Structure-antioxidant activity relationship of xylooligosaccharides obtained from carboxyl-reduced glucuronoarabinoxylans from bamboo shoots.

Xylooligosaccharides (XOs) have shown high potential as prebiotics with nutritional and health benefits. In this work, XOs were obtained from highly purified, carboxy-reduced glucuronoarabinoxylans by treatment with Driselase®. The mixtures were fractionated, and the structures were elucidated by methylation analysis and NMR spectroscopy. Antioxidant activity was determined by the methods of DPPH and ß-carotene/linoleic acid. It was found that the most active oligosaccharides (P3 and G3) comprised 4 or 5 xylose units, plus two arabinoses and one 4-O-methylglucose as side chains, their sequence of units was determined. The optimal concentration for their use as antioxidants was 2 mg/mL. The synthetic antioxidant butylated hydroxytoluene (BHT, 0.2 mg/mL) showed a percentage of inhibition 15% higher than P3. Although its concentration was ∼10 times higher, P3 is non-toxic, and could have great advantages as food additive. These results show that pure XOs exert significant antioxidant activity, only due to their carbohydrate nature.

Scutellarin-loaded pH/H2O2 dual-responsive polymer cyclodextrin mesoporous silicon framework nanocarriers for enhanced cancer therapy.

Stimulus-responsive nanomaterials, particularly with targeting capabilities, have garnered significant attention in the cancer therapy. However, the biological safety of these innovative materials in vivo remains unknown, posing a hurdle to their clinical application. Here, a pH/H2O2 dual-responsive and targeting nano carrier system (NCS) was developed using core shell structure of Fe3O4 mesoporous silicon (MSN@Fe3O4) as main body, scutellarin (SCU) as antitumor drug and polymer cyclodextrin (PCD) as molecular switch (denoted as PCD@SCU@MSN@Fe3O4, abbreviated as NCS). The NCS, with an average particle size of 100 nm, displayed exceptional SCU loading capacity, a result of its uniform radial channel structure. The in vitro investigation under condition of pH and H2O2 indicated that NCS performed excellent pH/H2O2-triggered SCU release behavior. The NCS displayed a higher cytotoxicity against tumor cells (Huh7 and HCT116) due to its pH/H2O2 dual-triggered responsiveness, while the PCD@MSN@Fe3O4 demonstrated lower cytotoxicity for both Huh7 and HCT116 cells. In vivo therapeutic evaluation of NCS indicates significant inhibition of tumor growth in mouse subcutaneous tumor models, with no apparent side-effects detected. The NCS not only enhances the bioavailability of SCU, but also utilizes magnetic targeting technology to deliver SCU accurately to tumor sites. These findings underscore the substantial clinical application potential of NCS.

Identifying Anti-NSCLC Bioactive Compounds in Scutellaria via 2D NMR-Based Metabolomic Analysis of Pharmacologically Classified Crude Extracts.

We presented a strategy utilizing 2D NMR-based metabolomic analysis of crude extracts, categorized by different pharmacological activities, to rapidly identify the primary bioactive components of TCM. It was applied to identify the potential bioactive components from Scutellaria crude extracts that exhibit anti-non-small cell lung cancer (anti-NSCLC) activity. Four Scutellaria species were chosen as the study subjects because of their close phylogenetic relationship, but their crude extracts exhibit significantly different anti-NSCLC activity. Cell proliferation assay was used to assess the anti-NSCLC activity of four species of Scutellaria. 1H-13C HSQC spectra were acquired for the chemical profiling of these crude extracts. Based on the pharmacological classification (PCA, OPLS-DA and univariate hypothesis test) were performed to identify the bioactive constituents in Scutellaria associated with the anti-NSCLC activity. As a result, three compounds, baicalein, wogonin and scutellarin were identified as bioactive compounds. The anti-NSCLC activity of the three potential active compounds were further confirmed via cell proliferation assay. The mechanism of the anti-NSCLC activity by these active constituents was further explored via flow cytometry and western blot analyses. This study demonstrated 2D NMR-based metabolomic analysis of pharmacologically classified crude extracts to be an efficient approach to the identification of active components of herbal medicine.

Hydrolysates containing xylooligosaccharides produced by different strategies: Structural characterization, antioxidant and prebiotic activities.

