ß-Galactosidase-activated near-infrared AIEgen for ovarian cancer imaging in vivo.

Near-infrared (NIR) aggregation induced-emission luminogens (AIEgens) circumvent the noisome aggregation-caused quenching (ACQ) effect in physiological milieu, thus holding high promise for real-time and sensitive imaging of biomarkers in vivo. ß-Galactosidase (ß-Gal) is a biomarker for primary ovarian carcinoma, but current AIEgens for ß-Gal sensing display emissions in the visible region and have not been applied in vivo. We herein propose an NIR AIEgen QM-TPA-Gal and applied it for imaging ß-Gal activity in vitro and in ovarian tumor model. After being internalized by ovarian cancer cells (e.g., SKOV3), the hydrophilic nonfluorescent QM-TPA-Gal undergoes hydrolyzation by ß-Gal to yield hydrophobic QM-TPA-OH, which subsequently aggregates into nanoparticles to turn NIR fluorescence "on" through the AIE mechanism. In vitro experimental results indicate that QM-TPA-Gal has a sensitive and selective response to ß-Gal with a limit of detection (LOD) of 0.21 U/mL. Molecular docking simulation confirms that QM-TPA-Gal has a good binding ability with ß-Gal to allow efficient hydrolysis. Furthermore, QM-TPA-Gal is successfully applied for ß-Gal imaging in SKOV3 cell and SKOV3-bearing living mouse models. It is anticipated that QM-TPA-Gal could be applied for early diagnosis of ovarian cancers or other ß-Gal-associated diseases in near future.

Betaine postpones hyperglycemia-related senescence in ovarian and testicular cells: Involvement of RAGE and ß-galactosidase.

The structural and functional disorders of the testis and ovary are one of the main complications of hyperglycemia. Betaine is a trimethyl glycine with antioxidant, antidiabetic, and anti-inflammatory potential. The aim of this study is to investigate the potential of betaine on the expression of aging and oxidative stress markers in ovarian and testicular cells under hyperglycemic conditions. Testicular and ovarian cells were subjected to four different conditions, including normal glucose and hyperglycemia, with or without betaine (5 mM). The cells with hyperglycemia saw an increase in malondialdehyde (MDA), methylglyoxal (MGO), expression of a receptor for AGE, and aging-related genes (ß-GAL), and a decrease in the activity of antioxidant enzymes including catalase, glutathione peroxidase, and superoxide dismutase. The treatment with betaine, in contrast, decreased the amount of MGO and MDA, and also downregulated aging-related signaling. Although hyperglycemia induces senescence in testicular and ovarian cells, the use of betaine may have a protective effect against the cell senescence, which may be useful in the management of infertility.

GV1001 reduces neurodegeneration and prolongs lifespan in 3xTg-AD mouse model through anti-aging effects.

GV1001, which mimics the activity of human telomerase reverse transcriptase, protects neural cells from amyloid beta (Aß) toxicity and other stressors through extra-telomeric function, as noted in our prior in vitro studies. As per a recent phase II clinical trial, it improves cognitive function in patients with moderate to severe dementia. However, the underlying protective mechanisms remain unclear. This study aimed to investigate the effects of GV1001 on neurodegeneration, senescence, and survival in triple transgenic Alzheimer's disease (3xTg-AD) mice. GV1001 (1 mg/kg) was subcutaneously injected into old 3xTg-AD mice thrice a week until the endpoint for sacrifice, and survival was analysed. Magnetic resonance imaging (MRI) and Prussian blue staining (PBS) were performed to evaluate entry of GV1001 entrance into the brain. Diverse molecular studies were performed to investigate the effect of GV1001 on neurodegeneration and cellular senescence in AD model mice, with a particular focus on BACE, amyloid beta1-42 (Aß1-42), phosphorylated tau, volume of dentate gyrus, ß-galactosidase positive cells, telomere length, telomerase activity, and ageing-associated proteins. GV1001 crossed the blood-brain barrier, as confirmed by assessing the status of ferrocenecarboxylic acid-conjugated GV1001 using magnetic resonance imaging and PBS. GV1001 increased the survival of 3xTg-AD mice. It decreased BACE and Aß1-42 levels, neurodegeneration (i.e., reduced CA1, CA3 and dentate gyrus volume, decreased levels of senescence-associated ß-galactosidase positive cells, and increased telomere length and telomerase activity), and levels of ageing-associated proteins. We suggest that GV1001 exerts anti-ageing effects in 3xTg-AD mice by reducing neurodegeneration and senescence, which contributes to improved survival.

