confirmed by immunochemical studies in which the presence of β-galactosidase was detected in the membrane and vesicles of the cells. [ABSTRACT FROM AUTHOR]

Background: Autologous mesenchymal stem cells (MSCs) have emerged as a therapeutic option for many diseases. Hypertensive kidney disease (HKD) might impair MSCs' reparative ability by altering the biomolecular properties, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, we found hypoxic preconditioning (HPC) enhanced angiogenesis and suppressed senescence gene expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment. Methods: MSC samples (n = 12 each) were collected from the abdominal fat of healthy kidney donors (HC), hypertensive patients (HTN), and patients with hypertensive kidney disease (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions. MSC functionality was measured by proliferation assays and cytokine released in conditioned media. Senescence was evaluated by senescence-associated beta-galactosidase (SA-beta-gal) activity. Additionally, transcriptome analysis using RNA-sequencing and quantitative PCR (qPCR) were performed. Results: At baseline, normoxic HTN-MSCs had higher proliferation capacity compared to HC. However, HPC augmented proliferation in HC. HPC did not affect the release of pro-angiogenic protein VEGF, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. Under HPC, SA-β-gal activity tended to decrease, particularly in HC group. HPC upregulated mostly the pro-angiogenic and inflammatory genes in HC and HKD and a few senescence genes in HKD. Conclusions: HPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and modest decrease in senescence, whereas it has little effect on HTN or HKD MSCs. Significance statement: In this study, we evaluate the effect of hypoxic preconditioning on human mesenchymal stem cells characteristics from healthy individuals and patients with hypertension with and without kidney disease providing adaptative mechanisms involving molecular and functional alterations when compared to normoxic conditions. The differential gene expression patterns highlight the tissue repair which is unique to each group. This research enhances our understanding of cellular and molecular dynamics in diseased states, and the effects of hypoxic preconditioning on MSC performance. [ABSTRACT FROM AUTHOR]

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage destruction and inflammation. CC chemokine receptor 1 (CCR1), a member of the chemokine family and its receptor family, plays a role in the autoimmune response. The impact of BX471, a specific small molecule inhibitor of CCR1, on CCR1 expression in cartilage and its effects on OA remain underexplored. Methods: This study used immunohistochemistry (IHC) to assess CCR1 expression in IL-1β-induced mouse chondrocytes and a medial meniscus mouse model of destabilization of the medial meniscus (DMM). Chondrocytes treated with varying concentrations of BX471 for 24 h were subjected to IL-1β (10 ng/ml) treatment. The levels of the aging-related genes P16INK4a and P21CIP1 were analyzed via western blotting, and senescence-associated β-galactosidase (SA-β-gal) activity was measured. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), aggrecan (AGG), and the transcription factor SOX9 were determined through western blotting and RT‒qPCR. Collagen II, matrix metalloproteinase 13 (MMP13), and peroxisome proliferator-activated receptor (PPAR)-γ expression was analyzed via western blot, RT‒qPCR, and immunofluorescence. The impact of BX471 on inflammatory metabolism-related proteins under PPAR-γ inhibition conditions (using GW-9662) was examined through western blotting. The expression of MAPK signaling pathway-related molecules was assessed through western blotting. In vivo, various concentrations of BX471 or an equivalent medium were injected into DMM model joints. Cartilage destruction was evaluated through Safranin O/Fast green and hematoxylin–eosin (H&E) staining. Results: This study revealed that inhibiting CCR1 mitigates IL-1β-induced aging, downregulates the expression of iNOS, COX-2, and MMP13, and alleviates the IL-1β-induced decrease in anabolic indices. Mechanistically, the MAPK signaling pathway and PPAR-γ may be involved in inhibiting the protective effect of CCR1 on chondrocytes. In vivo, BX471 protected cartilage in a DMM model. Conclusion: This study demonstrated the expression of CCR1 in chondrocytes. Inhibiting CCR1 reduced the inflammatory response, alleviated cartilage aging, and retarded degeneration through the MAPK signaling pathway and PPAR-γ, suggesting its potential therapeutic value for OA. [ABSTRACT FROM AUTHOR]