This study explores the structural characterization, antioxidant and prebiotic activities of hydrolysates containing xylooligosaccharides (XOS) produced by different strategies: direct fermentation of beechwood xylan (FermBX) and enzymatic treatment of beechwood (EnzBX) and rice husk (EnzRH) xylans. EnzBX and EnzRH showed XOS with a backbone of (1 â†’ 4)-linked-xylopyranosyl residues and branches of arabinose, galactose, and uronic acids. FermBX presented the highest content of total phenolic compounds (14 mg GAE/g) and flavonoids (0.6 mg QE/g), which may contribute to its antioxidant capacity -39.1 µmol TE/g (DPPH), 45.7 µmol TE/g (ABTS), and 79.9 µmol Fe II/g (FRAP). The fermentation of hydrolysates decreased the abundance of microorganisms associated with intestinal diseases from Eubacteriales, Desulfovibrionales and Methanobacteriales orders, while stimulating the growth of organisms belonging to Bacteroides, Megamonas and Limosilactobacillus genera. The production of short-chain fatty acids, ammonia, and CO2 suggested the prebiotic potential. In conclusion, hydrolysates without previous purification and obtained from non-chemical approaches demonstrated promising biological activities for further food applications.

Evaluation of nutraceutical application of xylooligosaccharide enzymatically produced from cauliflower stalk for its value addition through a sustainable approach.

There is increasing attention on the exploration of waste feedstocks as economically viable substrates for the production of prebiotic oligosaccharides, especially xylooligosaccharides, as excellent candidates for the maintenance and promotion of gut microbiota. XOS, an emerging prebiotic that has several functional attributes and beneficial health effects, is mainly produced by different processes, especially enzymatic hydrolysis through the valorisation of xylan enriched lignocellulosic materials. The present study deals with the enzymatic production of xylooligosaccharide (XOS) from xylan rich cauliflower stalk, a novel source. Delignification with alkali (NaOH) was found to be more efficient than acid and autohydrolysis, resulting in a higher extraction yield of xylan (18.42%). Alkaline extraction for 120 minutes at 1.25 M alkali concentration produced maximum xylan yield. FTIR analysis of xylan extracted from cauliflower stalk by an alkaline (NaOH) pretreatment method showed typical absorption bands at 1729 cm-1 that correspond to acetyl groups exhibiting the typical xylan specific band. Enzymatic hydrolysis was carried out with indigenously produced crude endoxylanase obtained from Aspergillus niger MTCC 9687 and the effects of substrate concentration, enzyme concentration, pH, time and temperature were investigated. High resolution MS analysis showed the presence of xylobiose as the major XOS. The major 1H spectral signals of XOS liberated from enzymatically hydrolysed alkali extracted cauliflower stalk xylan showed the presence of ß-anomeric protons in the spectral region of 4.0-4.7 ppm. Prebiotic efficacy of cauliflower stalk derived XOS alone and synbiotic combinations with known probiotic strains (Lactiplantibacillus plantarum, Bifidobacterium bifidum, Lactobacillus delbrueckii ssp. Helveticus) were evaluated. Butyrate was found to be the major short chain fatty acid produced by XOS supplemented fermentation media. All the synbiotic combinations showed significantly higher antioxidant and antimicrobial activities and reduced the viability of human bone cancer MG-63 cells. The individual profiles of antimicrobial components of XOS were identified as dihydroxy benzoic acid and aspartic acid by HPLC coupled to a photodiode array detector.

Scutellarin Suppresses RPMI7951 Melanoma Cell Proliferation by Targeting TOPK.

BACKGROUND: T-LAK cell-Originated Protein Kinase (TOPK) belongs to the serine/threonine protein kinase family. It is highly expressed in RPMI7951 melanoma cells. Scutellarin (SCU) is an active ingredient extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. Its main physiological functions are related to its anti-inflammatory and antitumour activities. METHODS: The relationship between SCU and TOPK was assessed by molecular docking, an in vitro binding assay and an in vitro kinase assay. The effect of SCU on RPMI7951 cells was detected by MTS and soft agar assays. TOPK knockdown was induced by lentiviral infection. The TOPK downstream signalling pathway was detected by western blot and immunohistochemical analyses in vitro and in vivo. RESULTS: SCU was found to directly bind with TOPK and inhibit TOPK activity in vitro. SCU inhibited the proliferation and colony formation of RPMI7951 cells in a dose-dependent manner. Silencing TOPK decreased the sensitivity of colon cancer cells to SCU. SCU inhibited the phosphorylation levels of Extracellular Regulated protein Kinases 1/2 (ERK1/2) and histone H3 in a time- and dose-dependent manner in RPMI7951 cells. In addition, SCU inhibited the growth of xenograft tumours of RPMI7951 cells and decreased the phosphorylation levels of extracellular regulated protein kinases 1/2 and histone H3 in vivo. CONCLUSION: The results showed that SCU exerts promising antitumour effects on human RPMI7951 cells by inhibiting the activity of TOPK.

