Glucuronidase Activity Research Articles

Anemarrhena asphodeloides Bunge total saponins lower lipid via modulating MAOA activity to enhance defense mechanisms in mice and C. elegans

Ethnopharmacological relevanceWithin Anemarrhena asphodeloides Bunge (AAB), the pivotal bioactive constituents are identified as Anemarrhena asphodeloides Bunge total saponins (ABS). In traditional pharmacology, ABS has exhibited notable anti-inflammatory, hypoglycemic, and cardioprotective properties. Despite these observed effects, the specific protective mechanisms of ABS against metabolic diseases and improving the endocrine system remain largely uncharted. Aim to studyThis work intends to shed light on the effects and intrinsic mechanisms of ABS on metabolic diseases. Materials and methodsThe characterization of ABS components was achieved through High-Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS). To evaluate ABS's anti-inflammatory efficacy, mouse macrophages underwent analysis using the Griess method. Induced differentiation of mouse fibroblasts was assessed through Oil Red O staining. In an obesity model with C57BL/6 N mice, ABS administration prompted measurements of glucose and insulin tolerance. Western blot analysis quantified lipolysis and anti-inflammatory protein expression. Nile red staining gauged body fat content in C. elegans post-ABS treatment. The mechanism of ABS action was elucidated through mRNA sequencing, further validated using RNA interference technology, and nematode mutants. ResultsABS showcased the ability to diminish Nitric Oxide (NO) production in inflammatory macrophages and shrink adipocyte lipid droplets. In mice experiments, ABS was effective in alleviating fat accumulation and affecting serum lipid metabolism in diabetic mice. It enhanced oral glucose tolerance and insulin tolerance while increasing lipolysis-associated protein expression. ABS notably reduced fat content in C. elegans. Mechanistically, ABS downregulated NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and monoamine oxidase A (MAOA) expression while enhancing UGT, ilys-2, and ilys-3. Lipolysis emerged as a pivotal pathway for ABS in the therapeutic intervention of metabolic diseases. ConclusionsOur investigation has revealed that ABS exert a role in combating metabolic diseases by enhancing the body's defense mechanisms. ABS activate the NLRP3-neurotransmitter-visceral adipose pathway in mice, thereby bolstering resistance and diminishing fat accumulation. In C. elegans, ABS downregulated the expression of MAOA, bolstered resistance, and augmented glucuronidase activity, consequently leading to a reduction in fat content.

Endogenous crude Scutellaria baicalensis polysaccharide robustly enhances one-pot extraction and deglycosylation of baicalin

With the extensive application of baicalein in the treatment of cardiovascular and cerebrovascular diseases, its clinical and market demand has gradually expanded. But the natural yield of baicalein is very low, and it is mainly prepared by the deglycosylation of baicalin. However, the insolubility of baicalin in water significantly limits the deglycosylation of it under biocatalysis. To make biocatalysis of baicalin more efficient and environmental, a strategy was designed to enhance its water solubility through the solubilization mechanism of endogenous biological macromolecules, and the effect on the activity of glucuronidase was further explored. The results showed that wrapping with Scutellaria baicalensis polysaccharide (SBP) significantly improved the solubility of baicalin in water (the water solubility of baicalin increased by 23 times, BI/SBP = 1/12, w/w). It was not only contributed to the efficient production of baicalein by one-pot method, but also effectively improved the deglycosylation rate of baicalin (increase by 47.04 % in aqueous solution). With the help of the solubilization of endogenous polysaccharide on baicalin in aqueous solution, a green, low-cost and efficient method (one-pot method) was designed to simultaneously extract and enzymatic hydrolyze baicalin to prepare baicalein. Under the same conditions, the yield of one-pot method is 87.17 %, which was much higher than that of the conventional method (29.38 %). In addition, one-pot method with the aid of endogenous polysaccharide could simply and conveniently prepare aglycone of other insoluble natural flavonoids, which has a wide range of industrial application value.

