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Senescence is an important cellular program occurring in development, tissue repair, cancer, and aging. Increased senescence is also associated with disease states, including obesity and Type 2 diabetes (T2D). Characterizing and quantifying senescent cells at a single cell level has been challenging and particularly difficult in large primary cells, such as human adipocytes. In this study, we present a novel approach that utilizes reflected light for accurate senescence‐associated beta‐galactosidase (SABG) staining measurements, which can be integrated with immunofluorescence and is compatible with primary mature adipocytes from both human and mouse, as well as with differentiated 3T3‐L1 cells. This technique provides a more comprehensive classification of a cell's senescent state by incorporating multiple criteria, including robust sample‐specific pH controls. By leveraging the precision of confocal microscopy to detect X‐gal crystals using reflected light, we achieved superior sensitivity over traditional brightfield techniques. This strategy allows for the capture of all X‐gal precipitates in SABG‐stained samples, revealing diverse X‐gal staining patterns and improved detection sensitivity. Additionally, we demonstrate that reflected light outperforms western blot analysis for the detection and quantification of senescence in mature human adipocytes, as it offers a more accurate representation of SABG activity. This detection strategy enables a more thorough investigation of senescent cell characteristics and specifically a deeper look at the relationship between adipocyte senescence and obesity associated disorders, such as T2D. [ABSTRACT FROM AUTHOR]

Beta-galactosidase (EC 3.2.1.23), also known as lactase, is an exoglycosidase enzyme responsible for hydrolyzing the β-glycosidic bond between galactose and its organic moiety. This study focused on extracting, purifying, characterizing and immobilizing β-galactosidase from tomato leaves. The enzyme was successfully extracted using a 0.2 M sodium phosphate buffer at pH 6. To evaluate its chemical properties, the effects of temperature, pH, enzyme concentration and substrate concentration on enzyme activity were examined. The optimal temperature for β-galactosidase activity was found to be 50°C and the enzyme demonstrated the highest solubility at pH 6. The kinetic parameters of the free enzyme were determined, revealing a Michaelis constant (Km) of 0.1 mM and a maximum reaction velocity (Vmax) of 48.78 ìmol/min. These findings provide valuable insights into the enzyme's efficiency and behaviour under various conditions, which are essential for optimizing its application in industrial processes. [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]

In this research, qualitative characteristics were studied under different post-harvest treatments in Hass and Fuerte cultivars of avocado (Persea americana) fruits. The post-harvest treatments performed in fruits of these cultivars comprised Ethrel application and plastic film (membrane) covering. The measurements of qualitative characteristics were related to color; flesh consistency; measurements of titratable acidity, total soluble solids, percentage of total phenolic contents, and ascorbic peroxidase activity; and the real-time (quantitative) polymerase chain reaction (qPCR) of gene expression and enzyme activities of phenylalanine ammonia-lyase (PAL) and beta-galactosidase (β-gal). The experiments found that the application of plastic film has excellent results in retaining qualitative characteristics and enzyme activities via maintaining firmness in higher levels. The plastic film covering appeared to delay ripening without the use of chemicals and, therefore, it has the potential to extend the duration of the post-harvest life of the avocado fruit. Variations between the two cultivars were found in the measurements of total soluble solids (Fuerte cultivar showed an increase of 22%, whereas Hass cultivar showed an increase of 120% in Brix values) and total phenolic contents (Fuerte cultivar showed a decrease of 16% and Hass cultivar showed an increase of 29%). It is worth noting that PAL's activity increased significantly (over 44%), as compared to other treatments, and β-galactosidase's activity decreased, as compared to other treatments. In conclusion, plastic film covering results in a decrease in the activity of β-galactosidase, as shown by the reaction of hydrolysis (enzyme activity) but also from the expression of the related genes. [ABSTRACT FROM AUTHOR]

Lactose intolerance, which affects about 65–75% of the world's population, is caused by a genetic post-weaning deficiency of lactase, the enzyme required to digest the milk sugar lactose, called lactase non-persistence. Symptoms of lactose intolerance include abdominal pain, bloating and diarrhea. Genetic variations, namely lactase persistence, allow some individuals to metabolize lactose effectively post-weaning, a trait thought to be an evolutionary adaptation to dairy consumption. Although lactase non-persistence cannot be altered by diet, prebiotic strategies, including the consumption of galactooligosaccharides (GOSs) and possibly low levels of lactose itself, may shift the microbiome and mitigate symptoms of lactose consumption. This review discusses the etiology of lactose intolerance and the efficacy of prebiotic approaches like GOSs and low-dose lactose in symptom management. [ABSTRACT FROM AUTHOR]

The growing global market of dairy products has led to the need for alternative approaches regarding whey valorization, which is the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, thereby employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. The maximum yields of galactooligosaccharides in concentrated sweet whey (15% w/v initial lactose) and raw acid whey (3.1% w/v initial lactose) reached 34.4 and 14.7% with lactase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for lactase from Aspergillus oryzae were equal to 27.4 and 24.8% in the most concentrated sweet and acid whey, respectively, using enzyme loads of 2 U/mL in sweet whey and 1 U/mL in acid whey. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyzed lactose more rapidly and resulted in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae, and achieved in both cases a polymerization degree of up to six. [ABSTRACT FROM AUTHOR]

Fruit development and softening play pivotal roles in determining fruit quality and post-harvest shelf life in Chinese bayberry (Myrica rubra). However, the specific role of beta (β)-galactosidase, particularly β-galactosidase of M. rubra (MrBGAL), in facilitating fruit softening remains unclear. In this study, we aimed to address this gap by investigating the involvement of MrBGALs genes in fruit softening. We identified all 15 MrBGALs and conducted a comprehensive analysis, including phylogenetic relationships, gene structure, protein motifs, co-linearity, and expression patterns. Using phylogenetic analysis, we classified all MrBGALs into five distinct groups. Additionally, cis-element prediction and comparative genome analysis provided insightful clues about the functionality of MrBGALs. Transcriptome data revealed unique expression patterns of MrBGALs throughout various fruit development stages. These findings introduce valuable candidate genes that can contribute to unraveling the functions and molecular mechanisms governing fruit development and softening in Chinese bayberry. [ABSTRACT FROM AUTHOR]