Cyclodextrin pendant polymer as an efficient drug carrier for scutellarin.

A novel ß-cyclodextrin pendant polymer (ε-PL-CD), composed of poly(ε-lysine) (ε-PL) main chain and glycine-ß-cyclodextrin (Gly-CD) side chains, was prepared by a simple two-step procedure. The ε-PL-CD was investigated as a drug carrier of hydrophobic drug scutellarin (SCU). The characterization and complexation mode of the SCU:ε-PL-CD were researched in both solution and solid state by means of photoluminescence spectroscopy, 1H and 2D NMR, X-Ray powder diffraction (XRPD), thermal gravimetric analysis, Particle size and Zeta potential. The solubility test indicated that the solubilizing ability of SCU:ε-PL-CD was significantly improved compared with SCU:ß-CD and free SCU. Besides, in vitro cell experiment, it was found that SCU:ε-PL-CD has a strong inhibitory effect on the growth and invasion of tumor cells. The present study provides useful information for ε-PL-CD as a drug carrier material.

Inhibition of glucuronomannan hexamer on the proliferation of lung cancer through binding with immunoglobulin G.

The anti-lung cancer activity of oligosaccharides derived from glucuronomannan was investigated. The inhibition of A549 cell proliferation by glucuronomannan (Gn) and its oligomers (dimer (G2), tetramer (G4) and hexamer (G6)) were concentration dependent. In vivo activities on the A549-derived tumor xenografts showed the tumor inhibition of G2, G4 and G6 were 17 %, 40 % and 46 %, respectively. Organ coefficients in nude mice showed an increase in the kidney with G4, the brain with G6, and the spleen with G6. An advanced tandem mass tag labeled proteomics approach was performed. A significant differential expression was found in 59 out of the 4371 proteins, which involved the immune system. Surface plasmon resonance (SPR) studies revealed G6 was strongly bound to immunoglobulin G. This suggests that glucuronomannan hexamer inhibits the proliferation of lung cancer through its binding to immunoglobulin.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan.

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.

Influence of temperature fluctuations on growth and recrystallization of ice crystals in frozen peeled shrimp (Litopenaeus vannamei) pre-soaked with carrageenan oligosaccharide and xylooligosaccharide.

Temperature fluctuation is a common problem in the frozen storage of shrimp products. This study investigated the influence of carrageenan oligosaccharide (CO) and xylooligosaccharide (XO) on the growth and recrystallization of ice crystals in frozen peeled shrimp exposed to temperature fluctuations. Shrimp soaked with water and 3.0% (w/v) Na4P2O7 solution were designated as the negative and positive controls, respectively. Our data revealed that both CO- and XO-soaked shrimp had significant improvements in thawing and cooking loss, myofibrillar protein content, Ca2+-ATPase activity, and textural variables when exposed to temperature fluctuations compared to control samples. Microstructural imaging indicated that soaking the shrimp in CO and XO slowed the progression of damage caused to tissue myofibrils by large ice crystals, as well as inhibited the growth and recrystallization of ice crystals in muscle tissues. SDS-PAGE analysis confirmed that treatment with the oligosaccharides exhibited marked effects on the stability of muscle proteins and inhibited the degradation of muscle proteins affected by the temperature fluctuations. Based on these data, we hypothesize that the incorporated CO and XO may bind to muscle proteins and capture water molecules in the myofibrillar network through hydrogen bonding, thereby suppressing the myofibrillar denaturation and tissue structure destruction induced by the growth and recrystallization of ice crystals.

In Silico and In Vitro Anti-Helicobacter Pylori Effects of Combinations of Phytochemicals and Antibiotics.