Transcription Factors ZjMYB39 and ZjMYB4 Regulate Farnesyl Diphosphate Synthase- and Squalene Synthase-Mediated Triterpenoid Biosynthesis in Jujube

Jujube (Ziziphus jujuba Mill.) is rich in valuable bioactive triterpenoids. However, the regulatory mechanism underlying triterpenoid biosynthesis in jujube remains poorly studied. Here, we characterized the triterpenoid content in wild jujube and cultivated jujube. The triterpenoid content was higher in wild jujube than in cultivated jujube, triterpenoids were most abundant in young leaves, buds, and later stages of development. The transcriptome analysis and correlation analysis showed that differentially expressed genes (DEGs) were enriched in the terpenoid synthesis pathways, and triterpenoids content was strongly correlated with farnesyl diphosphate synthase (ZjFPS), squalene synthase (ZjSQS), and transcription factors ZjMYB39 and ZjMYB4 expression. Gene overexpression and silencing analysis indicated that ZjFPS and ZjSQS were key genes in triterpenoid biosynthesis and transcription factors ZjMYB39 and ZjMYB4 regulated triterpenoid biosynthesis. Subcellular localization experiments showed that ZjFPS and ZjSQS were localized to the nucleus and endoplasmic reticulum and ZjMYB39 and ZjMYB4 were localized to the nucleus. Yeast one-hybrid, glucuronidase activity, and dual-luciferase activity assays suggested that ZjMYB39 and ZjMYB4 regulate triterpenoid biosynthesis by directly binding and activating the promoters of ZjFPS and ZjSQS. These findings provide insights into the underlying regulatory network of triterpenoids metabolism in jujube and lay theoretical and practical foundation for molecular breeding.

BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis

Nitrogen (N) is a major limiting factor for plant growth and vegetable production. Understanding the regulatory mechanisms of N uptake, transport, and assimilation is key to improving N use efficiency in plants. Ammonium transporters (AMTs) play an important role in plant N metabolism. In this study, we isolated an important AMT1 subfamily member (BcAMT1;5) with a highly conserved signatural AMT1 subfamily motif from flowering Chinese cabbage. Based on functional complementation in yeast mutant 31019b and overexpression of BcAMT1;5 in Arabidopsis, BcAMT1;5 is a functional AMT. Tissue expression analysis showed that BcAMT1;5 was mainly expressed in roots and showed multiple N regime transcript patterns to respond to varying nutritional conditions. This was up-regulated by N-deficiency and down-regulated by supplying NH4+. The glucuronidase (GUS) activities of BcAMT1;5pro::GUS showed a similar change in response to different N conditions. Overexpression of BcAMT1;5 accelerated the growth of transgenic seedlings, increased NH4+ net influxes, and enhanced the content and accumulation of NH4+ and NO3− at low N concentrations. Additionally, it increased the transcript levels of N assimilation-related genes in shoots. These results indicate that BcAMT1;5 may participate in N uptake and assimilation under various N conditions in flowering Chinese cabbage, but it was differed obviously from other AMT1s.

The potential of near real-time monitoring of β-d-glucuronidase activity to establish effective warning systems in urban recreational waters

The enzymatic activity of E. coli in urban surface waters gives a sharp but short response to contamination events.

Gut Microbiota, Metabolic Disorders and Breast Cancer: Could Berberine Turn Out to Be a Transversal Nutraceutical Tool? A Narrative Analysis.

