Acid Β-glucosidase Research Articles

Effect of zeolite application on soil enzyme activity of potted sandy soil cultivated with Swiss chard and cabbage

A zeolite pot experiment was conducted at the Agricultural Research Council Infruitec-Nietvoorbij in Stellenbosch, South Africa, under greenhouse conditions. The experiment aimed to investigate the impact of zeolite application on soil enzyme activities in sandy soils cultivated with Swiss chard (Beta vulgaris Var. cicla) and cabbage (Brassica oleracea Var. capitata L.) over two years (2018-2019). Different zeolite-to-soil ratios (0:1, 1:9, 2:8, and 3:7 w/w) were used, with each pot containing 12 kg of soil. The experiment involved 72 pots for each vegetable, arranged in a randomized complete block design (RCBD). Soil enzyme activities, including acid phosphatase, β-glucosidase, and urease, as well as soil chemical properties (pH, total plant-available nitrogen, organic carbon, and phosphorus), were analyzed. Key findings indicate that the effect of zeolite application on enzyme activities varied between the vegetable species. Zeolite application significantly increased (P

Acidovorax bellezanensis sp. nov., a novel bacterium from uranium mill tailings repository sites with selenium bioremediation capabilities

A Gram-stain-negative bacterial strain designated Be4T, belonging to the genus Acidovorax, was isolated from mining porewaters sampled in uranium mill tailings repository sites, located in Bellezane, near Bessines-sur-Gartempe (Limousin, France). Cells were facultative anaerobic, rod-shaped, non-endospore-forming and motile with flagella. The mean cell size was 1.25–1.31 μm long and 0.70–0.73 μm wide. Colonies were light yellow, opaque, circular, convex with smooth margins, and 1–2 mm in diameter. Growth occurs at 4–37 °C and between pH 5.5–9.0. It differed from its phylogenetically related strains by phenotypic and physiological characteristics such as growth at 4 °C, presence of acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-glucosidase enzymatic activities, and fermentation of l-xylose and esculin. The major fatty acids were C16:0, C16:1 ω7c/C16:1 ω6c, C17:0 cyclo and C18:1 ω7c. Phylogenetic analysis based on 16S rRNA and 938 core genes, confirmed its placement within the genus Acidovorax as a novel species. Strain Be4T showed highest 16S rRNA sequence similarity to Acidovorax antarcticus (98.2 %), Acidovorax radicis (97.9 %), Acidovorax temperans (97.8 %) and Acidovorax facilis (97.7 %). The genome of strain Be4T is 5,041,667 bp size with a DNA G + C content of 65.15 %. By automatic annotation numerous sequences involved in the interaction with metals/metalloids including some genes related to Se uptake and selenite resistance were detected in its genome. The average nucleotide identity (ANI) values calculated from whole genome sequences between strain Be4T and the most closely related strains A. radicis and A. facilis were below the threshold value of 95 %. Thus, the data from the phylogenetic, physiological, biochemical, and genomic analyses clearly indicates that strain Be4T represents a novel species with the suggested name Acidovorax bellezanensis sp. nov. The type strain is Acidovorax bellezanensis Be4T (=DSM116209T = CECT30865T). This novel species, due to its unique isolation source, genomic analysis, and preliminary laboratory tests where it was able to reduce toxic Se(IV) to less harmful Se(0) in the form of nanoparticles, holds great potential for further investigation in bioremediation, particularly concerning Se.

Characterization of Novel Human β-glucocerebrosidase Antibodies for Parkinson's Disease Research.

Mutations in GBA1, which encodes the lysosome enzyme β-glucocerebrosidase (also referred to as acid β-glucosidase or GCase), are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies. This study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays. Two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme developed to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays. The characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration. The hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.