Helicobacter pylori infection is a WHO class 1 carcinogenic factor of gastric adenocarcinoma. In the past decades, many studies have demonstrated the increasing trend of antibiotic resistance and pointed out the necessity of new effective treatment. This study was aimed at identifying phytochemicals that can inhibit H. pylori and possibly serve as adjuvant treatments. Here, in silico molecular docking and drug-like properties analyses were performed to identify potential inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase. These three enzymes are targets of the treatment of H. pylori. Susceptibility and synergistic testing were performed on the selected phytochemicals and the positive control antibiotic, amoxicillin. The in-silico study revealed that oroxindin, rosmarinic acid and verbascoside are inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase, respectively, in which, oroxindin has the highest potency against H. pylori, indicated by a minimum inhibitory concentration (MIC) value of 50 µg/mL. A combination of oroxindin and amoxicillin demonstrated additive effects against H. pylori, as indicated by a fractional inhibitory concentration (FIC) value of 0.75. This study identified phytochemicals that deserve further investigation for the development of adjuvant therapeutic agents to current antibiotics against H. pylori.

Sensitive and reliable gas chromatography tandem mass spectrometry assay for ethyl glucuronide in neonatal meconium.

Prenatal exposure to maternal ethanol leads to serious physical and mental irreversible disabilities. Ethyl glucuronide (EtG) is a direct metabolite of alcohol and its measurement in neonatal meconium has been established as the best biomarker to assess prenatal exposure to social and excessive gestational ethanol. We developed and validated the first gas chromatography tandem mass spectrometry method to quantify EtG extracted from meconium by a simple solid phase extraction pretreatment. The method was linear from limit of quantification (2 ng/g) to 200 ng/g matrix with good determination coefficient (r2 = 0.99). Recovery of EtG from meconium was always higher than 70% and intra-assay and inter-assay precision and accuracy were always better than 10%. Robustness of the developed GC-MS/MS method was tested by analysing 150 real samples coming from a previous national epidemiological project pre-screened through an ultra-chromatography tandem mass spectrometry assay obtaining a good comparability of results obtained by the two methods.

Identification of equol-7-glucuronide-4'-sulfate, monoglucuronides and monosulfates in human plasma of 2 equol producers after administration of kinako by LC-ESI-MS.

Equol is a product formed during the biotransformation of the naturally occurring isoflavone daidzein by intestinal bacteria. The role of equol in the prevention of several hormone-dependent diseases such as prostate cancer and osteoporosis as well as vasomotor symptoms has been extensively investigated. Equol primarily occurs in the form of major metabolites such as glucuronides and sulfates, while intact equol has been detected at only ca. 1% in human plasma. However, to date, conjugated metabolites have been evaluated by measuring the free equol obtained after selective enzymatic hydrolysis. Thus, the precise types of conjugates circulating in vivo and the position(s) of the conjugation sites on the equol skeleton have yet to be clarified. Our study describes the identification of polar equol metabolites in the plasma of 2 equol-producers obtained at 8 hours after consuming 50 g of kinako (approximately 37 mg of daidzein). The structural identification of these conjugated metabolites in plasma was performed by comparison to the LC-ESI-MS n and 1H-NMR spectral data of the corresponding chemically synthesized compounds. The results of the LC-ESI-MS/MS analysis indicated that the main conjugated metabolite in plasma was (S)-equol-7-glucuronide-4'-sulfate along with lower amounts of 7- and 4'-monoglucuronides as well as 7- and 4'-monosulfates.

Impact of in vitro gastrointestinal digestion on the chemical composition, bioactive properties, and cytotoxicity of Vitis vinifera L. cv. Syrah grape pomace extract.

Grape pomace (GP) is a major byproduct worldwide, and it is well known for its bioactive compounds, such as fibers and phenolic compounds, that are popular for their impact upon human health, including gastrointestinal health. The objective of this work was to evaluate the chemical composition and biological activities of an enzymatic GP extract, as well as to investigate how gastrointestinal digestion (GID) modulates these properties. GP extract was previously produced using an enzymatic cocktail with xylanase activity and was then exposed to simulated conditions of GID, characterized for its chemical composition, and screened for antimicrobial, prebiotic, and antioxidant activities. The safety of this ingredient after GID was also assessed. GP extract presented high contents of dietary fiber and other carbohydrates, including xylooligosaccharides, in addition to minerals and phenolic compounds. In vitro simulated GID revealed that xylobiose was resistant to gastric conditions, unlike phenolic compounds. The use of 2% (w/v) of this ingredient proved to be a potential carbon source that could be fermented by Lactobacillus and Bifidobacterium spp, even after digestion. The extract also exhibited strong antioxidant and antimicrobial activities against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; however, after GID, the antioxidant capacity decreased, and the antimicrobial capacity was strongly reduced or lost. Furthermore, the extract safety was also guaranteed on Caco-2 intestinal cells. This novel and green GP extract proved to be composed of relevant bioactive molecules, including xylooligosaccharides, polyphenols, organic acids, and minerals, which provided different biological properties; it has potential applications in the food industry such that it can be used as an ingredient in the development of new functional foods.