Metabolic disorders, mainly characterized as the marked alteration of the lipid and carbohydrate profile, in addition to the clinical presence of the direct consequences of these alterations, are pathological conditions that have considerably increased in prevalence in recent years. They are directly linked to the onset of various pathologies, including cancer, particularly breast cancer, and are hormone-responsive. Alongside the known conditions responsible for this scenario, such as nutrition and lifestyle in general, the importance of both the colonic microbiota and the various organs and systems is becoming increasingly evident. In fact, it is now evident that microbial dysbiosis plays a fundamental role in the onset of these metabolic disorders, and therefore how these conditions are indirectly responsible for the onset and progression of neoplasms. Indirect mechanisms such as an altered Firmicutes/Bacteroidetes ratio; the formation of metabolites such as short-chain fatty acids (SCFAs), in particular, butyrate, which is capable of acting as a tumor suppressor; and the glucuronidase activity of estroboloma (bacteria responsible for estrogen metabolism) are just some of the most important mechanisms that contribute to the history of breast cancer. It is therefore understandable that in clinical terms, it is essential to associate the modulation of metabolic disorders and the microbial conditions that contribute to generating them with common therapies, preferably using compounds and solutions that are effective and acceptable for the patient without side effects. Nutraceuticals such as berberine (active both in metabolic scenarios and in the microbiota) and interventions modulating the microbial structure such as the use of probiotics and prebiotics seem to be ideal solutions for these preventive and no-longer-ignorable strategies in the light of numerous data now present in the literature.

Monitoring Farmed Fish Welfare by Measurement of Cortisol as a Stress Marker in Fish Feces by Liquid Chromatography Coupled with Tandem Mass Spectrometry.

The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid–liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R2 > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

α-Lipoic Acid Reduces Ceramide Synthesis and Neuroinflammation in the Hypothalamus of Insulin-Resistant Rats, While in the Cerebral Cortex Diminishes the β-Amyloid Accumulation.

BackgroundOxidative stress underlies metabolic diseases and cognitive impairment; thus, the use of antioxidants may improve brain function in insulin-resistant conditions. We are the first to evaluate the effects of α-lipoic acid (ALA) on redox homeostasis, sphingolipid metabolism, neuroinflammation, apoptosis, and β-amyloid accumulation in the cerebral cortex and hypothalamus of insulin-resistant rats.MethodsThe experiment was conducted on male cmdb/outbred Wistar rats fed a high-fat diet (HFD) for 10 weeks with intragastric administration of ALA (30 mg/kg body weight) for 4 weeks. Pro-oxidant and pro-inflammatory enzymes, oxidative stress, sphingolipid metabolism, neuroinflammation, apoptosis, and β-amyloid level were assessed in the hypothalamus and cerebral cortex using colorimetric, fluorimetric, ELISA, and HPLC methods. Statistical analysis was performed using three-way ANOVA followed by the Tukey HSD test.ResultsALA normalizes body weight, food intake, glycemia, insulinemia, and systemic insulin sensitivity in HFD-fed rats. ALA treatment reduces nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase activity, increases ferric-reducing antioxidant power (FRAP) and thiol levels in the hypothalamus of insulin-resistant rats. In addition, it decreases myeloperoxidase, glucuronidase, and metalloproteinase-2 activity and pro-inflammatory cytokines (IL-1β, IL-6) levels, while in the cerebral cortex ALA reduces β-amyloid accumulation. In both brain structures, ALA diminishes ceramide synthesis and caspase-3 activity. ALA improves systemic oxidative status and reduces insulin-resistant rats’ serum cytokines, chemokines, and growth factors.ConclusionALA normalizes lipid and carbohydrate metabolism in insulin-resistant rats. At the brain level, ALA primarily affects hypothalamic metabolism. ALA improves redox homeostasis by decreasing the activity of pro-oxidant enzymes, enhancing total antioxidant potential, and reducing protein and lipid oxidative damage in the hypothalamus of HFD-fed rats. ALA also reduces hypothalamic inflammation and metalloproteinases activity, and cortical β-amyloid accumulation. In both brain structures, ALA diminishes ceramide synthesis and neuronal apoptosis. Although further study is needed, ALA may be a potential treatment for patients with cerebral complications of insulin resistance.