This study aimed was to covalently immobilize β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes. In this study, a two-level factorial design was employed to investigate the impact of seven continuous variables (activation pH, glutaraldehyde molarity, activation time (0–8 h), buffer solution pH (8-0), buffer solution molarity, MWCNT-NH2-glutaraldehyde quantity, and stabilization time (0–180 h)) on the immobilization efficiency and enzymatic activity of protease and β-galactosidase. Furthermore, the effect of time on the percentage of enzymatic activity was examined during specific intervals (24, 48, 72, 96, and 120 h) of the immobilization process. The analysis of variance results for protease enzymatic activity revealed a notable influence of the seven variables on immobilization efficiency and enzymatic activity. Additionally, the findings indicate that activation time, buffer pH, MWCNT-NH2-glutaraldehyde quantity, and stabilization time significantly affect the activity of the protease enzyme. The interplay between buffer pH and stabilization time is also significant. Indeed, both activation time and the quantity of MWCNT-NH2-glutaraldehyde exert a reducing effect on enzyme activity. Notably, the influence of MWCNT-NH2-glutaraldehyde quantity is more significant (p

The growing global market of dairy products has led to the need for alternative approaches regarding whey valorization, which is the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, thereby employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. The maximum yields of galactooligosaccharides in concentrated sweet whey (15% w/v initial lactose) and raw acid whey (3.1% w/v initial lactose) reached 34.4 and 14.7% with lactase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for lactase from Aspergillus oryzae were equal to 27.4 and 24.8% in the most concentrated sweet and acid whey, respectively, using enzyme loads of 2 U/mL in sweet whey and 1 U/mL in acid whey. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyzed lactose more rapidly and resulted in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae, and achieved in both cases a polymerization degree of up to six.

Purpose: Meningiomas are tumours originating from meningothelial cells, the majority belonging to grade 1 according to the World Health Organization classification of the tumours of the Central Nervous System. Factors contributing to the progression to the higher grades (grades 2 and 3) have not been elucidated yet. Senescence has been proposed as a potential mechanism constraining the malignant transformation of tumours. Senescence-associated beta-galactosidase (SA-β-GAL) and inhibitors of cyclin-dependent kinases p16 and p21 have been suggested as senescence markers. Methods: We analysed 318 meningiomas of total 343 (178 grade 1, 133 grade 2 and 7 grade 3). Tissue microarrays were constructed and stained immunohistochemically, using antibodies for SA-β-GAL, p16 and p21. Results: The positive correlation of the tumour grade with the expression of p16 (p = 0.016) and SA-β-GAL (p = 0.002) was observed. The expression of p16 and SA-β-GAL was significantly higher in meningiomas grade 2 compared to meningiomas grade 1 (p = 0.006 and p = 0.004, respectively). SA-β-GAL positivity positively correlated with p16 and p21 in the whole cohort. In grade 2 meningiomas, a positive correlation was only between SA-β-GAL and p16. Correlations of senescence markers in meningiomas grade 2 were not present. Conclusion: Our findings suggest the senescence activation in meningiomas grade 2 as a potential mechanism for the restraining of tumour growth and give hope for applying of promising senolytic therapy. [ABSTRACT FROM AUTHOR]

Galacto-oligosaccharides (GOS) are products of transgalactosylation reaction of β-galactosidase when lactose is used as the substrate. These carbohydrates are considered prebiotics which stimulate beneficial effects to human health. In the present work, Saphera, a commercial preparation of β-galactosidase from Bifidobacterium bifidum, was biochemically characterised for production of GOS. Using o-nitrophenyl-β-Dgalactopyranoside (oNPG) as the substrate, optimal activity for the enzyme was found to be at pH 6.0 and at 45°C. Ten (10) mM each of either Na+ or K+ enhanced enzyme activity by 10%, while Cu2+, Zn2+, Fe2+, and EDTA showed inhibitory effect on the enzyme activity. When incubated in 50 mM sodium phosphate and pH 6.5, the enzyme was found to have half-life time of 136 ± 6 and 2.1 ± 0.2 h at 30 and 50°C, respectively. The hydrolysis activity of the enzyme predominated when the initial lactose concentration used was 5% (w/v). When initial lactose concentration was increased to 20% (w/v), maximum GOS yield obtained was 10% (w/w) achieved at 86% lactose conversion. Analysis using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the major oligosaccharides produced by the enzyme were 3'-galactosylglucose, 3'-galactosyllactose, 3'-galactobiose, and allolactose, thus showing that this enzyme would prefer to form β-(1→3)- and β-(1→6)-linked GOS. Despite its predominantly hydrolytic activity, Saphera synthesised prebiotic GOS which could be interesting to dairy industry. [ABSTRACT FROM AUTHOR]

Thyroid receptor signaling controls major physiological processes and disrupted signaling can cause severe disorders that negatively impact human life. Consequently, methods to detect thyroid receptor ligands are of great toxicologic and pharmacologic importance. Previously, we reported thyroid receptor ligand detection with cell-free protein synthesis of a chimeric fusion protein composed of the human thyroid receptor beta (hTRβ) receptor activator and a β-lactamase reporter. Here, we report a 60% reduction in sensing cost by reengineering the chimeric fusion protein biosensor to include a reporter system composed of either the full-length beta galactosidase (β-gal), the alpha fragment of β-gal (β-gal-α), or a split alpha fragment of the β-gal (split β-gal-α). These biosensor constructs are deployed using E. coli XL1-Blue cell extract to (1) avoid the β-gal background activity abundant in BL21 cell extract and (2) facilitate β-gal complementation reporter activity to detect human thyroid receptor ligands. These results constitute a promising platform for high throughput screening and potentially the portable detection of human thyroid receptor ligands. [ABSTRACT FROM AUTHOR]

Lactose intolerance, which affects about 65–75% of the world’s population, is caused by a genetic post-weaning deficiency of lactase, the enzyme required to digest the milk sugar lactose, called lactase non-persistence. Symptoms of lactose intolerance include abdominal pain, bloating and diarrhea. Genetic variations, namely lactase persistence, allow some individuals to metabolize lactose effectively post-weaning, a trait thought to be an evolutionary adaptation to dairy consumption. Although lactase non-persistence cannot be altered by diet, prebiotic strategies, including the consumption of galactooligosaccharides (GOSs) and possibly low levels of lactose itself, may shift the microbiome and mitigate symptoms of lactose consumption. This review discusses the etiology of lactose intolerance and the efficacy of prebiotic approaches like GOSs and low-dose lactose in symptom management.