Commercial β-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [ K M ] for lactose) and product inhibition (inhibitor constant [ K i ] for galactose). An in silico screening of gene sequences was done and identified a putative β-galactosidase (Paenibacillus wynnii β-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low β-galactosidase activities of a maximum of 150 nkat per liter of medium with o -nitrophenyl-β- d -galactopyranoside (o NPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 μkat o NPGal /L culture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the K M value of BgaPw with lactose was as low as 0.63 ± 0.045 m M in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the β-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known β-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield). [ABSTRACT FROM AUTHOR]

Summary: In this study, an α‐galactosidase (MPG) was produced by submerged fermentation from Monascus purpureus, a food‐grade fungus used for making red yeast rice. MPG was purified from intracellular extract which showed a subunit molecular mass of 95 kDa. MPG was optimally active at pH 4.0 and 50 °C and was stable between pH 3.5–8.0 and at temperatures up to 50 °C. The enzyme retained its activity in the presence of most of the metal ions except Cu2+ and Fe2+. MPG exhibited remarkable resistance to the proteases, pepsin, trypsin and proteinase K as well as to the sugars, galactose and sucrose. Moreover, it retained complete activity under simulated gastric conditions. The enzyme displayed specificity for the substrates, melibiose, raffinose and stachyose and was able to hydrolyze these oligosaccharides. It was effective in the removal of non‐digestible raffinose family oligosaccharides (RFOs) from soybeans, chickpeas and kidney beans. MPG was immobilised onto chitosan and the immobilised enzyme (iMPG) displayed higher optimal temperature (60 °C), better thermostability and substrate specificity as compared to MPG. iMPG was also capable of hydrolyzing RFOs in chickpeas and could be efficiently reused six times. In view of their superior properties, MPG and iMPG may have great prospects in the food and feed industries for the removal of RFOs from soy and other legumes. [ABSTRACT FROM AUTHOR]

Many different strains of Streptococcus thermophilus are commonly used as starter cultures, and search for new safe strains with desired industrial and probiotic properties is an important issue. In this article, genomes of two new S. thermophilus strains—Str16t and Str159—were sequenced, and the main genome characteristics were determined. In silico analysis of the sequenced genomes revealed the absence of transmissible antibiotic resistance genes, virulence genes associated with pathogenicity, and integrated plasmids; gene clusters encoding class I and class II bacteriocins were found. In vitro tests showed phosphatase, peptidase, β-galactosidase, and esterase activity of both strains. Both strains were capable of fermenting glucose, lactose, sucrose, and ribose; in addition, Str16t fermented mannose. In conclusion, it was demonstrated that both Str16t and Str159 are promising strains for application as starter and probiotic cultures. [ABSTRACT FROM AUTHOR]

Fabry disease is an invalidating multisystemic disorder affecting α-Galactosidase, a rate-limiting hydrolase dedicated to lipid catabolism. Non-metabolized substrates, such as Globotriaosylceramide and its derivatives trigger the direct or indirect activation of inflammatory events and endothelial dysfunction. In spite of the efficacy demonstrated by enzyme replacement therapy or pharmacological chaperones in delaying disease progression, few studies have analyzed whether these treatments can improve the pro-inflammatory state of FD patients. Therefore, the aim of this work was to assess cytokines and cardiovascular risk-related proteins detectable in plasma from FD patients, whether treated or not with ERT, to evaluate the reliability of these markers in monitoring disease stage and treatment effects. We identified inflammatory and endothelial dysfunction markers (ADAMTS-13, TNF-α, GDF-15, MIP-1β, VEGFA, MPO, and MIC-1) that cooperate in a common pathway and are increased in FD patients' plasma samples. As shown by the assessment of these proteins over time, they can help to evaluate the risk of higher severity in FD, as well as ERT effects. Even though the analyzed proteins cannot be considered as proper biomarkers due to their non-specificity to FD, taken together they can provide a signature of reference molecules with prognostic value for early diagnosis, and evaluation of disease progression and treatment efficacy, using blood samples. [ABSTRACT FROM AUTHOR]