Association of Gut Bacterial Beta Glucuronidase Activity with Mycophenolate Mofetil Induced Cytopenia

<h3>Purpose</h3> Mycophenolate mofetil (MMF) is the anti-metabolite of choice in heart transplantation (HT). MMF tolerability is limited by its toxicity, including bone marrow suppression. MMF metabolism is dependent on gastrointestinal flora, particularly pathogens carrying β-D-glucuronidases (GUS). We investigated association between cytopenia and gut microbial composition focusing on GUS bacteria in HT pts. <h3>Methods</h3> 45 HT pts (53±12.9yo, 78.6% M) with 1-month post HT stool samples were included. Stool samples were analyzed using 16S rRNA sequencing. Gut alpha-diversity was defined using Shannon Index (SI). GUS bacteria abundance was computed as sum of individual known GUS-producing bacteria, matched by genus and species. The DESeq2 package and linear regression models were used to identify differentially abundant taxa and analyze difference in SI and GUS abundance by cytopenia occurrence. <h3>Results</h3> Leukopenia and neutropenia occurred in 30(66.7%) and 26(57.8%) pts. Mean SI±SD was similar in pts with leukopenia vs not (5.78±0.59 vs 5.58±0.58, p=0.29) and neutropenia vs not (5.73±0.57 vs 5.68±0.62, p=0.76). In DESeq2 analyses, 8 bacteria species were found to be significantly more abundant among those who had leukopenia, including two known GUS producing taxa <i>Bacteroides dorei</i> and <i>Bacteroides cellulosilyticus</i> (<b>Fig 1A</b><i>).</i>Seven bacteria species were significantly more abundant among those who had neutropenia, including <i>Bacteroides dorei</i> (<b>Fig 1B</b>)<i>.</i> Patients with leukopeniat had higher abundance of GUS bacteria 0.21±0.16 vs 0.12±0.18 (p=0.09) (<b>Fig 1C</b>). This difference became significant following adjustment for valcyte (p=0.04). Patients who experienced neutropenia vs not had a similar abundance of GUS bacteria 0.17±0.14 and 0.18±0.20 (p=0.88) (<b>Fig 1D</b>). <h3>Conclusion</h3> GUS bacteria were expanded among HT recipients with cytopenia, potentially highlighting mechanisms of MMF toxicity. Robust clinical trials delineating the effects of gut microbiome on MMF metabolism are warranted.

β-Glucuronidase Pattern Predicted From Gut Metagenomes Indicates Potentially Diversified Pharmacomicrobiomics.

β-glucuronidases (GUS) of intestinal bacteria remove glucuronic acid from glucoronides, reversing phase II metabolism of the liver and affecting the level of active deconjugated metabolites deriving from drugs or xenobiotics. Two hundred seventy-nine non-redundant GUS sequences are known in the gut microbiota, classified in seven structural categories (NL, L1, L2, mL1, mL2, mL1,2, and NC) with different biocatalytic properties. In the present study, the intestinal metagenome of 60 healthy subjects from five geographically different cohorts was assembled, binned, and mined to determine qualitative and quantitative differences in GUS profile, potentially affecting response to drugs and xenobiotics. Each metagenome harbored 4–70 different GUS, altogether accounting for 218. The amount of intestinal bacteria with at least one GUS gene was highly variable, from 0.7 to 82.2%, 25.7% on average. No significant difference among cohorts could be identified, except for the Ethiopia (ETH) cohort where GUS-encoding bacteria were significantly less abundant. The structural categories were differently distributed among the metagenomes, but without any statistical significance related to the cohorts. GUS profiles were generally dominated by the category NL, followed by mL1, L2, and L1. The GUS categories most involved in the hydrolysis of small molecules, including drugs, are L1 and mL1. Bacteria contributing to these categories belonged to Bacteroides ovatus, Bacteroides dorei, Bacteroides fragilis, Escherichia coli, Eubacterium eligens, Faecalibacterium prausnitzii, Parabacteroides merdae, and Ruminococcus gnavus. Bacteria harboring L1 GUS were generally scarcely abundant (<1.3%), except in three metagenomes, where they reached up to 24.3% for the contribution of E. coli and F. prausnitzii. Bacteria harboring mL1 GUS were significantly more abundant (mean = 4.6%), with Bacteroides representing a major contributor. Albeit mL1 enzymes are less active than L1 ones, Bacteroides likely plays a pivotal role in the deglucuronidation, due to its remarkable abundance in the microbiomes. The observed broad interindividual heterogeneity of GUS profiles, particularly of the L1 and mL1 categories, likely represent a major driver of pharmacomicrobiomics variability, affecting drug response and toxicity. Different geographical origins, genetic, nutritional, and lifestyle features of the hosts seemed not to be relevant in the definition of glucuronidase activity, albeit they influenced the richness of the GUS profile.