The present study aimed to evaluate the probiotic potential, α-amylase and α-glucosidase inhibitory effects, and β-galactosidase production of 19 non haemolytic lactic acid bacteria and bifidobacteria previously identified and isolated from honey bee gastrointestinal tract (BGIT) of Apis mellifera intermissa, honey, propolis and bee bread. The isolates were screened according to their high resistance to lysozyme and potent antibacterial activity. Our results indicated that among the 19 isolates, Limosilactobacillus fermentum BGITE12.2, Lactiplantibacillus plantarum BGITEC13, Limosilactobacillus fermentum BGITEC5.1 and Bifidobacterium asteroides BGITOB8, isolated from BGIT exhibited a good tolerance to 100 mg/mL lysozyme (> 82%), excellent tolerance to 0.5% bile salt [survival rate (SR) ≥ 83.19% ± 0.01], and a high SR (≥ 80.0%) under gastrointestinal tract conditions. The auto-aggregation ability was high (auto-aggregation index ranging from 67.14 ± 0.16 to 92.8% ± 0.03) for L. fermentum BGITE12.2, L. plantarum BGITEC13, and B. asteroides BGITOB8, and moderate for L. fermentum BGITEC5.1 (39.08% ± 0.11). Overall, the four isolates showed moderate co-aggregation capacity with pathogenic bacteria. They exhibited from moderate to high hydrophobicity towards toluene and xylene. The safety assessment revealed that the four isolates lacked gelatinase and mucinolytic activities. Also, they were susceptible to ampicillin, clindamycin, erythromycin, and chloramphenicol. Interestingly, the four isolates showed α-glucosidase and α-amylase inhibitory activities ranging from 37.08 ± 0.12 to 57.57% ± 0.1 and from 68.30 ± 0.09 to 79.42% ± 0.09, respectively. Moreover, L. fermentum BGITE12.2, L. plantarum BGITEC13, L. fermentum BGITEC5.1 isolates exhibited β-galactosidase activity over a wide range of 52.49 ± 0.24–746.54 ± 0.25 Miller Units. In conclusion, our findings suggest that the four isolates could be potential candidates for probiotics with interesting functional properties. [ABSTRACT FROM AUTHOR]

Droplet-based microfluidic systems have emerged as powerful alternatives to conventional high throughput screening platforms, due to their operational flexibility, high-throughput nature and ability to efficiently process small fluid volumes. However, the challenges associated with performing bespoke operations on user-defined droplets often limit their utility in screening applications that involve complex workflows. To this end, the marriage of droplet- and valve-based microfluidic technologies offers the prospect of balancing the controllability of droplet manipulations and analytical throughput. In this spirit, we present a microfluidic platform that combines the capabilities of integrated microvalve technology with droplet-based sample compartmentalization to realize a highly adaptable programmable fluid handling functionality. The microfluidic device consists of a programmable formulator linked to an automated droplet generation device and storage array. The formulator leverages multiple inputs coupled to a mixing ring to produce combinatorial solution mixtures, with a peristaltic pump enabling titration of reagents into the ring with picoliter resolution. The platform allows for the execution of user-defined reaction protocols within an array of storage chambers by consecutively merging programmable sequences of pL-volume droplets containing specified reagents. The precision in formulating solutions with small differences in concentration is perfectly suited for the accurate estimation of kinetic parameters. The utility of our platform is showcased through the performance of enzymatic kinetic measurements of beta-galactosidase and horseradish peroxidase with fluorogenic substrates. The presented platform provides for a range of automated manipulations and paves the way for a more diverse range of droplet-based biological experiments. [ABSTRACT FROM AUTHOR]

β-galactosidase also known as lactase (glycoside hydrolase enzyme) has potential uses in the dairy, food, and pharmaceutical industries. Yeast β-galactosidase is an intracellular enzyme which has been isolated from various sources. In this study, different isolates of yeast were assessed for their β-galactosidase productivity, and Kluyveromyces marxianus Y72 resulted in the highest production. β-galactosidase enzyme, which hydrolyses lactose and is largely dependent on its stability under different physical conditions, such as pH, temperature and incubation time. In the present study, the yeast, Kluyveromyces marxianus Y72, was isolated from curd and showed intracellular β-galactosidase activity during the growth phase. To understand further, response surface methodology (RSM) was carried out to optimize the process parameters such as carbon source, nitrogen source, and the growth of Kluyveromyces marxianus Y72 with respect to temperature, pH, and inoculum size. The enzyme activity was maximum (2.65 U/mg dcw) at 30 ˚C, pH 6.0 and at an incubation time of 24 h with galactose (4%) as the carbon source. In conclusion, the current study revealed β-galactosidase from Kluyveromyces marxianusY72 is having significant potential for possible use in various industries. [ABSTRACT FROM AUTHOR]

Background: Previous studies have shown that bone morphogenetic protein 9 (BMP9) is almost exclusively produced in the liver and reaches tissues throughout the body as a secreted protein. However, the mechanism of BMP9 action and its role in aging-associated liver injury and inflammation are still unclear. Results: Aging significantly aggravates acetaminophen (APAP)-induced acute liver injury (ALI). Increased expression of CCAAT/enhancer binding protein α (C/EBPα) and BMP9 was identified in aged livers and in hepatocytes and macrophages (MФs) isolated from aged mice. Further analysis revealed that excess BMP9 was directly related to APAP-induced hepatocyte injury and death, as evidenced by activated drosophila mothers against decapentaplegic protein 1/5/9 (SMAD1/5/9) signaling, an increased dead cell/total cell ratio, decreased levels of ATG3 and ATG7, blocked autophagy, increased senescence‐associated beta‐galactosidase (SA‐β‐Gal) activity, and a higher rate of senescence‐associated secretory phenotype (SASP) acquisition. In contrast, Bmp9 knockout (Bmp9−/−) partially alleviated the aforementioned manifestations of BMP9 overexpression. Moreover, BMP9 expression was found to be regulated by C/EBPα in vitro and in vivo. Notably, BMP9 also downregulated autophagy through its effect on autophagy-related genes (ATG3 and ATG7) in MΦs, which was associated with aggravated liver injury and SASP acquisition. Conclusions: In summary, the present study highlights the crucial roles played by C/EBPα-BMP9 crosstalk and provides insights into the interrelationship between hepatocytes and MΦs during acute liver injury. [ABSTRACT FROM AUTHOR]