Chitosan-based oral hydrogel formulations of ß-galactosidase to improve enzyme supplementation therapy for lactose intolerance.

ß-Galactosidase supplementation plays an important role in the life of people with lactose intolerance. However, these formulations are rendered ineffective by the low pH and pepsin in the stomach and pancreatic proteases in the intestine. Therefore, it is necessary to develop oral transport systems for carrying this enzyme in the active form up to the intestine, where the lactose digestion occurs. In this research, a new hydrogel was developed that could potentially be used for enzyme supplement therapy. In this regard, the chitosan-based ß-Gal formulations described in the manuscript are an alternative long-acting preparation to the so far available preparations that allow for enzyme protection and mucosal targeting. These hydrogels were prepared from chitosan and polyethylene glycol and contained a covalently immobilized ß-galactosidase from Aspergillus oryzae. The ß-galactosidase in the hydrogel was protected from degradation in a gastric medium at a pH of 2.5 and retained 75 % of its original activity under subsequent intestinal conditions. In the case of a simulated gastric fluid with a pH of 1.5, a copolymer containing methacrylic acid functional groups was sufficient to protect the hybrid hydrogel from the extremely acidic pH. In addition, the surface of the hydrogel was chemically modified with thiol and amidine groups, which increased the binding to intestinal mucin by 20 % compared with the unmodified hydrogel. These results represent a promising approach for oral transport as a reservoir for ß-galactosidase in the small intestine to reduce the symptoms of hypolactasia.

Evaluation of Bis(2-ethylhexyl) phthalate toxicity on the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg9.

Bis(2-ethylhexyl) phthalate, generally known as DEHP is a synthetic compound mainly used as a plasticizer to make polyvinyl chloride products flexible and soft. The present work aimed to study the toxicity of Bis(2-ethylhexyl) phthalate on the third instar larvae of transgenic Drosophila melanogaster(hsp70-lacZ) Bg9. The hsp70 gene is associated with the ß-galactosidase in our present transgenic strain therefore, the more activity of ß-galactosidase will indirectly correspond to hsp70 expression. The third instar larvae were allowed to feed on the diet for 24 h having 0.001, 0.005, 0.01, and 0.02 M of Bis(2-ethylhexyl) phthalate at the final concentration. After the exposure of 24hrs, the larvae were subjected to ONPG assay, X-gal staining, trypan blue exclusion test, oxidative stress markers assays, and comet assay. A dose-dependent increase in hsp70 expression, tissue damage, Glutathione-S-transferase (GST) activity, lipid peroxidation, monoamine oxidase, caspase-9 & 3, protein carbonyl content (PCC), DNA damage and decrease in the glutathione (GSH) content, delta-aminolevulinic acid dehydrogenase (ẟ-ALD-D) and acetylcholinesterase activity were observed in the larvae exposed to 0.005, 0.01, 0.02 M of Bis-(2-ethylhexyl) phthalate. The dose of 0.001 M of Bis(2-ethylhexyl) phthalate did not showed any toxic effects and hence can be considered as No Observed Adverse Effect Level (NOAEL) for Bis(2-ethylhexyl) phthalate. The study supports the use of Drosophila for the evaluation of possible toxic effects associated with synthetic compounds.

Oncogene-induced senescence in meningiomas-an immunohistochemical study.

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-ß-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-ß-GAL, p16 and p21. RESULTS: The positive correlation of the tumour grade with the expression of p16 (p = 0.016) and SA-ß-GAL (p = 0.002) was observed. The expression of p16 and SA-ß-GAL was significantly higher in meningiomas grade 2 compared to meningiomas grade 1 (p = 0.006 and p = 0.004, respectively). SA-ß-GAL positivity positively correlated with p16 and p21 in the whole cohort. In grade 2 meningiomas, a positive correlation was only between SA-ß-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.

The Potential of Mesenchymal Stem/Stromal Cell Therapy in Mustard Keratopathy: Discovering New Roads to Combat Cellular Senescence.

Mesenchymal stem/stromal cells (MSCs) are considered a valuable option to treat ocular surface disorders such as mustard keratopathy (MK). MK often leads to vision impairment due to corneal opacification and neovascularization and cellular senescence seems to have a role in its pathophysiology. Herein, we utilized intrastromal MSC injections to treat MK. Thirty-two mice were divided into four groups based on the exposure to 20 mM or 40 mM concentrations of mustard and receiving the treatment or not. Mice were clinically and histopathologically examined. Histopathological evaluations were completed after the euthanasia of mice after four months and included hematoxylin and eosin (H&E), CK12, and beta-galactosidase (ß-gal) staining. The treatment group demonstrated reduced opacity compared to the control group. While corneal neovascularization did not display significant variations between the groups, the control group did register higher numerical values. Histopathologically, reduced CK12 staining was detected in the control group. Additionally, ß-gal staining areas were notably lower in the treatment group. Although the treated groups showed lower severity of fibrosis compared to the control groups, statistical difference was not significant. In conclusion, it seems that delivery of MSCs in MK has exhibited promising therapeutic results, notably in reducing corneal opacity. Furthermore, the significant reduction in the ß-galactosidase staining area may point towards the promising anti-senescence potential of MSCs.