Whey is a byproduct of dairy industries, the aqueous portion which separates from cheese during the coagulation of milk. It represents approximately 85–95% of milk's volume and retains much of its nutrients, including functional proteins and peptides, lipids, lactose, minerals, and vitamins. Due to its composition, mainly proteins and lactose, it can be considered a raw material for value-added products. Whey-derived products are often used to supplement food, as they have shown several physiological effects on the body. Whey protein hydrolysates are reported to have different activities, including antihypertensive, antioxidant, antithrombotic, opioid, antimicrobial, cytomodulatory, and immuno-modulatory. On the other hand, galactooligosaccharides obtained from lactose can be used as prebiotic for beneficial microorganisms for the human gastrointestinal tract. All these compounds can be obtained through physicochemical, microbial, or enzymatic treatments. Particularly, enzymatic processes have the advantage of being highly selective, more stable than chemical transformations, and less polluting, making that the global enzyme market grow at accelerated rates. The sources and different products associated with the most used enzymes are particularly highlighted in this review. Moreover, we discuss metagenomics as a tool to identify novel proteolytic enzymes, from both cultivable and uncultivable microorganisms, which are expected to have new interesting activities. Finally enzymes for the transformation of whey sugar are reviewed. In this sense, carbozymes with ß-galactosidase activity are capable of lactose hydrolysis, to obtain free monomers, and transgalactosylation for prebiotics production. Key points: • Whey can be used to obtain value-added products efficiently through enzymatic treatments • Proteases transform whey proteins into biopeptides with physiological activities • Lactose can be transformed into prebiotic compounds using ß-galactosidases [ABSTRACT FROM AUTHOR]

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. Key points: • β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima β-glucosidase has high activity and high thermostability. • Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages. [ABSTRACT FROM AUTHOR]

Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-β-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-β-Gal showed exo- β-galactosidase activity with regiopreference for hydrolyzing β- d -(1,6) glycosidic linkages. Notably, it exhibited a preference for d -glucopyranosyl (d -Glc p) over d -galactopyranosyl (d -Gal p) and d -fructofuranosyl (d -Fru f) at the reducing end of the investigated disaccharides. In addition, we determined the high resolution crystal structure of BD-β-Gal, thus providing the first structural characterization of a GH154 enzyme. Surprisingly, this revealed an (α/α) 6 topology, which has not been observed before for β-galactosidases. BD-β-Gal displayed low structural homology with characterized CAZymes, but conservation analysis suggested that the active site was located in a central cavity, with conserved E73, R252, and D253 as putative catalytic residues. Interestingly, BD-β-Gal has a tetrameric structure and a flexible loop from a neighboring protomer may contribute to its reaction specificity. Finally, we showed that the founding member of GH154, BT3677 from Bacteroides thetaiotaomicron , described as β-glucuronidase, displayed exo -β-galactosidase activity like BD-β-Gal but lacked a tetrameric structure. [Display omitted] • New β-galactosidase activity in Glycoside Hydrolase family 154 (GH154). • BD-β-Gal shows β-galactosidase activity with preference for β-(1,6)-linkages. • First crystal structure of GH154 with novel (α/α) 6 topology for β-galactosidase. • Novel active site topology for BD-β-Gal, different from canonical β-galactosidases. • BD-β-Gal shows transglycosylation activity on lactose. [ABSTRACT FROM AUTHOR]