Synergistic effect of biochar and plant growth promoting bacteria improve the growth and phytostabilization potential of Sorghum bicolor in Cd and Zn contaminated soils

The synergetic effect of biochar (BC) and plant growth promoting bacteria (PGPB) on phytoremediation potential of plants in heavy metal (HM) contaminated soils was not well understood. A pot experiment was carried out to evaluate the effects of banana pith derived BC (0, 2.5 and 5%, w/w) as soil amendment together with inoculation of PGPB Bacillus thuringiensis SE1C2 on the growth, physiological response and phytoremediation potential of Sorghum bicolor (L.) Moench in Cd and Zn contaminated soils. Combined treatment of BC 5% and SE1C2 increased plant shoot length, root length, fresh weight and dry weight but decreased Cd and Zn accumulation as compared to other treatments. The BC 5% treatment also increased SE1C2 colonization in rhizosphere soils and tissue interior of S. bicolor even under HM stress. In addition, the plants treated with BC 5% and SE1C2 also had increased chlorophyll, carotenoid and antioxidant enzymes while lower leaf malondialdehyde and proline contents which suggest their potential in improving HM stress tolerance in plants. Similarly, the combined BC 5% and SE1C2 treatment considerably improved enzymatic activities (acid phosphatases, alkaline phosphatases, β-glucosidase and urease) in HM contaminated soils. Thus, this study concludes that the combined treatment of Bacillus thuringiensis SE1C2 and banana pith BC could be exploited as an effective strategy for improving the plant growth and phytostabilization potential in HM contaminated soils.

Proteomics analysis of the brain from a Gaucher disease mouse identifies pathological pathways including a possible role for transglutaminase 1.

Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by the defective activity of acid β-glucosidase (GCase) which results from mutations in GBA1. Neurological forms of GD (nGD) can be generated in mice by intra-peritoneal injection of conduritol B-epoxide (CBE) which irreversibly inhibits GCase. Using this approach, a number of pathological pathways have been identified in mouse brain by RNAseq. However, unlike transcriptomics, proteomics gives direct information about protein expression which is more likely to provide insight into which cellular pathways are impacted in disease. We now perform non-targeted, mass spectrometry-based quantitative proteomics on brains from mice injected with 50 mg/kg body weight CBE for 13 days. Of the 5038 detected proteins, 472 were differentially expressed between control and CBE-injected mice of which 104 were selected for further analysis based on higher stringency criteria. We also compared these proteins with differentially expressed genes (DEGs) identified by RNAseq. Some lysosomal proteins were up-regulated as was interferon signaling, whereas levels of ion channel related proteins and some proteins associated with neurotransmitter signaling were reduced, as was cholesterol metabolism. One protein, transglutaminase 1 (TGM1), which is elevated in a number of neurodegenerative diseases, was absent from the control group but was found at high levels in CBE-injected mice, and located in the extracellular matrix (ECM) in layer V of the cortex and intracellularly in Purkinje cells in the cerebellum. Together, the proteomics data confirm previous RNAseq data and add additional mechanistic understanding about cellular pathways that may play a role in nGD pathology.

Gaucher disease prevalence in 600 patients affected by monoclonal gammopathy of undetermined significance.

Gaucher disease (GD) is a rare autosomal recessive inherited disorder caused by the lysosomal enzyme acid β-glucosidase deficiency. Many patients experience a critical delay in the diagnosis of up to 8-10 years due to its rarity and variability in signs and symptoms, with the consultation of several specialists. This prospective observational study analyzed the prevalence of GD in 600 patients with monoclonal gammopathy of uncertain significance (MGUS) from January 2018 until February 2022. The mean age of participants was 66 years, with a mean monoclonal component of 0.58 g/dL. In 433 MGUS patients with available data, anemia (hemoglobin level < 10 g/dL) was present in 31 patients (7%), and thrombocytopenia (platelet count <100.000/mm3 ) in 24 (5.5%). Of 600 MGUS patients tested for acid β-glucosidase enzyme activity, 7 patients (1.2%) had activity below 2.5 nmol/h/mL. In comparison, GBA gene analysis was executed in 110 patients. It revealed 4 patients (0.7%) affected by GD (3 patients with compound heterozygous mutation and 1 with homozygous mutation), with a prevalence of 1 every 150 MGUS patients. Furthermore, 12 out of the remaining 106 evaluated patients (11%) were carriers of a single heterozygous mutation while having regular enzyme activity. The clinical heterogeneity of GD and frequent lack of awareness among physicians often lead to diagnostic delays and severe clinical manifestations. The role of MGUS in the presence of at least one clinical sign, such as low platelet count, organomegaly, bone pain, or bleeding tendency, could aid in initiating GD screening with DBS, thus reducing the period between symptom onset and the diagnosis of this rare disease.

Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology.

Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid β-glucosidase gene (GBA1), leading to a deficiency in the β-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1β and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD.

Ecoenzymatic Stoichiometry in the Rhizosphere and Bulk Soil of a Larix principis-rupprechtii Plantation in North China

Soil extracellular enzymes play an important role in ecosystem energy conversion and material cycling. Ecoenzymatic stoichiometry can reflect the relationship between the soil’s microbial nutrient cycle and nutrient limitation. However, there have been few studies on the differences in ecoenzymatic stoichiometry and nutrient limitation between rhizosphere soil and bulk soil. This study examined soil nutrients and enzyme activities in rhizosphere soil and bulk soil in a Larix principis-rupprechtii plantation in north China. The results showed that the levels of soil organic carbon (C), total nitrogen (N), and available nutrients in the rhizosphere soil were significantly higher than those in the bulk soil, whereas the total potassium (TK) level was significantly lower. The soil C:N, C:P, and N:P ratios of the rhizosphere soil also exceeded those of the bulk soil. The acid phosphatase (ACP), urease (UE), and β-glucosidase (β-GC) activities in the rhizosphere soil exceeded those in the bulk soil, whereas the activities of N-acetyl-β-D-glucosidase (NAG), aminopeptidase (LAP), and nitrogenase (NA) were lower. The ratios of C, N, and P acquisition activities changed from 1:1.7:1 in the rhizosphere soil to 1:2:1 in the bulk soil. Redundancy analysis showed that the available K and soil water content in the rhizosphere soil were the most important soil factors affecting soil enzyme activities and ecoenzymatic stoichiometry; those in the bulk soil were soil N:P and soil water content. These results suggest that not all soil enzyme activities present rhizosphere effects and that bulk soil is more susceptible to N limitation in Larix principis-rupprechtii plantations. Plant roots play an important role in regulating soil nutrients and soil activities, and future studies should examine the underlying mechanisms in more detail.

Soil enzymatic activity in Brazilian biomes under native vegetation and contrasting cropping and management

Enzymatic activity is a useful indicator for the evaluation of soil biological components. However, a detailed analysis of such indicators in Brazilian soils is lacking. In this study, the activity of soil enzymes was evaluated using data from 73 publications covering 57 locations across all six biomes and eight soil orders out of the 13 that occur in Brazil. The results indicated a wide variation of enzymatic activity in soils, where the means in μg p-nitrophenol g−1 soil h−1 were 569, 116, 112, and 80 for acid phosphatase, alkaline phosphatase, β-glucosidase, and arylsulfatase, respectively. Likewise, values of 148 μg reducing sugars g−1 soil day−1 for cellulase, 57 μg triphenyl tetrazolium formazan g−1 soil day−1 for dehydrogenase, and 38 μg N-NH4+ g−1 soil h−1 for urease were recorded. Soil organic matter and microbial biomass were the best attributes correlated with enzymatic activity, followed by clay content and pH. A more detailed analysis of the acid phosphatase, arylsulfatase, and β-glucosidase activities showed that land-use effects were more pronounced in the Atlantic Forest and Cerrado than in the Caatinga biome. Soils under non-tillage and integrated crop-livestock systems showed higher enzymatic activity than soils under conventional tillage. Soils housing higher biodiversity and delivering higher crop yields also presented higher enzymatic activity, especially in terms of arylsulfatase and β-glucosidase. Enzymatic activity has been shown to be a versatile indicator of soil quality, as it can elucidate how to achieve high-quality standards in tropical and subtropical soils.