Cloning, Expression, Purification, and Characterization of a Novel ß-Galactosidase/α-L-Arabinopyranosidase from Paenibacillus polymyxa KF-1.

Glycosidases are essential for the industrial production of functional oligosaccharides and many biotech applications. A novel ß-galactosidase/α-L-arabinopyranosidase (PpBGal42A) of the glycoside hydrolase family 42 (GH42) from Paenibacillus polymyxa KF-1 was identified and functionally characterized. Using pNPG as a substrate, the recombinant PpBGal42A (77.16 kD) was shown to have an optimal temperature and pH of 30 °C and 6.0. Using pNPαArap as a substrate, the optimal temperature and pH were 40 °C and 7.0. PpBGal42A has good temperature and pH stability. Furthermore, Na+, K+, Li+, and Ca2+ (5 mmol/L) enhanced the enzymatic activity, whereas Mn2+, Cu2+, Zn2+, and Hg2+ significantly reduced the enzymatic activity. PpBGal42A hydrolyzed pNP-ß-D-galactoside and pNP-α-L-arabinopyranoside. PpBGal42A liberated galactose from ß-1,3/4/6-galactobiose and galactan. PpBGal42A hydrolyzed arabinopyranose at C20 of ginsenoside Rb2, but could not cleave arabinofuranose at C20 of ginsenoside Rc. Meanwhile, the molecular docking results revealed that PpBGal42A efficiently recognized and catalyzed lactose. PpBGal42A hydrolyzes lactose to galactose and glucose. PpBGal42A exhibits significant degradative activity towards citrus pectin when combined with pectinase. Our findings suggest that PpBGal42A is a novel bifunctional enzyme that is active as a ß-galactosidase and α-L-arabinopyranosidase. This study expands on the diversity of bifunctional enzymes and provides a potentially effective tool for the food industry.

Validation of a highly sensitive HaloTag-based assay to evaluate the potency of a novel class of allosteric ß-Galactosidase correctors.

Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology is a disease-agnostic drug discovery tool that can be applied to any protein target of interest with a known three-dimensional structure. We used this proprietary technology to identify and characterize the therapeutic potential of structurally targeted allosteric regulators (STARs) of the lysosomal hydrolase ß-galactosidase (ß-Gal), which is deficient due to gene mutations in galactosidase beta 1 (GLB1)-related lysosomal storage disorders (LSDs). The biochemical HaloTag cleavage assay was used to monitor the delivery of wildtype (WT) ß-Gal and four disease-related ß-Gal variants (p.Ile51Thr, p.Arg59His, p.Arg201Cys and p.Trp273Leu) in the presence and absence of two identified STAR compounds. In addition, the ability of STARs to reduce toxic substrate was assessed in a canine fibroblast cell model. In contrast to the competitive pharmacological chaperone N-nonyl-deoxygalactonojirimycin (NN-DGJ), the two identified STAR compounds stabilized and substantially enhanced the lysosomal transport of wildtype enzyme and disease-causing ß-Gal variants. In addition, the two STAR compounds reduced the intracellular accumulation of exogenous GM1 ganglioside, an effect not observed with the competitive chaperone NN-DGJ. This proof-of-concept study demonstrates that the SEE-Tx® platform is a rapid and cost-effective drug discovery tool for identifying STARs for the treatment of LSDs. In addition, the HaloTag assay developed in our lab has proved valuable in investigating the effect of STARs in promoting enzyme transport and lysosomal delivery. Automatization and upscaling of this assay would be beneficial for screening STARs as part of the drug discovery process.

Purification of Galacto-oligosaccharide (GOS) by fermentation with Kluyveromyces lactis and Interaction between GOS and casein under simulated acidic fermentation conditions.