The Salvia L. genus (Lamiaceae) is largely used in the pharmaceutical and food industry. Several species of biological relevance are extensively employed in traditional medicine, including Salvia aurea L. (syn. S. africana-lutea L.), which is used as a traditional skin disinfectant and in wounds as a healing remedy; nevertheless, these properties have not been validated yet. The aim of the present study is to characterise S. aurea essential oil (EO), unveiling its chemical composition and validating its biological properties. The EO was obtained by hydrodistillation and subsequently analysed by GC-FID and GC-MS. Different biological activities were assessed: the antifungal effect on dermatophytes and yeasts and the anti-inflammatory potential by evaluating nitric oxide (NO) production and COX-2 and iNOS protein levels. Wound-healing properties were assessed using the scratch-healing test, and the anti-aging capacity was estimated through the senescence-associated beta-galactosidase activity. S. aurea EO is mainly characterised by 1,8-cineole (16.7%), β-pinene (11.9%), cis-thujone (10.5%), camphor (9.5%), and (E)-caryophyllene (9.3%). The results showed an effective inhibition of the growth of dermatophytes. Furthermore, it significantly reduced protein levels of iNOS/COX-2 and simultaneously NO release. Additionally, the EO exhibited anti-senescence potential and enhanced wound healing. Overall, this study highlights the remarkable pharmacological properties of Salvia aurea EO, which should be further explored in order to develop innovative, sustainable, and environmentally friendly skin products. [ABSTRACT FROM AUTHOR]

On February 23rd 1936, a boy-child (“Kn”) died in an asylum near Munich after years of severe congenital dis-ease, which had profoundly impaired his development leading to inability to walk, talk and see as well as to severe epilepsy. While a diagnosis of “Little’s disease” was made during life, his postmortem brain investiga-tion at Munich neuropathology (“Deutsche Forschungsanstalt für Psychiatrie”) revealed the diagnosis of “amaurotic idiocy” (AI). AI, as exemplified by Tay-Sachs-Disease (TSD), back then was not yet understood as a specific inborn error of metabolism encompassing several disease entities. Many neuropathological studies were performed on AI, but the underlying processes could only be revealed by new scientific techniques such as biochemical analysis of nervous tissue, deciphering AI as nervous system lipid storage diseases, e.g. GM2-gangliosidosis. In 1963, Sandhoff & Jatzkewitz published an article on a “biochemically special form of AI” reporting striking differences when comparing their biochemical observations of hallmark features of TSD to tissue composition in a single case: the boy Kn. This was the first description of “GM1-Gangliosidosis”, later understood as resulting from genetically determined deficiency in beta-galactosidase. Here we present illus-trative materials from this historic patient, including selected diagnostic slides from the case “Kn” in virtual microscopy, original records and other illustrative material available. Finally, we present results from genetic analysis performed on archived tissue proving beta-galactosidase-gene mutation, verifying the 1963 interpre-tation as correct. This synopsis shall give a first-hand impression of this milestone finding in neuropathology.

Steroid-induced ocular hypertension can be seen even after trabecular meshwork (TM) bypass/ablation. Thus, the purpose was to investigate steroid-response in cells distal to the TM by using primary scleral fibroblasts. Primary scleral cell cultures were generated using mid-depth scleral wedges from human donor corneo-scleral rims (n = 5) after corneal transplantation. Cells were treated with dexamethasone (DEX; 100 nM) and compared to media (MED)/vehicle (DMSO) controls. Cell size, shape, and migration were studied using the IncuCyte Live-Cell Analysis System. Cytoskeleton was compared using Alexa Fluor-568 Phalloidin and senescence tested by evaluating beta-galactosidase. Western blot comparison was performed for α-SMA, FKBP-51, fibronectin, phospho-myosin light chain, and myocilin. Scleral fibroblasts upregulated FKBP-51 in response to DEX indicating the existence of steroid-responsive pathways. Compared to controls, DEX-treated cells proliferated slower (~50%; p

Enzymatic hydrolysis of lactose is a crucial step to improve the efficiency and selectivity of membrane-based separations toward the recovery of milk oligosaccharides free from simple sugars. Response surface methodology was used to investigate the effects temperature (25.9 to 54.1 °C) and amount of enzyme (0.17 to 0.32% w/w) at 1, 2, and 4 h of reaction on the efficiency of lactose hydrolysis by Aspergillus oryzae β-galactosidase, preservation of major goat whey oligosaccharides, and on the de-novo formation of oligosaccharides. Lactose hydrolysis above 99% was achieved at 1, 2, and 4 h, not being significantly affected by temperature and amount of enzyme within the tested conditions. Formation of 4 Hexose (Hex) and 4 Hex 1 Hex and an increased de-novo formation of 2 Hex 1 N-Acetyl-Neuraminic Acid (NeuAc) and 2 Hex 1 N-Glycolylneuraminic acid (NeuGc) was observed in all treatments. Overall, processing conditions using temperatures ≤40 °C and enzyme concentration ≤0.25% resulted in higher preservation/formation of goat whey oligosaccharides.

Bovine milk oligosaccharides (BMOs) that resemble human milk oligosaccharides are found in whey permeate, indicating that dairy streams can be used as a potential source of bioactive oligosaccharides. Recovery of oligosaccharides from whey permeate is hindered by their low abundance and high concentration of lactose. In the present work, lactose in bovine colostrum whey permeate was hydrolyzed by Aspergillus oryzae β-galactosidase to facilitate subsequent monosaccharide removal by membrane separation. Chromatographic separation coupled with high-resolution mass spectrometry revealed β-galactosidase degradation of several β-linkage-containing BMOs and production of novel oligosaccharides that ranged in size from 5 to 11 monosaccharide units containing several galactose repeating units and N-acetylhexosamine at their reducing ends. Optimization of BMO hydrolysis and separation methodology could generate high amounts of hetero-oligosaccharides for improved recovery of potentially biotherapeutic oligosaccharides.

Trees can control their shape and resist gravity by producing tension wood (TW), which is a special wood that results from trees being put under stress. TW is characterized by the presence of a gelatinous layer (G layer) and the differential distribution of cell wall polymers. In this study, we investigated whether or not gravistimulation in N. cadamba resulted in TW with an obvious G layer. The results revealed an absence of an obvious G layer in samples of the upper side of a leaning stem (UW), as well as an accumulation of cellulose and a decrease in lignin content. A negligible change in the content of these polymers was recorded and compared to untreated plant (NW) samples, revealing the presence of a G layer either in much lower concentrations or in a lignified form. A transcriptomic investigation demonstrated a higher expression of cell wall esterase- and hydrolase-related genes in the UW, suggesting an accumulation of noncellulosic sugars in the UW, similar to the spectroscopy results. Furthermore, several G-layer-specific genes were also downregulated, including fasciclin-like arabinogalactan proteins (FLA), beta-galactosidase (BGAL) and chitinase-like proteins (CTL). The gene coexpression network revealed a strong correlation between cell-wall-synthesis-related genes and G-layer-synthesis-specific genes, suggesting their probable antagonistic role during G layer formation. In brief, the G layer in N. cadamba was either synthesized in a very low amount or was lignified during an early stage of growth; further experimental validation is required to understand the exact mechanism and stage of G layer formation in N. cadamba during gravistimulation. [ABSTRACT FROM AUTHOR]