Soil Enzyme Activity Response to Substrate and Nutrient Additions on Undisturbed Forest Subsoil Samples

Previous studies have found that C turnover is bound to hotspots of microbial activity. The objective of this study was to analyze the effects of pure energy substrate (glucose), nutrient (mineral N or P) and combined substrate and nutrient (glucose + N, glucose + P, sterile DOC, artificial root exudate extract) additions to enzyme activity inside and outside hotspots as a proxy for microbial C turnover in a subsoil. By means of different substrate and nutrient additions, we tested how the limitations of our site were distributed on a small scale and depth-dependently to contribute to an increase in knowledge of subsoil mechanistics. The study site is a sandy Dystric Cambisol under an over 100-year-old beech forest stand in Lower Saxony, Germany. Forty-eight undisturbed soil samples from two depth increments (15–27 cm and 80–92 cm) of three profiles were sprayed homogeneously with easily available C, N and P sources to investigate the impacts of substrates and nutrients on three enzyme activities (acid phosphatase, β-glucosidase and N-acetylglucosaminidase) by using the soil zymography approach. Comparisons of upper and lower subsoils showed significantly fewer and smaller hotspots in the lower subsoil but with a high degree of spatial variation in comparison to the upper subsoil. Different patterns of enzyme distribution between upper and lower subsoil suggest microbial communities with a lower diversity are found in deeper soil regions of the site. Both substrate and nutrient additions stimulated enzyme activities significantly more outside the initial hotspots than within. Because of this, we conclude that microorganisms in the initial hotspots are less limited than in the surrounding bulk soil. Changes in enzyme activities owing to both substrate and nutrient addition were stronger in the lower subsoil than in the upper subsoil, showing differences in limitations and possible changes in microbial community structure with increasing depth. The results of our study emphasize the need to consider spatial factors in microbial turnover processes, especially in lower subsoil regions where stronger substrate and nutrient limitations occur.

Reaction Mechanism of Glycoside Hydrolase Family 116 Utilizes Perpendicular Protonation.

Retaining glycoside hydrolases use acid/base catalysis with an enzymatic acid/base protonating the glycosidic bond oxygen to facilitate leaving-group departure alongside attack by a catalytic nucleophile to form a covalent intermediate. Generally, this acid/base protonates the oxygen laterally with respect to the sugar ring, which places the catalytic acid/base and nucleophile carboxylates within about 4.5-6.5 Å of each other. However, in glycoside hydrolase (GH) family 116, including disease-related human acid β-glucosidase 2 (GBA2), the distance between the catalytic acid/base and the nucleophile is around 8 Å (PDB: 5BVU) and the catalytic acid/base appears to be above the plane of the pyranose ring, rather than being lateral to that plane, which could have catalytic consequences. However, no structure of an enzyme-substrate complex is available for this GH family. Here, we report the structures of Thermoanaerobacterium xylanolyticum β-glucosidase (TxGH116) D593N acid/base mutant in complexes with cellobiose and laminaribiose and its catalytic mechanism. We confirm that the amide hydrogen bonding to the glycosidic oxygen is in a perpendicular rather than lateral orientation. Quantum mechanics/molecular mechanics (QM/MM) simulations of the glycosylation half-reaction in wild-type TxGH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed 4C1 chair at the -1 subsite. Nevertheless, the reaction can still proceed through a 4H3 half-chair transition state, as in classical retaining β-glucosidases, as the catalytic acid D593 protonates the perpendicular electron pair. The glucose C6OH is locked in a gauche, trans orientation with respect to the C5-O5 and C4-C5 bonds to facilitate perpendicular protonation. These data imply a unique protonation trajectory in Clan-O glycoside hydrolases, which has strong implications for the design of inhibitors specific to either lateral protonators, such as human GBA1, or perpendicular protonators, such as human GBA2.

