Acid Β-glucosidase Research Articles

Prevalence of Gaucher Disease in Patients of Unknown Cause of Splenomegaly and/or Thrombocytopenia in Saudi Arabia

Rationale: Gaucher disease (GD) is the most common amongst the lysosomal storage disorders. Prevalence of GD in Saudi Arabia is not available in published literature and it is expected to be high and remains undiagnosed. In 2004, a Saudi study reported that GD accounts for 6% of all genetic metabolic disorders. While acid sphingomyelinase deficiency (ASMD) is a rare progressive genetic disorder with no clear data about its incidence in Saudi Arabia. Consequently, this study proposes to determine the prevalence of GD and ASMD in outpatient settings in Saudi Arabia by screening patients with unknown causes of splenomegaly and/or thrombocytopenia. These data will change the local practice and increase the awareness towards GD and ASMD in Saudi Arabia. Objectives: To determine the prevalence of GD in high risk group (defined as patients with splenomegaly and/or thrombocytopenia of unknown cause) and describe their characteristics. We also aim to determine the prevalence of ASMD in high risk group. Methods: A multi-center, observational, study will be conducted in 25 specialty care centres across Saudi Arabia, these centres are mainly focusing on pediatric hematology, adult hematology and hemato-pathology. Female or male patients (aged 2-75 years) will deemed eligible if they have clinical, instrumental or laboratory signs of splenomegaly or thrombocytopenia over a period of 12 months without definitive cause. These patients will be tested for acid β-glucosidase and acid sphingomyelinase enzymes activity on dried blood spot (DBS) samples. Patients with hematological malignancies, hemolytic anemia, and/or thalassemia (except sickle cell disease) will be excluded. A total of 400 patients from Saudi Arabia who fulfill the eligibility criteria will be enrolled in the study. All patient data will be collected in a single visit. Each enrolled patient will visit the investigator for a baseline visit. The investigator will contact them later for sharing the investigation blood test results. During the baseline visit, data and blood sample for enzyme tests and genotyping will be collected by the investigator/designated person at the site. Discussion: Arab world represents one of the leading regions in terms of the incidence of congenital and genetic disorders; a growing body of published literature reported a notable trends towards higher incidence of congenital and genetic diseases, compared to other parts of the world(1). High consanguinity rates which reach up to 60% in some regions, high prevalence of haemoglobinopathies and metabolic disorders, relatively high maternal and parental age, and lack of proper genetic screening were reported as contributing factors for this high prevalence of genetic disorders in the Arab world(1-3). In Saudi Arabia, the situation appears to be no different as previous retrospective studies showed a relatively high incidence of genetic diseases such as inborn error of metabolism. The data of the present study will change the local practice and increase the awareness towards GD and ASMD in Saudi Arabia.

Pregnancy outcome in women with Gaucher disease type 1 who had unplanned pregnancies during eliglustat clinical trials.

Gaucher disease type 1 (GD1) is an inherited lysosomal storage disorder caused by deficient enzymatic activity of acid β‐glucosidase, resulting in accumulation of its substrate glucosylceramide, leading to debilitating visceral, hematologic, and skeletal manifestations. Women with GD1 are at increased risk for complications during pregnancy, delivery, and postpartum. Treatment with enzyme replacement therapy is generally recommended before and during pregnancy to reduce risks. Eliglustat, an oral substrate‐reduction therapy, is a first‐line treatment for adults with GD1 adults who have extensive, intermediate, or poor CYP2D6‐metabolizer phenotypes (>90% of patients). We report on pregnancy outcomes among women in eliglustat trials who had unplanned pregnancies and female partners of men in the trials. In four phase 2 and 3 eliglustat trials of 393 adults with GD1, women of childbearing potential were required to use contraception, have monthly pregnancy tests, and discontinue eliglustat promptly if pregnant. In phase 2 and 3 trials, 18 women had 19 pregnancies, resulting in 14 healthy infants from 13 pregnancies (one set of twins), three elective terminations, one ectopic pregnancy, one spontaneous abortion, and one in utero death. Median estimated eliglustat exposure duration during pregnancy was 38 days. In phase 1 trials (non‐GD1 subjects), one woman had a spontaneous abortion. Partners of 16 eliglustat‐treated men with GD1 had 18 pregnancies, all resulting in healthy infants. Eliglustat is not approved during pregnancy due to limited data. Guidelines for clinicians and patients with GD that address use of eliglustat in women of childbearing potential are needed.