Delineating thermophilic xylanase from Bacillus licheniformis DM5 towards its potential application in xylooligosaccharides production.

In this present study novel endoxylanase producing Bacillus licheniformis DM5 isolated, identified based on 16S rDNA from Garampani hotspring, Assam, India and enzyme was purified. RNA secondary structure predicted the similarity of B. licheniformis DM5 with B. licheniformis ATCC14580. Highest production of xylanase from B. licheniformis DM5 was achieved in the TY medium with cell densities 12 g/l and extracellular protein concentration containing xylanase 400 mg/l. Partially purified extracellular xylanase displayed optimum pH 6.5 and temperature 50 °C. Thermostability of the xylanase at the elevated temperature showed stability between 50 and 60 °C retaining its 99% activity. Kinetic parameters of thermophilic xylanase revealed Km 1.5 ± 0.2 mg/ml, Vmax 2.7 ± 0.2 U/ml and and Kcat 1.8 ± 0.2 s-1 against beechwood xylan but ruled out any exo-acting activity against synthetic pNP-xylopyranoside substrate. Time dependent enzymatic hydrolysis of beechwood xylan and preprocessed agrowaste corncob exhibited the release of xylotriose and xylobiose oligosaccharide (XOS) significantly high. Xylobiose and xylotriose exhibited higher binding affinities with BIAXP transporter protein of probiotic bacteria explaining their easy uptake by the cells. Mixed oligosaccharides also exhibited better prebiotic activity by promoting growth of Bifidobacterium infantis and Lactobacillus delbrueckii. Mixed XOS when tested for their cytotoxicity on Hela cell lines in in vitro MTT assay displayed significant lowering of cell viability after 48 h and 24 h at 100 µg/ml to 60% and 50%, respectively. In contrast, cytotoxicity wasn't observed against normal cervical cell line (VK2/E6E7-ATCC-CRL-2616). Therefore, thermophilic endoxylanase from B. licheniformis DM5 could be attributed for the production of prebiotic and anti-inflammatory XOS from agrowaste.

Recent structural advances in constrained helical peptides.

Given the ubiquity of the ⍺-helix in the proteome, there has been much research in developing mimics of ⍺-helices, and most of this study has been toward developing protein-protein interaction inhibitors. A common strategy for mimicking ⍺-helices has been through the use of constrained, helical peptides. The addition of a constraint typically provides for conformational and proteolytic stability and, in some cases, cell permeability. Some of the most well-known strategies included are lactam formation and hydrocarbon "stapling." Beyond those strategies, there have been many recent advances in developing constrained peptides. The purpose of this review is to highlight recent advances in the development of new helix-stabilizing technologies, constraint diversification strategies, tether diversification strategies, and combination strategies that create new bicyclic helical peptides.

Subcritical Water-Carbon Dioxide Pretreatment of Oil Palm Mesocarp Fiber for Xylooligosaccharide and Glucose Production.

The present work aimed to investigate the pretreatment of oil palm mesocarp fiber (OPMF) in subcritical H2O-CO2 at a temperature range from 150⁻200 °C and 20⁻180 min with CO2 pressure from 3⁻5 MPa. The pretreated solids and liquids from this process were separated by filtration and characterized. Xylooligosaccharides (XOs), sugar monomers, acids, furans and phenols in the pretreated liquids were analyzed by using HPLC. XOs with a degree of polymerization X2⁻X4 comprising xylobiose, xylotriose, xylotetraose were analyzed by using HPAEC-PAD. Enzymatic hydrolysis was performed on cellulose-rich pretreated solids to observe xylose and glucose production. An optimal condition for XOs production was achieved at 180 °C, 60 min, 3 MPa and the highest XOs obtained was 81.60 mg/g which corresponded to 36.59% of XOs yield from total xylan of OPMF. The highest xylose and glucose yields obtained from pretreated solids were 29.96% and 84.65%, respectively at cellulase loading of 10 FPU/g-substrate.