Objective(s): Investigating the impact of cadmium (Cd) on annulus fibrosus (AF) cells and its potential mechanism was the purpose of the current study. Materials and Methods: Cd was cultivated in different concentrations (0, 1, 5, 10, and 20 μM) on AF cells and the potential effects of the metal were assessed. Using the CCK-8 method, cell viability and proliferation were identified. Using transcriptome analysis, the annulus fibrosus cells were sequenced both with and without cadmium chloride. The EdU method was used to determine the rate of cell proliferation; senescence-associated β-galactosidase (SA-β-Gal) staining was used to determine the number of positive cells; and western blot, RT-PCR, and immunofluorescence were used to determine the protein and mRNA expression of senescence-associated proteins (p16, p21, and p53) and c-Jun N-terminal kinase (JNK). Results: According to the findings, Cd has the ability to increase the production of senescenceassociated genes (p16 and p21) and senescence-associated secreted phenotype (SASP), which includes IL-1β and IL-6. Through the JNK/p53 signal pathway, Cd exposure simultaneously accelerated AF cell senescence and promoted SASP. Following JNK inhibitor (SP600125) treatment, the expression of p53, JNK, and senescence-associated indices were all down-regulated. Conclusion: By activating the JNK/p53 signaling pathway, Cd can induce oxidative stress damage and AF cell senescence. These findings could provide a new approach for treating and preventing intervertebral disc degeneration (IVDD) caused by Cd exposure. [ABSTRACT FROM AUTHOR]

Hierarchical self‐assembly from simple building blocks to complex polymers is a feasible approach to constructing multi‐functional smart materials. However, the polymerization process of polymers often involves challenges such as the design of building blocks and the drive of external energy. Here, a hierarchical self‐assembly with self‐driven and energy conversion capabilities based on p‐aminophenol and diethylenetriamine building blocks is reported. Through β‐galactosidase (β‐Gal) specific activation to the self‐assembly, the intelligent assemblies (oligomer and superpolymer) with excellent photothermal and fluorescent properties are dynamically formed in situ, and thus the sensitive multi‐mode detection of β‐Gal activity is realized. Based on the overexpression of β‐Gal in ovarian cancer cells, the self‐assembly superpolymer is specifically generated in SKOV‐3 cells to achieve fluorescence imaging. The photothermal therapeutic ability of the self‐assembly oligomer (synthesized in vitro) is evaluated by a subcutaneous ovarian cancer model, showing satisfactory anti‐tumor effects. This work expands the construction of intelligent assemblies through the self‐driven cascade assembly of small molecules and provides new methods for the diagnosis and treatment of ovarian cancer. [ABSTRACT FROM AUTHOR]

Fabry disease, a rare X-linked genetic disorder, results from pathogenic variants in GLA, leading to deficient lysosomal α-galactosidase A enzyme activity and multiorgan manifestations. Since 2001, enzyme replacement therapy (ERT), using agalsidase alfa or agalsidase beta, has been the mainstay treatment, albeit with limitations such as rapid clearance and immunogenicity. Pegunigalsidase alfa, a novel PEGylated recombinant alpha-galactosidase, offers promise as an alternative. Produced in plant cells, pegunigalsidase alfa exhibits enhanced stability, prolonged half-life, and reduced immunogenicity due to pegylation. A phase 1/2 clinical trial demonstrated Gb3 clearance from renal capillary endothelial cells and its 48-month extension study revealed notable outcomes in renal function preservation. Three phase 3 clinical trials (BRIDGE, BRIGHT, and BALANCE) have shown favorable efficacy and safety profile, although caution is warranted in interpreting the results of BRIDGE and BRIGHT which lacked control groups. In BALANCE, the pivotal phase 3 trial comparing pegunigalsidase alfa with agalsidase beta, an intention-to-treat analysis of the eGFR decline over 2 years showed that the intergroup difference [95%confidence interval] in the median slope was -0.36 mL/min/ 1.73 m²/year [-2.44; 1.73]. The confidence interval had a lower limit above the prespecified value of -3 mL/min/1.73 m2/year and included zero. Despite challenges such as occasional hypersensitivity reactions and immunecomplex- mediated glomerulonephritis, pegunigalsidase alfa approval by the European Medicines Agency and the Food and Drug Administration represents a significant addition to Fabry disease therapeutic landscape providing an option for patients in whom enzyme replacement therapy with current formulations is poorly tolerated or poorly effective. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are condensed intracellular phases that are formed by liquid-liquid phase separation (LLPS) of proteins, either in the absence or presence of nucleic acids. These condensed phases regulate various biochemical reactions by recruitment of enzymes and substrates. Developments in the field of LLPS facilitated new insights on the regulation of compartmentalized enzymatic reactions. Yet, the influence of condensate chemical composition on enzymatic reactions is still poorly understood. Here, by using peptides as minimalistic condensate building blocks and β-galactosidase as a simple enzymatic model we show that the reaction is restricted in homotypic peptide condensates, while product formation is enhanced in peptide-RNA condensates. Our findings also show that condensate composition affects the recruitment of substrate, the spatial distribution, and the kinetics of the reaction. Thus, these findings can be further employed for the development of microreactors for biotechnological applications. Peptide condensates are known to regulate compartmentalized enzymatic reactions, however, the influence of condensate chemical composition on enzymatic reactions is still poorly understood. Here, the authors study β-galactosidase as a simple enzymatic model and reveal that product formation is enhanced in heterotypic peptide-RNA condensates, but the reaction is restricted in homotypic peptide condensates. [ABSTRACT FROM AUTHOR]