Background: Texture quality is impotent for melon (Cucumis melo L.) fruit. β-galactosidase (β-Gal, EC 3.2.1.23) is an important cell wall glycosyl hydrolase involved in fruit softening, However, the β-Gal gene (BGALs) family hasn't been identified genome-wide in melon. Thus, it's necessary to conduct an in-depth bioinformatic analysis on melon BGALs family and to seek out the key members who participated in melon fruit softening. Results: A total of 21 BGALs members designated as CmBGAL1-CmBGAL21 were identified genome-wide in melon, clustered into A-G seven clades. Among them, three duplications CmBGAL1:CmBGAL3, CmBGAL19:CmBGAL21, and CmBGAL20:CmBGAL21 happened. For conserved domains, besides the Glyco_hydro_35 domain (PF01301), all the members also contained the GHD domain (PF17834) except for CmBGAL12, and the Gal_Lectin (PF02140) domain existed in most CmBGALs at the C-termini. Motifs, protein secondary and tertiary structure analysis showed that the CmBGAL12 is a unique member. Moreover, protein-protein association network analysis showed that the CmBGAL12 is the only node protein. Furthermore, spatiotemporal expression pattern analysis by quantitative real-time PCR (qRT-PCR) suggested that most of CmBGALs expressed in tissues with vigorous cell wall remodeling/disassembly. In addition, cis-acting regulatory elements analysis in promoters inferred that CmBGALs might participate in diverse responsiveness to phytohormone, biotic and abiotic signaling. Conclusions: A novel clade of CmBGAL members (Clade F) related to melon fruit softening was discovered, since their expression showed a specific surge in the mature fruit of 'HPM' with mealy texture (softening sharply), but not in 'HDB' with crisp texture (softening bluntly). The homologous CmBGAL7–11 in Clade F exhibited identical spatiotemporal expression patterns may multiple genes leading to melon fruit softening. [ABSTRACT FROM AUTHOR]

This study aimed to assess metabolic responses and senescent cell burden in young female mice induced to estropause and treated with senolytic drugs. Estropause was induced by 4-vinylcyclohexene diepoxide (VCD) injection in two-month-old mice. The senolytics dasatinib and quercetin (D + Q) or fisetin were given by oral gavage once a month from five to 11 months of age. VCD-induced estropause led to increased body mass and reduced albumin concentrations compared to untreated cyclic mice, without affecting insulin sensitivity, lipid profile, liver enzymes, or total proteins. Estropause decreased catalase activity in adipose tissue but had no significant effect on other redox parameters in adipose and hepatic tissues. Fisetin treatment reduced ROS levels in the hepatic tissue of estropause mice. Estropause did not influence senescence-associated beta-galactosidase activity in adipose and hepatic tissues but increased senescent cell markers and fibrosis in ovaries. Senolytic treatment did not decrease ovarian cellular senescence induced by estropause. Overall, the findings suggest that estropause leads to minor metabolic changes in young females, and the senolytics D + Q and fisetin had no protective effects despite increased ovarian senescence. [ABSTRACT FROM AUTHOR]

Lactococcus lactis is an important industrial microorganism and a widely used model object for research in the field of lactic acid bacteria (LAB) biology. The development of new L. lactis and related LAB strains with improved properties, including phage-resistant strains for dairy fermentation, LAB-based vaccines or strains with altered genotypes for research purposes, are hindered by the lack of genome-editing tools that allow for the easy and straightforward incorporation of a significant amount of the novel genetic material, such as large genes or operons, into the chromosomes of these bacteria. We recently employed a suggested system based on the CRISPR–Cas-associated transposon for the editing of the L. lactis genome. After the in-depth redesign of the system, we were able to achieve the stable incorporation of the fragments that were sized up to 10 kbp into the L. lactis beta-galactosidase gene. The efficiency of editing under the optimized conditions were 2 × 10−4 and 4 × 10−5 for 1 kbp and 10 kbp, respectively, which are sufficient for fast and easy modifications if a positive selection marker can be used. [ABSTRACT FROM AUTHOR]

β‐galactosidase (β‐gal) is one of the most prevalent markers of gene expression. Its activity can be monitored via optical and fluorescence microscopy, electrochemistry, and many other ways after slight modification using protein engineering. Here, we have constructed a chimeric version that incorporates a streptococcal protein G domain at the N‐terminus of β‐gal that binds immunoglobins, namely IgG. This protein G: β‐galactosidase fusion enables β‐gal‐based spectrophotometric and electrochemical measurements of IgG. Moreover, our results show linearity over an industrially relevant range. We demonstrate applicability with rapid spectroelectrochemical detection of IgG in several formats including using an electrochemical sensing interface that is rapidly assembled directly onto electrodes for incorporation into biohybrid devices. The fusion protein enables sensitive, linear, and rapid responses, and in our case, makes IgG measurements quite robust and simple, expanding the molecular diagnostics toolkit for biological measurement. [ABSTRACT FROM AUTHOR]

BackgroundThe complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain.MethodsTo identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1+ neurons in both wild type mice and a mouse model of Alzheimer's disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis.ResultsWhile C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1+ neurons, the brains of C1qa FL/FL :Cx3cr1 CreERT2 mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 CreERT2/WganJ mice. C1q expression in C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice relative to controls, and C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice had minimal, if any, reduction in plasma C1q.ConclusionsThus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 CreERT2/WganJ deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodegenerative disorders.