Herbal Compounds with Special Reference to Gastrodin as Potential Therapeutic Agents for Microglia Mediated Neuroinflammation.

BACKGROUND: Activated microglia play a pivotal role neurodegenerative diseases by producing a variety of proinflammatory mediators including tumor necrosis factor-alpha (TNF-α), interleukin- 1beta (IL-1ß) and nitric oxide (NO) that are toxic to neurons and oligodendrocytes. METHODS: In view of the above, suppression of microglia mediated neuroinflammation is deemed a therapeutic strategy for neurodegenerative diseases. Several potential Chinese herbal extracts have been reported to exert neuroprotective effects against neurodegenerative diseases targeting specifically at the activated microglia. In this connection, the phenolic glucoside gastrodin, a main constituent of the Chinese herbal medicine Gastrodia rhizoma, produced widely in the local community exhibits potential neuroprotective effects through suppression of neurotoxic proinflammatory mediators. RESULTS: Here, we first review the roles of activated microglia in different brain diseases. The effects of gastrodin on activated microglia are then considered. We have identified gastrodin as a putative therapeutic agent as it has been found to suppress microglial activation thus ameliorating neuroinflammation. More importantly, gastrodin downregulates the expression of renin angiotensin system (RAS) and production of proinflammatory mediators. Remarkably, gastrodin promotes Sirtuin 3 (Sirt3) up-regulation and nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX-2) down-regulation after ischemichypoxia in activated microglia mediated by AT1 or AT2 receptors which are angiotensin II receptors subtypes, indicating a possible molecular link between RAS and Sirt3 survival genes. CONCLUSION: This review summarizes the beneficial effects of gastrodin acting on activated microglia along with other herbal compounds. Its efficacy in neuroprotection is consistent with some common herbal products in China.

Rapid Detection of Escherichia coli in Water Using Sample Concentration and Optimized Enzymatic Hydrolysis of Chromogenic Substrates.

Methods for rapid detection of fecal indicator bacteria in water are important to ensure that water is safe for drinking, bathing, recreation, fishing and shellfish harvesting. In this study, we tested experimental conditions for bacterial hydrolysis of two promising enzymatic substrates, 5-Bromo-4-chloro-3-indolyl ß-D-glucuronide (X-Gluc) and Resorufin ß-D-glucuronide (REG), and optimized parameters such as temperature and pH to determine conditions for rapid reactions. We then innovated a membrane filter-based approach to facilitate more rapid enzyme-based detection of Escherichia coli in water based on the combination of an initial concentration step and optimized test conditions. For this approach, a water sample (10‒100 mL) is filtered through a 0.45-µm pore size filter with a diameter of 4 or 13 mm. After filtration, a newly designed rapid detection broth is added containing the enzymatic inducer Methyl-beta-D-Glucuronide sodium (MetGlu) and the substrate REG or X-Gluc. After a few (1‒7) hours of incubation at 35 °C, the filter shows pink color (for REG-containing broth) or green color (for X-Gluc containing broth) if E. coli is present. The study provides insights and approaches towards developing a simple, fast, and low-cost method to detect fecal indicator bacteria in water.

Highly Specific near-Infrared Fluorescent Probe for the Real-Time Detection of ß-Glucuronidase in Various Living Cells and Animals.

ß-Glucuronidase (GLU) is an important biomarker for primary cancers and intestinal metabolism of drugs or endogenous substances; however, an effective optical probe for near-infrared (NIR) monitoring in vivo is still lacking. Herein, we design an enzyme-activated off-on NIR fluorescent probe, HC-glu, based on a hemicyanine keleton, which is conjugated with a d-glucuronic acid residue via a glycosidic bond, for the fluorescent quantification and trapping of endogenous GLU activity in vitro and in vivo. The newly developed NIR probe exhibited prominent features including prominent selectivity, high sensitivity, and ultrahigh imaging resolution. It has been successfully used to detect and image endogenous GLU in various hepatoma carcinoma cells, tumor tissues, and tumor-bearing mouse models, for cancer diagnosis and therapy. Moreover, it could detect the in vivo activity of GLU in the intestinal tracts of animals including mice and zebrafish, where GLU performs a vital biological function and is mainly distributed. It could also evaluate real intestinal distribution and real-time variations of GLU in development and growth, all of which are very helpful to guide rational drug use in the clinic. Our results fully demonstrated that HC-glu may serve as a promising tool for evaluating the biological function and process of GLU in living systems.