The high incidence of restenosis after angioplasty has been the leading reason for the recurrence of coronary heart disease, substantially increasing the mortality risk for patients. However, current anti‐stenosis drug‐eluting stents face challenges due to their limited functions and long‐term safety concerns, significantly compromising their therapeutic effect. Herein, a stent‐free anti‐stenosis drug coating (denoted as Cur‐NO‐Gel) based on a peptide hydrogel is proposed. This hydrogel is formed by assembling a nitric oxide (NO) donor‐peptide conjugate as a hydrogelator and encapsulating curcumin (Cur) during the assembly process. Cur‐NO‐Gel has the capability to release NO upon β‐galactosidase stimulation and gradually release Cur through hydrogel hydrolysis. The in vitro experiments confirmed that Cur‐NO‐Gel protects vascular endothelial cells against oxidative stress injury, inhibits cellular activation of vascular smooth muscle cells, and suppresses adventitial fibroblasts. Moreover, periadventitial administration of Cur‐NO‐Gel in the angioplasty model demonstrate its ability to inhibit vascular stenosis by promoting reendothelialization, suppressing neointima hyperplasia, and preventing constrictive remodeling. Therefore, the study provides proof of concept for designing a new generation of clinical drugs in angioplasty. [ABSTRACT FROM AUTHOR]

Background: Fabry disease is a progressive, X chromosome-linked lysosomal storage disorder with multiple organ dysfunction. Due to the absence or reduced activity of alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide (Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous and currently routinely used fluorometry-based assays measuring mean activity mostly fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry assay to measure AGAL activity in individual cells. Methods: Conventional and multispectral imaging flow cytometry was used to detect AGAL activity. Specificity was validated using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin. The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with exome sequencing, gene expression and substrate accumulation. Results: Flow cytometric detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan blue solution is necessary for the specific detection of lysosomal substrate accumulation. Conclusion: A flow cytometry-based assay was developed for the quantitative detection of AGAL activity at the single-cell level, which may contribute to the diagnosis of Fabry patients. [ABSTRACT FROM AUTHOR]

Several studies have demonstrated that Moringa oleifera (MO) has different pharmacological properties, including neuroprotective effects. However, the role of MO in preventing brain impairment in high-fat, high-fructose diet (HFFD) remains unknown. This study aimed to investigate the neuroprotective effects of MO leaves aqueous extract (MOE) and moringa seed oil (MOO) against brain impairment in mice with HFFD. Twenty-eight male mice were randomly divided into four groups: normal diet, HFFD, HFFD + MOE 500 mg/kgBW, and HFFD + MOO 2 mL/kgBW. Cognitive function was assessed using the Y-maze and novel object recognition (NOR) tests. The p16, p21, and BDNF expressions were analyzed using the RT-PCR method. Senescence-associated beta-galactosidase (SA-β-gal) staining in the brain was also performed. The results showed that administration of MOE or MOO could increase the percentage of alternation and recognition of new objects, prevent the increase of p16 and p21 expression, and ameliorate SA-β-Gal activity in the brain. MOO, but not MOE, increased BDNF expression in senescence brains isolated from HFFD mice. The findings indicate that MOO and MOE possess neuroprotective properties, with MOO demonstrating a greater ability to inhibit the brain senescence process compared to MOE. [ABSTRACT FROM AUTHOR]