IPSC-derived neural precursor cells engineering GBA1 recovers acid β-glucosidase deficiency and diminishes α-synuclein and neuropathology

Mutations in GBA1, encoding the lysosomal acid β-glucosidase (GCase), cause neuronopathic Gaucher disease (nGD) and promote Parkinson disease (PD). The mutations on GBA1 include deletion and missense mutations that are pathological and lead to GCase deficiency in Gaucher disease. Both nGD and PD lack disease-modifying treatments and are critical unmet medical needs. In this study, we evaluated a cell therapy treatment using mouse iPSC-derived neural precursor cells (NPCs) engineered to overexpress GCase (termed hGBA1-NPCs). The hGBA1-NPCs secreted GCase that was taken up by adjacent mouse Gba -/- neurons and improved GCase activity, reduced GCase substrate accumulation, and improved mitochondrial function. Short-term invivo effects were evaluated in 9H/PS-NA mice, an nGD mouse model exhibiting neuropathology and α-synuclein aggregation, the typical PD phenotypes. Intravenously administrated hGBA1-NPCs were engrafted throughout the brain and differentiated into neural lineages. GCase activity was increased in various brain regions of treated 9H/PS-NA mice. Compared with vehicle, hGBA1-NPC-transplanted mice showed ∼50% reduction of α-synuclein aggregates in the substantia nigra, significant reduction of neuroinflammation and neurodegeneration in the regions of NPC migration, and increased expression of neurotrophic factors that support neural cell function. Together, these results support the therapeutic benefit of intravenous delivery of iPSC-derived NPCs overexpressing GCase in mitigating nGD and PD phenotypes and establish the feasibility of combined cell and gene therapy for GBA1-associated PD.

The Effect of Enzyme Activity on Carbon Sequestration and the Cycle of Available Macro- (P, K, Mg) and Microelements (Zn, Cu) in Phaeozems

The study objective was to determine the relationship of selected enzyme activities with carbon sequestration and N, P, K, Mg, Zn and Cu contents in Phaeozem soils. Soil samples were taken from a 10 ha area. A selection of their physical and chemical properties and the contents of the available forms of selected macro- and microelements were determined. The activities of dehydrogenases (DEH), catalase (CAT), peroxidases (PER), alkaline (AlP) and acid (AcP) phosphatase, β-glucosidase (BG) and proteases (PR) were also determined. The relationship between enzymatic soil fertility indices (AlP/AcP, BIF, GMea, TEI, BA12 and BA13) and selected soil parameters was also determined. The research used principal component analysis (PCA) to distinguish significantly correlated parameters of a Phaeozem used for agricultural purposes. The study area showed low TOC and K contents and average P and Mg contents. Significant positive correlations were found between the TOC content and activity of the tested enzymes, evidencing that soil enzymes are an important parameter in carbon sequestration and soil nutrient dynamics.

РЕДКИЕ ЗАБОЛЕВАНИЯ СИСТЕМЫ КРОВИ: БОЛЕЗНЬ ГОШЕ. ОПИСАНИЕ КЛИНИЧЕСКОГО СЛУЧАЯ

Gaucher disease is the most common form of hereditary enzymopathies included in the group of lysosomal storage diseases. The disease is based on a hereditary deficiency in the activity of the enzyme glucocerebrosidase (also called glucosylceramidase or acid β-glucosidase) which is involved in the degradation of cellular metabolic products. Gaucher disease is a systemic disease characterized by the same type of clinical manifestations (hepatosplenomegaly, cytopenias, lesions of the skeletal system) but an extremely heterogeneous clinical course. In the article, the authors describe their own clinical case of Gaucher disease in a teenage girl. A long past history of he¬terogeneous clinical symptoms, the complexity of differential diagnosis and interpretation of existing deviations within the course of the single systemic process were noted. The high effectiveness of modern enzyme replacement therapy has been demonstrated.

The activity of lysosomal enzymes in organs of the White Sea mussel Mytilus edulis L. under crude oil impact in different salinity conditions