Targeted quantification of B vitamins using ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry in faba bean seeds

Ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry (UPLC-SRM MS) was used for targeted quantification of water-soluble B vitamins from seeds of 30 genotypes of faba bean with contrasting anti-nutritional profiles. Sixteen vitamers from seven B vitamins were quantified from whole seeds, seed coats and cotyledons. A single-step extraction approach with the use of four enzymes (acid phosphatase, β-glucosidase, protease and rat serum) and 2 h incubation at 37 °C was used. The total B vitamin concentrations ranged from 23.08 to 39.26 μg/g in whole seeds, 11.51 to 30.31 μg/g in seed coats and 23.56 to 32.66 μg/g in cotyledons. Significant genotype by environment interaction effects were observed for all B vitamins. Our results indicated low-tannin genotypes had high B5 content, whereas tannin-containing genotypes had high B1, B2, B6, and B9 content, particularly in seed coats. The analysis of a large number of faba bean germplasm here suggests that its seeds are a rich source of vitamins B1, B2, B3 and B5. The increased consumption of faba bean seeds and ingredients in the human diet can provide enhanced dietary B vitamins.

Acid phosphomonoesterase and β-glucosidase activities in volcanic soils under permanent fertilized pastures: distribution profile and microbial effort toward P acquisition

ABSTRACT This work focused on the amounts and distribution of organic C (OC), organic phosphorus (Po), and acid phosphomonoesterase (AcP) and β-glucosidase (βG) activities in volcanic soils, as well as on the effect of inorganic P (Pi) on potential P-acquiring effort. The OC and Po contents are correlated with the active aluminum (Alox) and iron (Feox) and with the stratified gradients of the enzyme activities. The AcP activity does not seem to be suppressed by soil Pi content, showing a positive linear relationship with Pi. High values for the AcP:βG activity ratio are observed, which suggests a high energy allocation (C consumption) to ensure P demand. With the increase of Alox and Feox, the AcP:βG ratios increase and the potential C:P acquisition (ln(βG):ln(AcP)) values decrease. Nevertheless, the average C:P molar ratio of the six soils (115) are below the critical value, above which soils may present low P bioavailability. The results suggest that the effort for P acquisition (AcP:βG) and the potential C:P acquisition are both affected by the size of the Pi pools and by Alox and Feox contents. If Pi retention increases due to high contents of Alox and Feox, energy allocation shifts toward AcP production, in detriment of microbial population and plant nutrition. Considering the OC accumulation in the studied soils, several hypotheses may be considered such as the energy demand toward P acquisition, and the ability of Alox and Feox metal complexes to protect organic molecules to depress βG activity.

Cover crop biomass and species composition affect soil microbial community structure and enzyme activities in semiarid cropping systems

Cover crops are promoted to increase soil organic carbon (SOC) storage and improve soil biological health in agricultural systems. However, cover crop effects on soil microbial communities – key regulators of SOC and nutrients – and functioning are not clear in semiarid environments. This study investigated the response of soil microbial community structure and enzyme activities to cover crop integration under limited-irrigation winter wheat (Triticum aestivum)-sorghum (Sorghum bicolor)-fallow rotation. The study had a randomized complete block design with eight treatments and three replications. Treatments were pea (Pisum sativum); oat (Avena sativa); canola (Brassica napus L.); and mixtures of pea + oat (POmix), pea + canola (PCmix), pea + oat + canola (POCmix), and pea + oat + canola + hairy vetch (Vicia villosa) + forage radish (Raphanus sativus L.) + barley (Hordeum vulgare L.) (diverse-mix) as cover crops; and a fallow. Soil samples were collected in the summer of 2017 and 2018 from 0 to 15 cm depth of each plot established in fall 2015. Microbial community size and structure were evaluated via ester-linked fatty acid methyl ester (EL-FAME) analysis, which showed that total microbial community size and fungal community were similar between soils under oats and diverse-mix, and were 31% and 41% greater than fallow, respectively. The FAME marker for arbuscular mycorrhizal fungi (AMF) was 84% greater under oats than fallow. The combined enzyme activity of acid phosphatase, β-glucosidase, and β-glucosaminidase was 294 mg p-nitrophenol (PNP) kg−1 soil h−1 under diverse-mix, which was greater than fallow (204 mg PNP kg−1 soil h−1, p = 0.021) in 2018. Cover cropping during fallow period in a crop-fallow rotation could increase total microbial community size, fungal abundance, and enzyme activities associated with carbon (C) and nutrient cycling. Among cover crops, oat and its mixture with legumes (pea and hairy vetch) and brassicas (canola and forage radish) were most effective in improving soil health and biogeochemical cycling in a hot and dry semiarid climate.