Introduction. Studying the functional properties of practically important enzymes, in particular α-galactosidases, is a necessary step towards obtaining stable preparations with high selectivity of action to be used in the food industry for biodegradation and modification of raw materials. Materials and methods. Gel filtration and ion exchange chromatography on TSK-gels were used to purify the microbial α-galactosidase. To determine the activity and specificity of the enzymatic action, p-nitrophenyl-α-D-galactopyranoside, raffinose, stachyose, and galactomannan were used as substrates. Results and discussion. α-Galactosidase with a specific activity of 49.1 IU/mg was isolated from the culture liquid of Penicillium restrictum and purified to a homogeneous state. The molecular weight of the enzyme was 17 kDa according to the data of gel filtration on Sepharose 6B. The optimum pH for the enzyme action was 4.0, and the thermal optimum was 60 °C. Enzyme showed high stability in the temperature ranged from 40 to 60 °C. The half-life of α-galactosidase from Penicillium restrictum at 70°C increased in 2-fold in the presence of 20-40% glycerol and 2.3-fold in the presence of 4 M sorbitol. Km for the hydrolysis of p-nitrophenyl-α- galactopiranoside, raffinose, stachyose, and galactomannan were 0.9 mM, 2.2 mM, 2.4 mM, and 3.8 mM, respectively. It was shown that the rate and efficiency of hydrolysis of galactose-containing natural substrates by Penicillium restrictum α-galactosidase decreased with increasing their molecular weight. The enzyme was completely inhibited in the presence of 10-3 M Ag+, Hg2+, Ca2+, and Cd2+. α-Galactosidase from Penicillium restrictum was resistant to action of proteinase K, pronase E, and trypsin. Conclusions. The high thermal stability and efficiency of α-galactosidase from Penicillium restrictum for the modification of galactooligosaccharides suggests its broadrange applicability for food and animal feed processing. [ABSTRACT FROM AUTHOR]

The prevalence of periodontitis increases with physiological aging. However, whether bacteria associated with periodontal diseases foster aging and the mechanisms by which they may do so are unknown. Herein, we hypothesize that Fusobacterium nucleatum, a microorganism associated with periodontitis and several other age‐related disorders, triggers senescence, a chief hallmark of aging responsible to reduce tissue repair capacity. Our study analyzed the senescence response of gingival epithelial cells and their reparative capacity upon long‐term exposure to F. nucleatum. Specifically, we assessed (a) cell cycle arrest by analyzing the cyclin‐dependent kinase inhibitors p16INK4a and p14ARF and their downstream cascade (pRb, p53, and p21) at both gene and protein levels, (b) lysosomal mediated dysfunction by using assays targeting the expression and activity of the senescence‐associated β‐galactosidase (SA‐β‐Gal) enzyme, and (c) nuclear envelope breakdown by assessing the expression of Lamin‐B1. The consequences of the senescence phenotype mediated by F. nucleatum were further assessed using wound healing assays. Our results revealed that prolonged exposure to F. nucleatum promotes an aging‐like phenotype as evidenced by the increased expression of pro‐senescence markers (p16INK4a, p21, and pRb) and SA‐β‐Gal activity and reduced expression of the counter‐balancing cascade (p14ARF and p53) and Lamin‐B1. Furthermore, we also noted impaired wound healing capacity of gingival epithelial cells upon prolong bacterial exposure, which was consistent with the senescence‐induced phenotype. Together, our findings provide a proof‐of‐concept evidence that F. nucleatum triggers a pro‐senescence response in gingival epithelial cells. This might affect periodontal tissue homeostasis by reducing its repair capacity and, consequently, increasing susceptibility to periodontitis during aging. [ABSTRACT FROM AUTHOR]