The effect of the toxicant on the activity of lysosomal hydrolases in organs of the White Sea mussels Mytilus edulis (Linnaeus 1758) was studied in aquatic experiments simulating a crude oil spill in the tidal zone. The mollusks were exposed for 1, 5 and 10 days to three pollutant concentrations (0.05; 0.25, and 2.50 ml/l). The impact of oil pollution was studied in combination with salinity normal for White Sea surface water (25 ‰) and low (15 ‰), which corresponds to reality during oil spills on the coast and in estuaries. We determined the activity of six lysosomal enzymes (acid phosphatase, RNase, DNase, β-glucosidase, β-galactosidase, and β-glucuronidase) in the gills and hepatopancreas. Both factors (sea water desalination and oil impact) had a significant effect on the activity of acid hydrolases. The most pronounced lysosomal reaction to oil appeared in the gills under normal salinity at 0.25 ml/l concentration and exposure for 10 days, as evidenced by a several-fold increase in the activity of acid phosphatase, DNase, β-galactosidase, and β-glucuronidase compared to the control. In the hepatopancreas, the same dependence in enzyme activity was noted, but to a lesser extent. Seawater desalination to 15 ‰ in the control caused a slight increase in the activity of DNase, β-galactosidase, and β-glucuronidase in the gills, while the effect for the hepatopancreas was insignificant. The combined effect of desalination and oil pollution somewhat reduced the protective functions of the lysosomal apparatus in mussels organs, which was especially noticeable at high oil concentrations and prolonged exposure of mollusks to experimental conditions. Thus, the results of the study demonstrate active participation of the lysosomal apparatus of the gills and hepatopancreas of mollusks in the adaptive responses to the combined effects of oil pollution and low water salinity. Compensatory changes in the activity of the enzyme complex of lysosomes are aimed at the utilization, transformation and excretion of oil components from the body, elimination of the structures and macromolecules damaged by the toxicant, as well as maintaining the vital activity of the organism under adverse conditions.

Venglustat combined with imiglucerase for neurological disease in adults with Gaucher disease type 3: the LEAP trial.

Gaucher disease type 3 is a chronic neuronopathic disorder with wide-ranging effects, including hepatosplenomegaly, anaemia, thrombocytopenia, skeletal disease and diverse neurological manifestations. Biallelic mutations in GBA1 reduce lysosomal acid β-glucosidase activity, and its substrates, glucosylceramide and glucosylsphingosine, accumulate. Enzyme replacement therapy and substrate reduction therapy ameliorate systemic features of Gaucher disease, but no therapies are approved for neurological manifestations. Venglustat is an investigational, brain-penetrant, glucosylceramide synthase inhibitor with potential to improve the disease by rebalancing influx of glucosylceramide with impaired lysosomal recycling. The Phase 2, open-label LEAP trial (NCT02843035) evaluated orally administered venglustat 15 mg once-daily in combination with maintenance dose of imiglucerase enzyme replacement therapy during 1 year of treatment in 11 adults with Gaucher disease type 3. Primary endpoints were venglustat safety and tolerability and change in concentration of glucosylceramide and glucosylsphingosine in CSF from baseline to Weeks 26 and 52. Secondary endpoints included change in plasma concentrations of glucosylceramide and glucosylsphingosine, venglustat pharmacokinetics in plasma and CSF, neurologic function, infiltrative lung disease and systemic disease parameters. Exploratory endpoints included changes in brain volume assessed with volumetric MRI using tensor-based morphometry, and resting functional MRI analysis of regional brain activity and connectivity between resting state networks. Mean (SD) plasma venglustat AUC0-24 on Day 1 was 851 (282) ng•h/ml; Cmax of 58.1 (26.4) ng/ml was achieved at a median tmax 2.00 h. After once-daily venglustat, plasma concentrations (4 h post-dose) were higher compared with Day 1, indicating ∼2-fold accumulation. One participant (Patient 9) had low-to-undetectable venglustat exposure at Weeks 26 and 52. Based on mean plasma and CSF venglustat concentrations (excluding Patient 9), steady state appeared to be reached on or before Week 4. Mean (SD) venglustat concentration at Week 52 was 114 (65.8) ng/ml in plasma and 6.14 (3.44) ng/ml in CSF. After 1 year of treatment, median (inter-quartile range) glucosylceramide decreased 78% (72, 84) in plasma and 81% (77, 83) in CSF; median (inter-quartile range) glucosylsphingosine decreased 56% (41, 60) in plasma and 70% (46, 76) in CSF. Ataxia improved slightly in nine patients: mean (SD, range) total modified Scale for Assessment and Rating of Ataxia score decreased from 2.68 [1.54 (0.0 to 5.5)] at baseline to 1.55 [1.88 (0.0 to 5.0)] at Week 52 [mean change: -1.14 (95% CI: -2.06 to -0.21)]. Whole brain volume increased slightly in patients with venglustat exposure and biomarker reduction in CSF (306.7 ± 4253.3 mm3) and declined markedly in Patient 9 (-13894.8 mm3). Functional MRI indicated stronger connectivity at Weeks 26 and 52 relative to baseline between a broadly distributed set of brain regions in patients with venglustat exposure and biomarker reduction but not Patient 9, although neurocognition, assessed by Vineland II, deteriorated in all domains over time, which illustrates disease progression despite the intervention. There were no deaths, serious adverse events or discontinuations. In adults with Gaucher disease type 3 receiving imiglucerase, addition of once-daily venglustat showed acceptable safety and tolerability and preliminary evidence of clinical stability with intriguing but intrinsically inconsistent signals in selected biomarkers, which need to be validated and confirmed in future research.