Draft Genome Sequences of Buttiauxella spp. Isolates from Water and Gastropods with Putative β-d-Glucuronidase Activity.

ABSTRACTWe report the draft genome sequences of Buttiauxella spp. strains that were isolated from water and gastropods. Three isolates show fluorescence in the Colilert system, indicating unusual β-d-glucuronidase activity, and phylogenetic analyses suggest that they represent a novel species. Another strain, without β-d-glucuronidase activity, was assigned to the species Buttiauxella ferragutiae.

O27: MICROGLIAL CORRECTION AFTER FETAL THERAPY WITHOUT CONDITIONING IN MICE WITH MUCOPOLYSACCHARIDOSIS TYPE VII

Abstract Introduction Mucopolysaccharidosis type 7 (MPS7) is a lysosomal storage disorder typically fatal in utero. Postnatal enzyme replacement therapy (ERT) to replace missing glucuronidase (GUS) does not penetrate the blood-brain barrier (BBB). We investigated whether in utero ERT (IUERT) specifically targets brain microglia (natural GUS storehouses and key brain inflammation mediators) and whether in utero hematopoietic stem cell transplantation (IUHSCT) results in microglial engraftment as a strategy for permanent correction. Methods We performed IUERT by injecting GUS into MPS7 fetuses mid-gestation, and analyzed tissue homogenates (via colorimetric substrate) and brain microglia (via flow cytometry) for enzyme activity after 4-7 days. We performed IUHSCT by transplanting HSCs mid-gestation from CX3CR1-GFP donors. We examined blood, bone marrow, and brain for engraftment. We assessed brain inflammation by staining for CD68. We performed RNA sequencing to characterize engrafted microglia. Results IUERT resulted in detectable brain GUS activity. Flow cytometry showed that GUS activity after IUERT was near wild-type levels, and brains harvested in adulthood had decreased inflammation via CD68 immunohistochemistry. IUHSCT resulted in multilineage engraftment of hematopoietic cells in blood and bone. Confocal microscopy revealed multifocal engraftment of donor-derived microglia. RNA sequencing indicated that engrafted microglia were nearly identical to endogenous microglia. MPS7 chimeras had evidence of reduced brain inflammation near donor microglia. Conclusion Both IUERT and IUHSCT are complementary treatment modalities that can penetrate the BBB and ameliorate neurologic manifestations of diseases such as MPS7. These results lay the foundation for future studies using in utero molecular therapies for MPS7 as well as other storage disorders.

Impact of N-fertilization and peanut shell biochar on soil microbial community structure and enzyme activities in a Typic Haplustoll under different management practices

The application of biochar with N-fertilizer is one of the most attractive practices for increasing soil quality and fertility. However, few studies have examined the combined effects of peanut biochar and urea on soil microbial community structure and function. Phospholipid fatty acid (PLFA) analysis and various enzyme activities were used to monitor microbial community structure and function in a 20-week incubation experiment. Different rates of biochar (0, 1, and 3% dry wt. soil) and N-fertilizers (with or without urea addition at 0.2 g kg−1) were applied to a Typic Haplustoll under conventional (CV) and conservation (CS) land management practices. A pristine forest soil (CK) was also included as control. Overall, the lowest microbial activity and abundance of microbial PLFA bioindicators were recorded under CV, confirming that conservation management may improve soil quality. Our study also indicated that the effect of biochar and management practices on the microbial community structure was more significant than that of urea. Particularly under both CS and CK, biochar tended to increase glucosidase, glucuronidase, and phosphomonoesterase activities, being on average 65%, 43%, and 85% significantly higher in biochar 3% than in the control treatment, respectively. Thus, we found a strong correlation between soil macronutrients and enzymes such as glucosidase and urease. Although total PLFA content was poorly affected, biochar addition increased some individual PLFA biomarkers, suggesting that both the biochar rates (1% and 3%) may increase Gram-negative bacteria and fungal populations. Regardless of biochar addition, urea application significantly increased urease activity. We also found a positive correlation between fungal PLFAs (VAM and total fungi) and urease activity, indicating that biochar tended to increase fungal biomass. The combined application of biochar and urea to soil under appropriate management may thus be a feasible and effective strategy for improving soil microbial quality.