Prebiotic galacto‐oligosaccharides (GOSs) are synthesized by transgalactosylation of lactose using β‐galactosidases. Well‐established, large‐scale production of GOS is performed by stirred‐tank reactors (STR) operating in batch fashion. Main manufacturing expenses are attributed to enzyme purchase costs and the downstream operations required for subsequent enzyme inactivation and removal. In the last decades, several studies and patents have appeared on free enzyme membrane reactor (FEMR) as an economic alternative to STR due to enhanced enzyme recovery and reuse. In this review, we attempt to summarize the key design aspects, the catalytic performance and efficiency of FEMR compared to STR technology, and the main concerns related to stable product quality over long‐term operation. [ABSTRACT FROM AUTHOR]

The musculoskeletal system is one of the most affected organs by aging that correlates well with an accumulation of senescent cells as for other multiple age-related pathologies. The molecular mechanisms underpinning muscle impairment because of senescent cells are still elusive. The availability of in vitro model of skeletal muscle senescence is limited and restricted to a small panel of phenotypic features of these senescent cells in vivo. Here, we developed a new in vitro model of senescent C2C12 mouse myoblasts that, when subjected to differentiation, the resulting myotubes showed sarcopenic features. To induce senescence, we used SYUIQ-5, a quindoline derivative molecule inhibitor of telomerase activity, leading to the expression of several senescent hallmarks in treated myoblasts. They had increased levels of p21 protein accordingly with the observed cell cycle arrest. Furthermore, they had enhanced SA-βgalactosidase enzyme activity and phosphorylation of p53 and histone H2AX. SYUIQ-5 senescent myoblasts had impaired differentiation potential and the resulting myotubes showed increased levels of ATROGIN-1 and MURF1, ubiquitin ligases components responsible for protein degradation, and decreased mitochondria content, typical features of sarcopenic muscles. Myotubes differentiated from senescent myoblasts cultures release increased levels of MYOSTATIN that could affect skeletal muscle cell growth. Overall, our data suggest that a greater burden of senescent muscle cells could contribute to sarcopenia. This study presents a well-defined in vitro model of muscle cell senescence useful for deeper investigation in the aging research field to discover new putative therapeutic targets and senescence biomarkers associated with the aged musculoskeletal system. [ABSTRACT FROM AUTHOR]

Plants synthesize large amounts of stored and structural polysaccharides. Aspergillus oryzae is used in traditional Japanese fermentation and produces many types of plant polysaccharide degradation-related enzymes. The carbohydrate-active enzymes of A. oryzae are important in the fermentation process and biotechnological applications. Because plant polysaccharides have a complex structure, cooperative and synergistic actions of enzymes are crucial for the degradation of plant polysaccharides. For example, the cooperative action of isoprimeverose-producing oligoxyloglucan hydrolase, β-galactosidase, and α-xylosidase is important for the degradation of xyloglucan, and A. oryzae coordinates these enzymes at the expression level. In this review, I focus on the plant polysaccharide degradation-related enzymes identified in A. oryzae. [ABSTRACT FROM AUTHOR]