Rhodotorula mucilaginosa CAM4 improved selenium uptake in Spinacia oleracea L. and soil enzymatic activities under abiotic stresses.

This study aimed to examine selenium (Se) acquisition by spinach (Spinacia oleracea L.) plants growing under salinity and drought stress through the inoculation of Rhodotorula mucilaginosa strain CAM4. Under abiotic stress conditions, strain CAM4 with Se inoculation increased the shoot length, root length, shoot dry weight and root dry weight by 75.8-93.7%, 47.7-80.9%, 101.9-109.8% and 130.5-270.2%, respectively compared to uninoculated Se-treated plants grown under the same conditions. Under abiotic stresses, the Se-treated CAM4 inoculated plants showed a significant increase in Se concentration in the edible leaves of spinach, which was 227.3-234.5% higher than uninoculated Se-treated control plants. Likewise, strain CAM4 treatment significantly enhanced the plant nutrition of both micro and macro-nutrients. Under normal and abiotic stresses, CAM4 inoculation enhanced soil activities of acid phosphatase, alkaline phosphatase, dehydrogenase, β-glucosidase and urease in the post harvested soil up to 28-47.5%, 62.6-121.8%, 69-177.1%, 16.2-37.9% and 19.8-41.2%, respectively over corresponding uninoculated soil.

Inhibition of lysosomal acid β-glucosidase induces cell apoptosis via impairing mitochondrial clearance in pancreatic cancer

Sphingolipid metabolism plays an important role in the formation of cellular membranes and is associated with malignant potential and chemosensitivity of cancer cells. Sphingolipid degradation depends on multiple lysosomal glucosidases. We focused on acid β-glucosidase (GBA), a lysosomal enzyme the deficiency of which is related to mitochondrial dysfunction. We analyzed the function of GBA in pancreatic ductal adenocarcinoma (PDAC). Human PDAC cell lines (PANC-1, BxPC-3 and AsPC-1) were examined under conditions of GBA knockdown via the short interfering RNA (siRNA) method. We assessed the morphological changes, GBA enzyme activity, GBA protein expression, cell viability, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) and mitophagy flux of PDAC cells. The GBA protein level and enzyme activity differed among cell lines. GBA knockdown suppressed cell proliferation and induced apoptosis, especially in PANC-1 and BxPC-3 cells, with low GBA enzyme activity. GBA knockdown also decreased the MMP and impaired mitochondrial clearance. This impaired mitochondrial clearance further induced dysfunctional mitochondria accumulation and ROS generation in PDAC cells, inducing apoptosis. The antiproliferative effects of the combination of GBA suppression and gemcitabine were higher than those of gemcitabine alone. These results showed that GBA suppression exerts a significant antitumor effect and may have therapeutic potential in the clinical treatment of PDAC.