Bioremediation of Cd-contaminated soil by earthworms (Eisenia fetida): Enhancement with EDTA and bean dregs

The remediation of cadmium (Cd) contaminated soil has become a global problem due to its toxicity to living organisms. In this study, earthworm (Eisenia fetida) alone or combined with EDTA or bean dregs were used for Cd removal from soils. The total and available Cd in soils, soil physicochemical and biological (soil enzyme) properties, Cd accumulation in the earthworm and its antioxidant responses towards Cd, were determined during the 35 days of soil incubation experiment. Our results showed that earthworms were capable of removing Cd from soils, and the remediation process was accelerated by both EDTA and bean dregs. By translocation of Cd from soils, the content of Cd in earthworm steadily increased with the exposure time to 8.11, 12.80, and 9.26 mg kg−1 on day 35 for T2 (earthworm alone), T3 (EDTA enhancement), and T4 (bean dregs enhancement), respectively. Consequently, a great reduction in the Cd contents in soils was achieved in T3 (36.53%) and T4 (30.8%) compared with T2 (28.95%). The concentrations of water/DTPA extractable Cd were also reduced, indicating the low Cd mobility after amendment. Finally, the soil became more fertile and active after wermi-remediation. The soil pH, EC, NO3−-N, available P, and K contents increased, while soil SOM, DOC, and NH4+-N contents were decreased. There were higher soil enzyme activities (including acid phosphatase activity, β-glucosidase activity, and urease activity) among treatments with earthworms. Additionally, the operational taxonomic units (OTUs) increased by 100–150 units, and the higher chao1 and Shannon indexes indicated the enhanced microbial community after wermi-remediation, especially among treatment with EDTA and bean dregs. Therefore, we concluded that earthworms, alone or combined with EDTA and bean dregs, are feasible for the remediation of Cd-contaminated soil.

Genotype/phenotype relationship in Gaucher disease patients. Novel mutation in glucocerebrosidase gene.

Objectives Gaucher disease (GD) is the most common inherited lysosomal storage disease, caused by mutations in acid β-glucosidase (GBA) gene. This study aimed to identify mutations in Andalusia patients with GD and their genotype-phenotype correlation. Methods Descriptive observational study. University Hospital Virgen del Rocio patients diagnosed from GD from 1999 to 2019 were included. Demographic and clinical data, β-glucocerebrosidase activity, variants pathogenic in GBA gene and biomarkers for monitoring treatment were collected from digital medical record. Results Twenty-six patients with aged between 1day and 52 years were studied. A total of six mutations described as pathogenic and one mutation not described above [c.937T>C (p.Tyr313His)] were identified in the GBA gene, four patients were homozygotes and 22 compound heterozygotes. Twenty-four patients were diagnosed in non-neuropathic form (type 1) and two cases presented neurological involvement (type 2 or 3). The most common variant was c.1226A>G (p.Asn409Ser), which was detected in 24 patients, followed by c.1448T>C (p.Leu483Pro) variant, identified in 13 patients. The c.1448T>C (p.Leu483Pro) mutation has been presented in the most severe phenotypes with neurological involvement associated with type 2 and 3 GD, while c.1226A>G (p.Asn409Ser) mutation has not been associated with neurological alterations. Splenomegaly and bone disease were the most frequent clinical manifestations, and thrombocytopenia was the most common hematological disorder. Conclusions The c.1226A>G (p.Asn409Ser) and c.1448T>C (p.Leu483Pro) mutations were the most common. The c.937T>C (p.Tyr313His) was identified as a novel mutation. The c.1448T>C (p.Leu483Pro) mutation was associated with neurological alterations and c.1226A>G (p.Asn409Ser) mutation has not been associated it.