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac/LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild‐type LacI. An in‐depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild‐type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high‐value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven‐fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto‐oligosaccharides (GOS). [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is an inherited lysosomal storage disease caused by deficiency of α-galactosidase A (α-Gal A), an enzyme that hydrolyzes glycosphingolipids in lysosome. Accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in tissues, induces cellular dysfunction leading to multi-organ disorder. Gene therapy is a promising strategy that can overcome these problems, and virus vectors such as adeno-associated virus (AAV) have been used for study on gene therapy. We used human Gb3 synthetase-transgenic (TgG3S)/α-Gal A knockout (GLAko) mice. TgG3S/GLAko mice have elevated Gb3 accumulation in the major organs compared with GLAko mice, which have been widely used as a model for FD. At the age of 6 weeks, male TgG3S/GLAko were injected with 2 × 1012 vector genome AAV9 vectors containing human α-Gal A cDNA. Eight weeks after intravenous injection of AAV, α-Gal A enzymatic activity was elevated in the plasma, heart, and liver of TgG3S/GLAko mice to levels corresponding to 224%, 293%, and 105% of wild-type, respectively. Gb3 amount 8 weeks after AAV injection in the heart and liver of this group was successfully reduced to levels corresponding to 16% and 3% of untreated TgG3S/GLAko mice. Although the brain and kidney of AAV9-treated TgG3S/GLAko mice showed no significant increases in α-Gal A activity, Gb3 amount was smaller than untreated littermates (48% and 44%, respectively). In this study, systemic AAV administration did not show significant extension of the lifespan of TgG3S/GLAko mice compared with the untreated littermates. The timing of AAV injection, capsid choice, administration route, and injection volume may be important to achieve sufficient expression of α-Gal A in the whole body for the amelioration of lifespan. [ABSTRACT FROM AUTHOR]

α-Galactosidase is an important exoglycosidase belonging to the hydrolase class of enzymes, which has therapeutic and industrial potential. It plays a crucial role in hydrolyzing α-1,6 linked terminal galacto-oligosaccharide residues such as melibiose, raffinose, and branched polysaccharides such as galacto-glucomannans and galactomannans. In this study, Actinoplanes utahensis B1 was explored for α-galactosidase production, yield improvement, and activity enhancement by purification. Initially, nine media components were screened using the Plackett–Burman design (PBD). Among these components, sucrose, soya bean flour, and sodium glutamate were identified as the best-supporting nutrients for the highest enzyme secretion by A. Utahensis B1. Later, the Central Composite Design (CCD) was implemented to fine-tune the optimization of these components. Based on sequential statistical optimization methodologies, a significant, 3.64-fold increase in α-galactosidase production, from 16 to 58.37 U/mL was achieved. The enzyme was purified by ultrafiltration-I followed by multimode chromatography and ultrafiltration-II. The purity of the enzyme was confirmed by Sodium Dodecyl Sulphate–Polyacrylamide Agarose Gel Electrophoresis (SDS-PAGE) which revealed a single distinctive band with a molecular weight of approximately 72 kDa. Additionally, it was determined that this process resulted in a 2.03-fold increase in purity. The purified α-galactosidase showed an activity of 2304 U/mL with a specific activity of 288 U/mg. This study demonstrates the isolation of Actinoplanes utahensis B1 and optimization of the process for the α-galactosidase production as well as single-step purification. [ABSTRACT FROM AUTHOR]

Lactose in cheese whey is increasingly challenging to metabolise under normal conditions. The hydrolysis of whey lactose into glucose and galactose using enzymatic methods has been acclaimed to confer benefits like enhanced substrate availability for better degradation in anaerobic digestion. In the present study, whey lactose was subjected to hydrolysis using the enzyme β-galactosidase derived from Aspergillus oryzae fungus to reduce the difficulty of lipid and fat transformation in anaerobic digestion. The individual and combined effects of hydrolysis parameters, pH, enzyme load, reaction time and temperature were studied using Response Surface Methodology by Central Composite Design. The optimum conditions were determined based on variance analyses and surface plots; pH 4.63, temperature 40.47°C, reaction time 25.96 min and enzyme load 0.49%. Results showed a maximum lactose hydrolysis value of 86.21%, while the predicted value was 87.44%. Indeed, enzyme hydrolysis induced a change of soluble chemical oxygen demand around 24.6% and 75.8% reduction in volatile fatty acid concentration. Upon anaerobic digestion, the pre-hydrolysed whey revealed a 3.6-fold higher bio-methane production than that of raw hey, and a visible decrease in volatile fatty acid concentrations. The resultant data agreed with the Gompertz model, and lag phase times were significantly reduced for hydrolysed whey. [ABSTRACT FROM AUTHOR]