Comparison and application of the extraction method for the determination of enzymatic profiles in matured soybean koji

In this study, two methods of extracting crude enzymes from soy sauce koji were compared to investigate the effect of replacing the buffer extraction with distilled water extraction by measuring the enzyme activity. Results showed the enzyme activities of the two extracts in each koji sample were not significant differences. The stability of the different enzymes in the extracts varied. Acid protease, β-glucosidase and cellulase activities showed the best stability during the storage. Neutral protease, glucoamylase and aminopeptidase were moderately stable and their activities needed to be determined within 3 d of extract preparation, while xylanase activity was the least stable, decreasing by 40% on the second day of storage. Therefore, the recommended procedure for the analysis of koji enzyme profiles was as follows, xylanase was determined immediately after distilled water extraction, and then all other enzyme activity analyses need to be completed within 3 d to ensure the validity of enzyme analysis. This method was applied to the enzymatic analysis of 7 kojis made by different strains of Aspergillus oryzae, and to explore the association between the enzyme profile and the taste characteristics of seven fermented soy sauce. The results showed proteases were correlated with sour and richness, while carbohydrate-degrading enzymes were relatively more relevant to umami taste.

P931: PROSPECTIVE OBSERVATIONAL MULTICENTER STUDY OF GAUCHER DISEASE PREVALENCE IN PATIENTS AFFECTED BY MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE

Background: Gaucher disease (GD) is a rare, autosomal recessive genetic disorder, caused by deficiency of glucocerebrosidase, leading to glucosylceramide accumulation in tissue macrophages of hematological, visceral, and skeletal organ systems. Type 1 GD accounts for more than 90% all patients. Monoclonal gammopathy of undetermined significance is the most common plasma cell disorder, occurring in 3% of the population older than 50 years and is typically detected as an incidental finding. Type 1 GD It is frequently associated with polyclonal and monoclonal gammopathy (MGUS) and is hypothetically caused by long-term immune activation due to accumulated lysolipids, particularly glucosylsphingosine, although the cause remains unknown. Aims: We evaluated the prevalence of GD in MGUS patients, particularly when other signs and symptoms are present, in order to determine its potential usefulness as screening tool and inclusion in diagnostic framework of GD. Methods: Between January 2018 and February 2022, dried blood spots (DBS) samples were collected and tested for the acid β-glucosidase (glucocerebrosidase) enzyme activity from MGUS patients, both adult and pediatric, followed in seven hematology units of Sicily, Calabria and Naples. The glucocerebrosidase activity was measured with multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD® assay system, and pathological range was within 0.2 and 2.5 nmol/h/ml. In case of DBS positive result, a confirmatory test was carried over in order to confirm the diagnosis of GD. The study was approved by the local institutional review board and supported by Sanofi Genzyme. All patients provided informed consent for the prospective collection of their data. Results: A total of 600 patients with MGUS was enrolled at last study update. Around half of the study population was male, with median age of 65 years. Immunoglobulin G was most frequently isolated with serum immunofixation, median level of monoclonal protein was 0.66 g/dL, while median kappa and lambda values based on monoclonal serum free light chain type were 24.8 mg/L and 32.7 mg/L, respectively. Out of 161 patients with available complete blood count median hemoglobin value was 13.5 g/dL with 15 patients having less than 11 g/dL, median platelet count was 224.000/mmc with 10 patients having less than 100.000/mmc platelets and median neutrophil count of 4.280/mmc and seven patients with less than 2.000/mmc neutrophils. Median ferritin level was 77 ng/mL, with more than 400 ng/mL in 10 patients. The median glucocerebrosidase activity in the entire cohort was 6.9 nmol/h/ml (range 0.3-58.8), with thirteen patients having pathological enzyme activity (median value 1.8 nmol/h/ml, range 0.2-2.5). Sequence analysis of GBA gene evidenced compound heterozygous mutation of the GBA1 gene in three patients (c.1226A>G - N370S and c.1448T>C -L444P in two and c.1226A>G -N370S and c.355G>A -G119R in one respectively), and homozygous mutation (c.1226A>G -N370S) in one patient associated with glucocerebrosidase activity below normal limits. All three patients with compound heterozygous mutational status had signs of GD (hepatosplenomegaly and mild thrombocytopenia). As for the rest of the MGUS patients, in a total of 114 patients sequence analysis evidenced single heterozygous mutation in 16 patients. Summary/Conclusion: Although type 1 GD remains a rare lysosomal storage disorder, patients with the diagnosis of MGUS could be considered for GD screening with DBS, especially when other symptoms (thrombocytopenia, splenomegaly, hyperferritinemia) are present.