The enzyme β-galactosidase, commonly known as lactase has been isolated, purified and characterized from various microbial, plant, animal and recombinant sources. The enzyme is a tetrameric structure with four identical chains. β-galactosidase is of immense importance because of wide applications in food industries. The enzyme is used in the production of lactose-free milk and milk-related products. Deficiency of β-galactosidase in the human body may lead to lactose intolerance, therefore its intake can combat problems associated with lactose intolerance. The enzyme is not only employed for the manufacture of lactosefree products but also used for the production of galacto-oligosaccharides (GOSs), an important class of prebiotics. The enzyme can be exploited for biosensor development and other biotechnological applications. This review focuses on structure and sources of β-galactosidase, its properties, purification techniques and a panoramic view of various applications. The timeline is limited post-2010. [ABSTRACT FROM AUTHOR]

Goat milk is considered a suitable matrix for the successful incorporation of probiotics, also obtaining new lactose-free fermented products can expand its use. This study aimed to develop and characterize formulations of lactose-free probiotic fermented goat dairy beverages as well as to determine the most appropriate concentration of red jambo pulp to be added. The beverages were developed with different concentrations of lactose-free goat milk and frozen jambo pulp (12, 15 and 18% w/v) and lyophilized (3, 6 and 9% w/v), corresponding to formulations F1 to F6, respectively, as source of bioactive compounds. Probiotics counts decreased significantly (from 8.58 to 7.38 log CFU mL−1). The formulation with a higher proportion of lyophilized (F6) pulp showed the highest levels of phenolic compounds (72.08 mg GAE 100 g−1), anthocyanins (50.80 mg cyanidin-3-glycoside 100 g−1), ascorbic acid (41.68 mg 100 g−1), and antioxidant activity (16.21 μmol TE g−1) (P < 0.05). On the other hand, F3 presented the highest global acceptance and purchase intention (P < 0.05). However, the principal component analysis (PCA) indicated that the components related to bioactive compounds (PC1) stood out on sensory attributes (PC3 and PC4) and, therefore, F6 was most appropriate for obtaining a lactose-free goat probiotic fermented milk with improved bioactive properties targeting lactose intolerant consumers and those who are allergic to bovine milk proteins. [ABSTRACT FROM AUTHOR]

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Lactose intolerance affects ∼65% of the global adult population, leading to the demand for lactose-free products. The enzyme β-galactosidase (βG) is commonly used in the industry to produce such products, but its recovery after lactose hydrolysis is challenging. In this scenario, the study aims to encapsulate βG within capsules, varying in dimensions and wall materials, to ensure their suitability for efficient industrial recovery. The enzyme βG was encapsulated through ionic gelation using alginate and its blends with pectin, maltodextrin, starch, or whey protein as wall materials. The capsules produced underwent evaluation for encapsulation efficiency, release profiles, activity of the βG enzyme, and the decline in enzyme activity when reused over multiple cycles. Alginate at 5% wt/vol concentrations, alone or combined with polymers such as maltodextrin, starch, or whey protein, achieved encapsulation efficiencies of ∼98%, 98%, 80%, and 88%, respectively. The corresponding enzyme recovery rates were 34%, 19%, 31%, and 48%. Capsules made with an alginate-pectin blend exhibited no significant hydrolysis and maintained an encapsulation efficiency of 79%. Encapsulation with alginate alone demonstrated on poor retention of enzyme activity, showing a loss of 74% after just 4 cycles of reuse. Conversely, when alginate was mixed with starch or whey protein concentrate, the loss of enzyme activity was less than 40% after 4 reuses. These results highlight the benefits of combining encapsulation materials to improve enzyme recovery and reuse, offering potential economic advantages for the dairy industry., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Sphingobacterium multivorum is a ubiquitous environmental microbe in nature, particularly abundant in soil and water sources. Recently, S. multivorum has been considered to be a potential opportunistic pathogen in humans. We report the 5,9540,064-bp genome sequence of S. multivorum strain FW102 , isolated from drinking fountain water and predicted to have multiple copies of β-galactosidase genes., Competing Interests: The authors declare no conflict of interest.

ABSTRACT The interest and exploration of biodiversity in subsurface ecosystems have increased significantly during the last 2 decades. The aim of this study was to investigate the in vitro probiotic properties of spore-forming bacteria isolated from deep caves. Two hundred fifty spore-forming microbes were enriched from sediment samples from 10 different pristine caves in Algeria at different depths. Isolates showing nonpathogenic profiles were screened for their potential to produce digestive enzymes (gliadinase and beta-galactosidase) in solid and liquid media, respectively. Different probiotic potentialities were studied, including (i) growth at 37°C, (ii) survival in simulated gastric juice, (iii) survival in simulated intestinal fluid, and (iv) antibiotic sensitivity and cell surface properties. The results showed that out of 250 isolates, 13 isolates demonstrated nonpathogenic character, probiotic potentialities, and ability to hydrolyze gliadin and lactose in solution. These findings suggest that a selection of cave microbes might serve as a source of interesting candidates for probiotics. IMPORTANCE Previous microbial studies of subsurface ecosystems like caves focused mainly on the natural biodiversity in these systems. So far, only a few studies focused on the biotechnological potential of microbes in these systems, focusing in particular on their antibacterial potential, antibiotic production, and, to some extent, enzymatic potential. This study explores whether subsurface ecosystems can serve as an alternative source for microbes relevant to probiotics. The research focused on the ability of cave microbes to degrade two substrates (lactose and gliadin) that cause common digestive disorders. Since these enzymes may prove to be useful in food processing and in reducing the effect of lactose and gliadin digestion within intolerant patients, isolation of microbes such as in this study may expand the possibilities of developing alternative strategies to deal with these intolerances.

Cellular senescence is a hallmark of aging and a promising target for therapeutic approaches. The identification of senescent cells requires multiple biomarkers and complex experimental procedures, resulting in increased variability and reduced sensitivity. Here, we propose a simple and broadly applicable imaging flow cytometry (IFC) method. This method is based on measuring autofluorescence and morphological parameters and on applying recent artificial intelligence (AI) and machine learning (ML) tools. We show that the results of this method are superior to those obtained measuring the classical senescence marker, senescence-associated beta-galactosidase (SA-β-Gal). We provide evidence that this method has the potential for diagnostic or prognostic applications as it was able to detect senescence in cardiac pericytes isolated from the hearts of patients affected by end-stage heart failure. We additionally demonstrate that it can be used to quantify senescence "in vivo" and can be used to evaluate the effects of senolytic compounds. We conclude that this method can be used as a simple and fast senescence assay independently of the origin of the cells and the procedure to induce senescence. [ABSTRACT FROM AUTHOR]