The role of an urban park's tree stand in shaping the enzymatic activity, glomalin content and physicochemical properties of soil

Soil enzymes play a key role in the circulation of nutrients and the functioning of the ecosystem. The aim of the study was to assess how the tree species of urban agglomerations affect soil quality and enzymatic activity (dehydrogenases DEH, catalase CAT, alkaline AlP and acid AcP phosphatase, protease PR, β-glucosidase GLU, and urease UR). To this end, soil samples were taken from beneath nine park trees. The risk of soil contamination by selected heavy metals (Pb, Ni, Cd) was also investigated against the background of the selected physicochemical properties. Enzyme activity results were used to calculate multi-parametric indices of soil quality: availability factor (AF), enzymatic pH indicator (AlP/AcP), biological index of fertility (BIF), geometric mean (GMea), alternation index (Al3), biochemical soil activity (BA16 and BA17). The results showed statistically significant differences in physicochemical and enzymatic properties of soil depending on tree species. Correlation analysis showed that the content of total organic carbon (TOC), total nirogen (TN), total phosphorus (TP) and humus (OM) in soil significantly influenced the activity of the studied enzymes and glomalin content. AF coefficient values (1.84%–18.19%) suggest that the bioavailability of available phosphorus (AP) was sufficient. The Pb, Ni, Cd content results were found to be low and did not exceed the permissible concentrations. DEH, CAT and AlP activity were highest under common hawthorn, and AcP, GLU and PR under northern white cedar. The calculated enzymatic indicators proved to be a sensitive and accurate indicator of the dynamics of changes taking place in the city park soil. Based on the results, an attempt can be made to assess the planning of sustainable development of studied areas of urban parks.

CRISPR/Cas9 Editing for Gaucher Disease Modelling.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid β-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson’s disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid β-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1β, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.

Imino- and Azasugar Protonation Inside Human Acid β-Glucosidase, the Enzyme that is Defective in Gaucher Disease.

Gaucher disease is caused by mutations in human acid β-glucosidase or glucocerebrosidase (GCase), the enzyme responsible for hydrolysis of glucosyl ceramide in the lysosomes. Imino- and azasugars such as 1-deoxynojirimycin and isofagomine are strong inhibitors of the enzyme and are of interest in pharmacological chaperone therapy of the disease. Despite several crystal structures of the enzyme with the imino- and azasugars bound in the active site having been resolved, the actual acid-base chemistry of the binding is not known. In this study we show, using photoinduced electron transfer (PET), that 1-deoxynojirimycin and isofagomine derivatives are protonated by human acid β-glucosidase when bound, even if they are completely unprotonated outside the enzyme. While isofagomine derivative protonation to some degree was foreshadowed by earlier crystal structures, 1-deoxynojirimycin derivatives were not believed to act as basic amines in the enzyme.

Systemic enzyme delivery by blood-brain barrier-penetrating SapC-DOPS nanovesicles for treatment of neuronopathic Gaucher disease