In the enzymatic synthesis of galacto-oligosaccharide (GOS), the primary by-products include glucose, galactose and unreacted lactose. This This study was aimed to provide a method to to purify GOS by yeat fermentation and explore the interaction between GOS and CAS with a view for expanding the prospects of GOS application in the food industry. The crude GOS(25.70 g/L) was purified in this study using the fermentation method with Kluyveromyces lactis CICC 1773. Optimal conditions for purification with the yeast were 75 g/L of the yeast inoculation rate and 50 g/L of the initial crude GOS concentration for 12 h of incubation. After removing ethanol produced by yeast by low-temperature distillation, GOS content could reach 90.17%. A study of the interaction between GOS and casein (CAS) in a simulated acidic fermentation system by D-(+)-gluconic acid δ-lactone (GDL) showed that the GOS/CAS complexes with higher GOS concentrations, e.g., 4% and 6% (w/v), was more viscoelastic with higher water-holding capacity, but decreased hardness, elasticity, and cohesiveness at 6% (w/v) of GOS. The addition of GOS to CAS suspension significantly caused (p<0.05) decreased particle sizes of the formed GOS/CAS complexes, and the suspension system became more stable. FT-IR spectra confirmed the existence of different forms of molecular interactions between CAS and GOS, e.g., hydrogen bonding and hydrophobic interaction, and the change of secondary structure after CAS binding to GOS.

Expression and Characterization of a ß-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

ß-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal ß-D-galactose residues in ß1,3-, ß1,4- or ß1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first ß-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the ß-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal ß1,3- and ß1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the ß-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed ß-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of ß-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

Sialidase NEU3 action on GM1 ganglioside is neuroprotective in GM1 gangliosidosis.

GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal ß-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual ß-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack ß-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.

The development of a broad-spectrum retaining ß-exo-galactosidase activity-based probe.

Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.

An NIR Fluorescence Turn-on and MRl Bimodal Probe for Concurrent Real-time in vivo Sensing and Labeling of ß-Galactosidase.

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel ß-galactosidase (ß-gal) activated bimodal imaging probe that combines near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real-time visualization of activity in living organisms. Upon ß-gal activation, Gal-Cy-Gd-1 exhibits a remarkable 42-fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high-resolution MRI. The unique features of Gal-Cy-Gd-1 enable real-time and precise visualization of ß-gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self-immobilizing target enzymes or proteins provides a robust approach for visualizing ß-gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real-time in vivo imaging of enzyme activity and localization.

Micro- and nanochamber array system for single enzyme assays.

Arrays of small reaction containers, ranging from 624 femtoliters (10-15 L) to 270 attoliters (10-18 L), for capturing a single enzyme molecule and measuring the activity were developed along with a new reversible sealing system based on a pneumatic valve actuator made of polydimethylsiloxane (PDMS). The valve was actuated by PBS solution, effectively preventing evaporation of the solution from the micro- and nanochambers and allowing the assay to be performed over a long period of time. The hydrolysis rates of ß-D-galactosidase (ß-gal), kcat, were decreased according to the decrease of the chamber size, and the overall tendency seems to be symmetrically related to the specific surface area of the chambers even under the prevented condition of non-specific adsorption. The spatial localization of the protons in the chambers, which might could affect the dissociation state of the proteins, was also investigated to explain the decrease in the hydrolysis rate. The developed chamber system developed here may be useful for artificially reproducing the confined intracellular environment and molecular crowding conditions.

Dual 6Pß-Galactosidase/6Pß-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1.

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving ß-galactosidase (ß-gal) and 6Pß-galactosidase (6Pß-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pß-gal gene, the 11 GH1 family proteins, in which their 6Pß-glucosidase (6Pß-glc) activity was experimentally demonstrated,, were assayed for 6Pß-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pß-gal activity. The sequence comparison of this dual 6Pß-gal/6Pß-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pß-gal/6Pß-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pß-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pß-gal activity with possible relevance for lactose maldigestion management.

Efficient secreted expression of natural intracellular ß-galactosidase from Bacillus aryabhattai via non-classical protein secretion pathway in Bacillus subtilis.

In this study, the natural intracellular ß-galactosidase (lacZBa) from Bacillus aryabhattai was expressed extracellularly in Bacillus subtilis. Sec and Tat signal peptides from different secretion pathways were incorporated to facilitate extracellular secretion of lacZBa, resulting in a yield of only 0.54 U/mL. Interestingly, it was discovered that lacZBa could be efficiently expressed and secreted in B. subtilis via a non-classical secretory pathway without the need for a signal peptide. The extracellular activity and secretion ratio were 5.3 U/mL and 65 %, respectively. Compared to E. coli, the expression of lacZBa in B. subtilis resulted in increased acid resistance and higher pH stability and thermostability, with a 1.7-fold increase in half-life at 50 °C and pH 6.0. Additionally, we combined single-factor experiments and response surface methodology to enhance extracellular expression of ß-galactosidase in shake-flasks. The resulting optimal medium contained 4.46 % glucose, 1.47 % corn steep liquor, 1.5 % beef extract, 0.82 % CaCl2, and 0.1 % MgSO4. Under optimal conditions, the yield of extracellularly secreted ß-galactosidase at the shake flask level was 17.41 U/mL, representing a 32.2-fold increase in initial extracellular enzyme activity. This study represents the first successful report of natural intracellular ß-galactosidase being expressed through the non-classical secretory pathway in B. subtilis.