Ascorbic acid (AsA) or Vitamin C is an antioxidant molecule and plays an important role in many biological processes in plants. GDP-D-mannose pyrophosphorylase (GMP or VTC1) catalyzes the synthesis of GDP-D-mannose, which is a precursor for AsA production and is used for cell wall polysaccharide and glycoprotein synthesis. In rice, the OsVTC1 gene consists of three homologs, including OsVTC1-1, OsVTC1-3 and OsVTC1-8. In this study, we characterized wild type (WT) and OsVTC1-1 RNAi lines (RI1-2 and RI1-3) and showed that the transcript levels of most genes in the AsA synthesis pathway, AsA content and leaf anatomical parameters in RNAi lines were reduced, revealing that OsVTC1-1 is involved in AsA synthesis. To further study the role of OsVTC1-1 gene, cell wall monosaccharide composition, transcriptome and proteome were compared, with specific attention paid to their wild type and OsVTC1-1 RNAi lines. Mannose and galactose composition (mole%) were decreased in OsVTC1-1 RNAi lines. Additionally, reduction of cell wall-associated proteins, such as kinesin, expansin, beta-galactosidase and cellulose synthase were observed in OsVTC1-1 RNAi lines. Our results suggest that OsVTC1-1 gene plays an important role in AsA synthesis and in cell wall-related processes. [ABSTRACT FROM AUTHOR]

Beta-galactosidases (β-GALs) hold a key role in both fruit softening and the increase of total soluble solids during maturation. Despite determining both quality and potential postharvest longevity, β-GAL activity during ripening, with a special focus on the postharvest period, has not been adequately addressed in a spatial and temporal manner. This study focused on the regulation of gene expression in relation to the total β-GAL enzyme activity during the ripening of tomato fruit attached on the plant, as well as harvested fruit ripened for 5 d at 4, 10, or 25 °C. The transcription of genes coding for β-GAL isoenzymes was significantly affected by both the fruit maturation stage (unripe vs. red ripe) and postharvest storage temperature. Cold stressed tomatoes (4 °C) exhibited a remarkably higher transcription of most β-GAL genes compared to on-plant red ripe fruit and to fruit exposed to either 10 or 25 °C, indicating a low temperature response. However, enzymatic activity and water-soluble pectin content increased with elevated temperature exposure, peaking in fruit stored at 25 °C. β-GAL activity was present in the pericarp, while it was less detected in locular parenchyma. These findings highlight the dual role of β-GAL not only in maturation, but also in the metabolism during postharvest homeostasis and cold acclimation of tomato fruit. [ABSTRACT FROM AUTHOR]

Galactooligosaccharides (GOS), as a type of prebiotics, have excellent physical and chemical properties, and can be widely used in the pharmaceutical and food fields. Recently, the microbial β-galactosidases have gained widespread attentions in industrial GOS production. However, most β-galactosidases from microorganisms have low transgalactosylation activity, resulting in poor GOS yield of enzymatic transformation from lactose. In this paper, a brand new β-galactosidase derived from Enterobacter cloaca e Zjut HJ2001 was screened out from soil, and successfully overexpressed, characterized, and mutated by combinatorial alanine-scanning and site-saturation mutagenesis. Compared to the yield of 51.73 % obtained by wild-type β-galactosidase with lactose concentration of 380 g/L, the obtained mutant β-gal-H542V achieved a higher GOS yield of 67.08 %, which was the highest in the reported literature. These results suggested that the developed mutagenesis strategy could improve the transgalactosylation efficiency, and the mutant β-gal-H542V could be regarded as a prospective biocatalyst for GOS industrial manufacturing. [Display omitted] • A novel β-galactosidase derived from E. cloaca e Zjut HJ2001 was screened out from soil. • β-galactosidase was successfully overexpressed, characterized, and mutated. • The obtained mutant β-gal-H542V achieved a highest GOS yield of 67.08 %. • β-gal-H542V would be a promising biocatalyst for GOS production. [ABSTRACT FROM AUTHOR]

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence-associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.

Two developments have greatly facilitated the construction of CCN mutant mouse strains. The first is the availability of modified embryonic stem (ES) cells and mice developed through several large-scale government-sponsored research programs. The second is the advent of CRISPR/Cas9 technology. In this chapter, we describe the available mouse strains generated by gene targeting techniques and the CCN targeting vectors and genetically modified ES cells that are available for the generation of CCN mutant mice. Many of these mutant mouse lines and ES cells carry a β-galactosidase reporter that can be used to track CCN expression, facilitating phenotypic analysis and revealing new sites of CCN action. Therefore, we also describe a method for β-galactosidase staining.

Cellular senescence and ferroptosis are the two main, fine-tuned processes in tissue damage restraint; however, they can be overactivated in pathologies such as nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH), becoming dangerous stimuli. Senescence is characterized by a decline in cell division and an abnormal release of reactive oxygen species (ROS), and ferroptosis is represented by iron deposition associated with an excessive accumulation of ROS. ROS and cellular stress pathways are also drivers of NAFLD/NASH development. The etiology of NAFLD/NASH lies in poor diets enriched in fat and sugar. This food regimen leads to liver steatosis, resulting in progressive degeneration of the organ, with a late onset of irreversible fibrosis and cirrhosis. Few studies have investigated the possible connection between senescence and ferroptosis in NAFLD/NASH progression, despite the two events sharing some molecular players. We hypothesized a possible link between senescence and ferroptosis in a NAFLD background. To thoroughly investigate this in the context of “Western-style” diet (WSD) abuse, we used an amylin-modified liver NASH mouse model. The main NASH hallmarks have been confirmed in this model, as well as an increase in apoptosis, and Ki67 and p53 expression in the liver. Senescent beta-galactosidase-positive cells were elevated, as well as the expression of the related secretory molecules Il-6 and MMP-1. Features of DNA damage and iron-overload were found in the livers of NASH mice. Gpx4 (glutathione peroxidase 4) expression, counteracting ferroptotic cell death, was increased. Notably, an increased number of senescent cells showing overexpression of gpx4 was also found. Our data seem to suggest that senescent cells acquire a gpx4-mediated mechanism of ferroptosis resistance and thus remain in the liver, fostering the deterioration of liver fitness.

Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box–Behnken design to model and optimize the parameters of β-galactosidase entrapment in sol–gel-derived silica composites. We have also demonstrated the influence of polymer–polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on β-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum β-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of β-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey. [ABSTRACT FROM AUTHOR]

Detection and quantification of senescent cells remain difficult due to variable phenotypes and the absence of highly specific and reliable biomarkers. It is therefore widely accepted to use a combination of multiple markers and cellular characteristics to define senescent cells in vitro. The exact choice of these markers is a subject of ongoing discussion and usually depends on objective reasons such as cell type and treatment conditions, as well as subjective considerations including feasibility and personal experience. This study aims to provide a comprehensive comparison of biomarkers and cellular characteristics used to detect senescence in melanocytic systems. Each marker was assessed in primary human melanocytes that overexpress mutant BRAFV600E, as it is commonly found in melanocytic nevi, and melanoma cells after treatment with the chemotherapeutic agent etoposide. The combined use of these two experimental settings is thought to allow profound conclusions on the choice of senescence biomarkers when working with melanocytic systems. Further, this study supports the development of standardized senescence detection and quantification by providing a comparative analysis that might also be helpful for other cell types and experimental conditions. [ABSTRACT FROM AUTHOR]

O principal carboidrato do leite é a lactose e a sua absorção ocorre devido à hidrólise enzimática, gerando glicose e galactose. A intolerância à lactose é a redução da capacidade de hidrólise intestinal devido à hipolactasia, gerando a necessidade do consumo de alimentos lácteos com baixo teor deste carboidrato. As enzimas beta-galactosidase são utilizadas nas indústrias de laticínios para hidrolisar a lactose, proporcionando ao consumidor intolerante a possibilidade de ingerir os produtos lácteos sem prejuízos à saúde. Para quantificar o conteúdo de carboidratos resultante da hidrólise enzimática, são utilizados métodos analíticos alternativos e oficiais. O objetivo deste estudo foi avaliar a hidrólise enzimática de duas enzimas industriais distintas produzidas pelos microrganismos Bacillus licheniformis e Kluyveromyces lactis, através de três métodos analíticos: método enzimático, crioscopia e HPLC. A inteligência artificial foi utilizada para melhorar o controle dos processos industriais. Após a adição das enzimas ao leite desnatado, foi realizada a cinética de tempo coletando as amostras no tempo 0, a cada 10 minutos até completar 1 hora de reação e a cada 30 minutos até o fechamento das 5 horas de reação de hidrólise. Em 97% dos casos observados a diminuição da concentração de lactose por HPLC acompanhou o aprofundamento do ponto crioscópico. As medições de glicose por absorbância e HPLC foram correlacionadas (r = 0,79; p < 0,01), mas não concordantes (p < 0,01). Concluiu-se que, por meio da inteligência artificial, é possível estimar indiretamente a concentração de lactose a partir de um algoritmo que associa a crioscopia e a concentração de glicose.

Yeast’s beta-galactosidase is an intracellular enzyme, through which it is possible to determine in vivo its activity as a biocatalyst in the lactose hydrolysis. Permeabilization process was used for transforming the microorganisms cells into biocatalysts with an enhanced enzyme activity. The potential application of this enzyme technology in industrial process depends mainly on the enzyme activity. Beta-galactosidase enzyme that hydrolyzes lactose, for instance, is largely dependent on the reaction time and its stability under different physical conditions, such as pH, temperature and enzyme concentration. The objective of this study was to optimize the cellular permeabilization process of Kluyveromyces marxianus CCT 3172 and Saccharomyces fragilis CCT 7586 cultured in cheese whey for lactose hydrolysis. Box-Behnken design was carried out for cell permeabilization with three independent variables, ethanol concentration, permeabilization time and temperature. The best permeability conditions for K. marxianus CCT 3172 were 27% (v v-1) ethanol, 3 min at 20ºC, with specific enzymatic activity of 0.98 U mg-1. For S. fragilis CCT 7586, a specific enzymatic activity of 1.31 U mg-1 was achieved using 45% (v v-1) of ethanol, 17 min. of reaction under 17ºC. Thus, it was concluded that cellular permeabilization with ethanol is an efficient process to determine beta-galactosidase activity.

UnlabelledAs neural structures grow in size and increase metabolic demand, the CNS vasculature undergoes extensive growth, remodeling, and maturation. Signals from neural tissue act on endothelial cells to stimulate blood vessel ingression, vessel patterning, and acquisition of mature brain vascular traits, most notably the blood-brain barrier. Using mouse genetic and in vitro approaches, we identified retinoic acid (RA) as an important regulator of brain vascular development via non-cell-autonomous and cell-autonomous regulation of endothelial WNT signaling. Our analysis of globally RA-deficient embryos (Rdh10 mutants) points to an important, non-cell-autonomous function for RA in the development of the vasculature in the neocortex. We demonstrate that Rdh10 mutants have severe defects in cerebrovascular development and that this phenotype correlates with near absence of endothelial WNT signaling, specifically in the cerebrovasculature, and substantially elevated expression of WNT inhibitors in the neocortex. We show that RA can suppress the expression of WNT inhibitors in neocortical progenitors. Analysis of vasculature in non-neocortical brain regions suggested that RA may have a separate, cell-autonomous function in brain endothelial cells to inhibit WNT signaling. Using both gain and loss of RA signaling approaches, we show that RA signaling in brain endothelial cells can inhibit WNT-β-catenin transcriptional activity and that this is required to moderate the expression of WNT target Sox17. From this, a model emerges in which RA acts upstream of the WNT pathway via non-cell-autonomous and cell-autonomous mechanisms to ensure the formation of an adequate and stable brain vascular plexus.Significance statementWork presented here provides novel insight into important yet little understood aspects of brain vascular development, implicating for the first time a factor upstream of endothelial WNT signaling. We show that RA is permissive for cerebrovascular growth via suppression of WNT inhibitor expression in the neocortex. RA also functions cell-autonomously in brain endothelial cells to modulate WNT signaling and its downstream target, Sox17. The significance of this is although endothelial WNT signaling is required for neurovascular development, too much endothelial WNT signaling, as well as overexpression of its target Sox17, are detrimental. Therefore, RA may act as a "brake" on endothelial WNT signaling and Sox17 to ensure normal brain vascular development.

A novel enzyme-induced metallization colorimetric assay is developed to monitor and measure beta-galactosidase (β-gal) activity, and is further employed for colorimetric bacteriophage (phage)-enabled detection of Escherichia coli (E. coli). This assay relies on enzymatic reaction-induced silver deposition on the surface of gold nanorods (AuNRs). In the presence of β-gal, the substrate p-aminophenyl β-d-galactopyranoside is hydrolyzed to produce p-aminophenol (PAP). Reduction of silver ions by PAP generates a silver shell on the surface of AuNRs, resulting in the blue shift of the longitudinal localized surface plasmon resonance peak and multicolor changes of the detection solution from light green to orange-red. Under optimized conditions, the detection limit for β-gal is 128 pM, which is lower than the conventional colorimetric assay. Additionally, the assay has a broader dynamic range for β-gal detection. The specificity of this assay for the detection of β-gal is demonstrated against several protein competitors. Additionally, this technique is successfully applied to detect E. coli bacteria cells in combination with bacteriophage infection. Due to the simplicity and short incubation time of this enzyme-induced metallization colorimetric method, the assay is well suited for the detection of bacteria in low-resource settings.