Metal-organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the ...selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely β-glucosidase, invertase, β-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH
, or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes.
Summary
Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent ...cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.
Development of "smart" noninvasive bioimaging probes for trapping specific enzyme activities is highly desirable for cancer therapy in vivo. Given that β-galactosidase (β-gal) is an important ...biomarker for cell senescence and primary ovarian cancers, we design an enzyme-activatable ratiometric near-infrared (NIR) probe (DCM-βgal) for the real-time fluorescent quantification and trapping of β-gal activity in vivo and in situ. DCM-βgal manifests significantly ratiometric and turn-on NIR fluorescent signals simultaneously in response to β-gal concentration, which makes it favorable for monitoring dynamic β-gal activity in vivo with self-calibration in fluorescent mode. We exemplify DCM-βgal for the ratiometric tracking of endogenously overexpressed β-gal distribution in living 293T cells via the lacZ gene transfection method and OVCAR-3 cells, and further realize real-time in vivo bioimaging of β-gal activity in colorectal tumor-bearing nude mice. Advantages of our system include light-up ratiometric NIR fluorescence with large Stokes shift, high photostability, and pH independency under the physiological range, allowing for the in vivo real-time evaluation of β-gal activity at the tumor site with high-resolution three-dimensional bioimaging for the first time. Our work provides a potential tool for in vivo real-time tracking enzyme activity in preclinical applications.
Senescence-associated diseases have severely diminished the quality of life and health of patients. However, a sensitive assay of these diseases remains limited due to a lack of straightforward ...methods. Considering that senescence-associated β-galactosidase (SA-β-Gal) is overexpressed in senescent cells, the detection of SA-β-Gal in senescent cells and tissues might be a feasible strategy for the early diagnosis of SA diseases. In this study, a β-galactosidase-activatable nanoprobe
was developed for the imaging of senescent cells and vasculature in atherosclerotic mice via real-time monitoring of β-Gal.
was fabricated by encapsulating a newly designed NIR ratiometric probe
within a poly(lactic-
-glycolic) acid (PLGA) core. Nanoprobe
showed good accumulation in arteries, thus successfully visualizing senescent cells and vasculature in atherosclerotic mice by tail vein injection. Our findings indicated that nanoprobe
holds great potential for the early diagnosis and therapy of atherosclerosis and other aging-associated diseases.
Reactivity based fluorescent probes have been widely investigated as a powerful and noninvasive tool for disease diagnosis in recent years. β-Galactosidase (β-gal), one of the typical lysosomal ...glycosidases, is reported to be a vital biomarker overexpressed in primary ovarian cancer cells. Fluorescent probes with excellent performance for endogenous β-gal detection offer a unique option for visualization and diagnosis of primary ovarian cancer cells. Herein, a near-infrared fluorescent probe Lyso-Gal with lysosome-targeting ability was developed for lysosomal β-gal detection and imaging in ovarian cancer cells (SKOV-3 cells). Lyso-Gal exhibits weak fluorescence in aqueous solution but emits bright NIR fluorescence at 725 nm after incubation with β-gal. Highly selective imaging of ovarian cancer cells has been achieved upon incubation with Lyso-Gal for only 1 min. The detection time is extremely short. In comparison with a similar hemicyanine probe, Hx-Gal, without lysosome-targeting ability, Lyso-Gal realizes endogenous β-gal visualization in lysosomes and shows brighter fluorescence than Hx-Gal in SKOV-3 cells. This work demonstrates the potential of Lyso-Gal for detection of primary ovarian cancer cells by using β-gal as the biomarker.
In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and ...single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.
α-Galactosidase, (E.C. 3.2.1.22) is an exoglycosidase that target galactooligosaccharides such as raffinose, melibiose, stachyose and branched polysaccharides like galactomannans and ...galacto-glucomannans by catalysing the hydrolysis of α-1,6 linked terminal galactose residues. The enzyme has been isolated and characterized from microbial, plant and animal sources. This ubiquitous enzyme possesses physiological significance and immense industrial potential. Optimization of the growth conditions and efficient purification strategies can lead to a significant increase in the enzyme production. To boost commercial productivity, cloning of novel α-galactosidase genes and their heterologous expression in suitable host has gained popularity. Enzyme immobilization leads to its greater reutilization, superior thermostability, pH tolerance and increased activity. The enzyme is well explored in food industry in the removal of raffinose family oligosaccharides (RFOs) in soymilk and sugar crystallization process. It also improves animal feed quality and biomass processing. Applications of the enzyme is in the area of biomedicine includes therapeutic advances in treatment of Fabry disease, blood group conversion and removal of α-gal type immunogenic epitopes in xenotransplantation. With considerable biotechnological applications, this enzyme has been vastly commercialized and holds greater future prospects.
A bioactive synthetic porous shell was engineered to enable cells to survive in an oligotrophic environment. Eukaryotic cells (yeast) were firstly coated with a β‐galactosidase (β‐gal), before ...crystallization of a metal–organic framework (MOF) film on the enzyme coating; thereby producing a bioactive porous synthetic shell. The β‐gal was an essential component of the bioactive shell as it generated nutrients (that is, glucose and galactose) required for cell viability in nutrient‐deficient media (lactose‐based). Additionally, the porous MOF coating carried out other vital functions, such as 1) shielding the cells from cytotoxic compounds and radiation, 2) protecting the non‐native enzymes (β‐gal in this instance) from degradation and internalization, and 3) allowing for the diffusion of molecules essential for the survival of the cells. Indeed, this bioactive porous shell enabled the survival of cells in simulated extreme oligotrophic environments for more than 7 days, leading to a decrease in cell viability less than 30 %, versus a 99 % decrease for naked yeast. When returned to optimal growth conditions the bioactive porous exoskeleton could be removed and the cells regained full growth immediately. The construction of bioactive coatings represents a conceptually new and promising approach for the next‐generation of cell‐based research and application, and is an alternative to synthetic biology or genetic modification.
Thick skinned: Saccharomyces cerevisiae cells were coated with a bioactive nanoporous shell based on β‐galactosidase and a metal–organic framework (MOF). This bioactive shell enables biocatalyzed formation of glucose from environmental lactose, showing cell survival in simulated extreme nutrient‐depleted environments.
Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an ...X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified.
ClinicalTrials.gov NCT01196871.
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