Simple enumeration of Escherichia coli concentrations in river water samples by measuring β-d-glucuronidase activities in a microplate reader.

Monitoring of Escherichia coli concentrations in river water (RW) is essential to identify fecal pollution of the river. The objective of this study was to assess the suitability of a novel, simple and high throughput method developed in our laboratory to enumerate E. coli concentrations in RW samples. The method is based on the use of the synthetic substrate specific for the β-d-glucuronidase (GUS) produced by E. coli. GUS activities and E. coli concentrations were monitored at eight selected sites in rivers running through Sapporo, Japan. Because the fluorescence intensities of the synthetic substrate in the RW samples increased linearly over a 4-h incubation period, we could estimate the GUS activities of the RW samples. The GUS activities were highly correlated with E. coli concentrations at >100 most probable numbers 100 mL-1 with a correlation coefficient of 0.87. The GUS activities of the RW samples collected from all sampling sites fitted well to a single correlation equation, which indicates that it was applicable to the estimation of E. coli concentrations regardless of the sampling sites. This method is simple, rapid, reliable, inexpensive, and high throughput, and is therefore useful for monitoring E. coli in RW.

Detection and Quantification of Polychlorinated Biphenyl Sulfates in Human Serum

Polychlorinated biphenyls (PCBs) are persistent toxic chemicals with both legacy sources (e.g., Aroclors) and new sources (e.g., unintentional contaminants in some pigments and varnishes). PCB sulfates are derived from further metabolism of hydroxylated PCBs (OH-PCBs), which are oxidative metabolites of PCBs. While OH-PCBs and PCB sulfates are implicated in multiple toxicological effects, studies of PCB sulfates in human serum have been limited by available analytical procedures. We have now developed a method for extraction of PCB sulfates from serum followed by differential analysis with, and without, sulfatase-catalyzed hydrolysis to OH-PCBs. A sulfatase from Helix pomatia was purified by affinity chromatography, and it displayed broad specificity for PCB sulfates without contaminant glucuronidase activity. Following sulfatase-catalyzed hydrolysis of the PCB sulfates extracted from serum, the corresponding OH-PCBs were derivatized to methoxy-PCBs and quantitated by GC-MS/MS. In a pooled sample of human serum, we identified 10 PCB sulfates, with three PCB sulfate congeners exhibiting the highest concentrations from 1200 to 3970 pg/g of serum. In conclusion, we have developed a sensitive and specific method for the determination of PCB sulfates in human serum.

Nanozymes and Glucuronides: Glucuronidase, Esterase, and/or Transferase Activity.

Nanozymes are fundamentally interesting catalysts that are investigated as alternatives to fragile protein-enzymes for applications in biotechnology, for prodrug activation, and use in biomedicine, as well as the catalysts that contributed to the Origin of Life. However, until now, nanozymes mostly have been documented to exhibit activity as red/ox catalysts, whereas examples of activity outside this broad class of reactions are very few. Herein, activity of nanozymes on glucuronide prodrugs is investigated, specifically focusing on the mechanism of prodrug conversion reactions. The main finding of this work is that nanozymes exhibit glucuronide-like activity, but also catalyze prodrug conversion via esterase-like mechanism and facilitate group transfer reactions.