Tetracycline-mediated fluorescence correlates with passage number and ß-galactosidase activity in HeLa and HEK293T cells.

We report data consistent with tetracycline-mediated fluorescence having the potential to be an effective marker of senescence in immortalised cells. HeLa cells that had previously undergone more than 20 passages were transiently transfected with a plasmid encoding a novel tetracycline-inducible transgene featuring an open reading frame for green fluorescent protein. While characterising the performance of this plasmid and transfection procedure, HeLa cell fluorescence was observed to result from incubating cells with media containing 2 µg/ml tetracycline alone, without plasmid or transfection reagent. To investigate this phenomenon further, HeLa and HEK293T cells were purchased from a tissue culture collection and after cultivation over 4-23 passages, incubated with media containing 2 µg/ml tetracycline. For both cell lines, tetracycline-mediated fluorescence increase correlated with passage number increase. This effect in HeLa and HEK293T cells was also borne out by expression of ß-galactosidase activity, an imperfect but widely used marker of cellular senescence. These data suggest tetracycline may have utility as a marker of cellular senescence in immortal cells and can inform future investigation and validation of this previously unreported application for this reagent.

Glutaraldehyde-pea protein grafted polysaccharide matrices for functioning as covalent immobilizers.

Three polysaccharide matrices (κ-Carrageenan (Carr), gellan gum, and agar) were grafted via glutaraldehyde (GA) and pea protein (PP). The grafted matrices covalently immobilized ß-D-galactosidase (ß-GL). Nonetheless, grafted Carr acquired the topmost amount of immobilized ß-GL (iß-GL). Thus, its grafting process was honed via Box-Behnken design and was further characterized via FTIR, EDX, and SEM. The optimal GA-PP-Carr grafting comprised processing Carr beads with 10% PP dispersion of pH 1 and 25% GA solution. The optimal GA-PP-Carr beads acquired 11.44 Ug-1 iß-GL with 45.49% immobilization efficiency. Both free and GA-PP-Carr iß-GLs manifested their topmost activity at the selfsame temperature and pH. Nonetheless, the ß-GL Km and Vmax values were reduced following immobilization. The GA-PP-Carr iß-GL manifested good operational stability. Moreover, its storage stability was incremented where 91.74% activity was offered after 35 storage days. The GA-PP-Carr iß-GL was utilized to degrade lactose in whey permeate with 81.90% lactose degradation efficiency.

SRF inhibitors reduce prostate cancer cell proliferation through cell cycle arrest in an isogenic model of castrate-resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) is challenging to treat, despite improvements with next-generation anti-androgens such as enzalutamide, due to acquired resistance. One of the mechanisms of such resistance includes aberrant activation of co-factors of the androgen receptor (AR), such as the serum response factor (SRF), which was associated with prostate cancer progression and resistance to enzalutamide. Here, we show that inhibition of SRF with three small molecules (CCG-1423, CCG-257081 and lestaurtinib), singly and in combination with enzalutamide, reduces cell viability in an isogenic model of CRPC. The effects of these inhibitors on the cell cycle, singly and in combination with enzalutamide, were assessed with western blotting, flow cytometry and ß-galactosidase staining. In the androgen deprivation-sensitive LNCaP parental cell line, a synergistic effect between enzalutamide and all three inhibitors was demonstrated, while the androgen deprivation-resistant LNCaP Abl cells showed synergy only with the lestaurtinib and enzalutamide combination, suggesting a different mechanism of action of the CCG series of compounds in the absence and presence of androgens. Through analysis of cell cycle checkpoint proteins, flow cytometry and ß-galactosidase staining, we showed that all three SRF inhibitors, singly and in combination with enzalutamide, induced cell cycle arrest and decreased S phase. While CCG-1423 had a more pronounced effect on the expression of cell cycle checkpoint proteins, CCG-257081 and lestaurtinib decreased proliferation also through induction of cellular senescence. In conclusion, we show that inhibition of an AR co-factors, namely SRF, provides a promising approach to overcoming resistance to AR inhibitors currently used in the clinic.