BETA GLUCURONIDASE ACTIVITY IN DIABETES ASSOCIATED PERIODONTITIS

Diabetes mellitus is a risk factor for development of periodontal disease with progressive periodontal destruction seen in diabetic patients. Advances in oral and periodontal disease diagnostic research are moving towards methods whereby periodontal risk can be identified and quantified by objective measures such as biomarkers. The aim of this study was to identify the presence of beta glucuronidase activity as an enzymatic biomarker of periodontal tissue destruction in patients of Diabetes mellitus associated with Periodontitis. β Glucuronidase activitywas estimated in saliva, GCF (Gingival Crevicular Fluid) and serum in healthy subjects and patients with Diabetes mellitus, Periodontitis and Diabetes associated periodontitis. Enzyme activity was compared with clinical parameters like Probing pocket depth, Plaque Index and Gingival Index. It was maximally raised in diabetic cases with periodontitis. Enzyme activity was found to be highest in crevicular fluid compared to saliva and serum. However, serum Glucuronidase correlated significantly with clinical indices. β-glucuronidase may be employed on routine basis as a chair side test for screening and diagnosis of patients with periodontitis in diabetics.

Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and β-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

Tinkering Cis Motifs Jigsaw Puzzle Led to Root-Specific Drought-Inducible Novel Synthetic Promoters.

Following an in-depth transcriptomics-based approach, we first screened out and analyzed (in silico) cis motifs in a group of 63 drought-inducible genes (in soybean). Six novel synthetic promoters (SynP14-SynP19) were designed by concatenating 11 cis motifs, ABF, ABRE, ABRE-Like, CBF, E2F-VARIANT, G-box, GCC-Box, MYB1, MYB4, RAV1-A, and RAV1-B (in multiple copies and various combination) with a minimal 35s core promoter and a 222 bp synthetic intron sequence. In order to validate their drought-inducibility and root-specificity, the designed synthetic assemblies were transformed in soybean hairy roots to drive GUS gene using pCAMBIA3301. Through GUS histochemical assay (after a 72 h 6% PEG6000 treatment), we noticed higher glucuronidase activity in transgenic hairy roots harboring SynP15, SynP16, and SynP18. Further screening through GUS fluorometric assay flaunted SynP16 as the most appropriate combination of efficient drought-responsive cis motifs. Afterwards, we stably transformed SynP15, SynP16, and SynP18 in Arabidopsis and carried out GUS staining as well as fluorometric assays of the transgenic plants treated with simulated drought stress. Consistently, SynP16 retained higher transcriptional activity in Arabidopsis roots in response to drought. Thus the root-specific drought-inducible synthetic promoters designed using stimulus-specific cis motifs in a definite fashion could be exploited in developing drought tolerance in soybean and other crops as well. Moreover, the rationale of design extends our knowledge of trial-and-error based cis engineering to construct synthetic promoters for transcriptional upgradation against other stresses.

Simple and reliable enumeration of Escherichia coli concentrations in wastewater samples by measuring β-d-glucuronidase (GUS) activities via a microplate reader.

Monitoring of Escherichia coli concentrations at wastewater treatment plants (WWTPs) is important to ensure process performance and protect public health. However, conventional E. coli enumeration methods are complicated and time- and labor-consuming. Here, we report a novel simple and reliable method based on β-d-glucuronidase (GUS) activity assay to enumerate E. coli concentrations in wastewater (WW) samples. An aliquot (20μL) of the medium with fluorogenic enzyme substrate for E. coli and 180μL of a WW sample were added to one well of a 96-well microplate. The microplate was placed in a microplate reader at 37°C. To this end, the fluorescence intensity of a fluorogenic enzyme substrate for E. coli was measured every 10min over 3h to determine GUS activity. The linear increase in the fluorescence intensity representing the GUS activities showed a positive correlation with E. coli concentrations in wastewater samples. However, the correlation equations were specific to WWTPs, which could be due to the difference in the E. coli population structures among WWTPs. We observed that the wastewater matrix is not a limitation to measure the GUS activity, and a WWTP-specific correlation equation can be used as a calibration curve to estimate the E. coli concentrations in the samples collected from that site. A comparison of the results with those of culture-dependent Colilert method proved that the current method is simple and useful for the enumeration of E. coli concentrations in wastewater samples reliably.