BackgroundEnzyme replacement therapy (ERT) can positively affect the visceral manifestations of lysosomal storage diseases (LSDs). However, the exclusion of the intravenous ERT agents from the central nervous system (CNS) prevents direct therapeutic effects. MethodsUsing a neuronopathic Gaucher disease (nGD) mouse model, CNS-ERT was created using a systemic, non-invasive, and CNS-selective delivery system based on nanovesicles of saposin C (SapC) and dioleoylphosphatidylserine (DOPS) to deliver to CNS cells and tissues the corrective, functional acid β-glucosidase (GCase). FindingsCompared to free GCase, human GCase formulated with SapC-DOPS nanovesicles (SapC-DOPS-GCase) was more stable in serum, taken up into cells, mostly by a mannose receptor-independent pathway, and resulted in higher activity in GCase-deficient cells. In contrast to free GCase, SapC-DOPS-GCase nanovesicles penetrated through the blood-brain barrier into the CNS. The CNS targeting was mediated by surface phosphatidylserine (PS) of blood vessel and brain cells. Increased GCase activity and reduced GCase substrate levels were found in the CNS of SapC-DOPS-GCase-treated nGD mice, which showed profound improvement in brain inflammation and neurological phenotypes. InterpretationThis first-in-class CNS-ERT approach provides considerable promise of therapeutic benefits for neurodegenerative diseases. FundingThis study was supported by the National Institutes of Health grants R21NS 095047 to XQ and YS, R01NS 086134 and UH2NS092981 in part to YS; Cincinnati Children's Hospital Medical Center Research Innovation/Pilot award to YS and XQ; Gardner Neuroscience Institute/Neurobiology Research Center Pilot award to XQ and YS, Hematology-Oncology Programmatic Support from University of Cincinnati and New Drug State Key Project grant 009ZX09102-205 to XQ.

LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population.

Background Gaucher disease (GD), caused by a deficiency in acid β-glucosidase, leads to the accumulation of glucosylsphingosine (GluSph), which has been used as a powerful biomarker for the diagnosis and follow-up of GD. Our aim was to perform the first retrospective study of GluSph in Spanish patients, analyzing its relationship with classical biomarkers and other parameters of disease and its utility regarding treatment monitoring. Methods Classical biomarkers were evaluated retrospectively by standard methods in a total of 145 subjects, including 47 GD patients, carriers, healthy controls and patients suffering from other lysosomal lipidoses. GluSph was also measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed as part of the present study. Results The optimized method presented intra- and inter-assay variations of 3.1 and 11.5%, respectively, overall recovery higher than 96% and linearity up to plasma concentrations of 1000 ng/mL with 100% specificity and sensitivity. Only GD patients displayed GluSph levels above 5.4 ng/mL at diagnosis and this was significantly correlated with the classical biomarkers chitotriosidase (r = 0.560) and the chemokine CCL18/PARC (CCL18/PARC) (ρ = 0.515), as well as with the Spanish magnetic resonance imaging index (S-MRI, r = 0.364), whereas chitotriosidase correlated with liver volume (r = 0.372) and CCL18/PARC increased in patients with bone manifestations (p = 0.005). GluSph levels decreased with treatment in naïve patients. Conclusions Plasma GluSph is the most disease-specific biomarker for GD with demonstrated diagnostic value and responsiveness to therapy. GluSph in the present series of patients failed to demonstrate better correlations with clinical characteristics at onset than classical biomarkers.

Salinization effects on stream biofilm functioning

Secondary salinization threatens fresh waters worldwide leading to deleterious ecological and economic consequences. The goal of the present study was to assess the potential response of catabolic functions (respiration rate, activities of acid phosphatase and β-glucosidase) of stream biofilms to salinization. Biofilm assemblages from five streams were incubated under controlled conditions in microcosms in spring water under four different treatments: no salt addition (0) and adding 0.25, 1, and 4 g NaCl l−1. Respiration rate at 0.25 g NaCl l−1 was higher than that at 1 and 4 g l−1, the latter concentration presenting the lowest value. Acid phosphatase activity was highest in the treatment without salt addition. The activity of β-glucosidase was highest in the non-salted treatment and at 1 g NaCl l−1. Results reveal salinization as an important threat for catabolism-related functions of stream biofilms, which can compromise nutrient cycling in small shaded streams and consequently the ecosystem services that they provide.

Decreased expression of GBA3 correlates with a poor prognosis in hepatocellular carcinoma patients.

Beta-glucosidase (GBA), also known as acid β-glucosidase, exhibits an activity of glucosylceramidase (EC 3.2.1.45). Three main isoforms of β-glucosidases have been identified in mammals: GBA1, GBA2, and GBA3. The deficiency of these enzymes leads to glucosylceramide accumulation, resulting in Gaucher's disease. The present study is focused on the cytosolic β-glucosidase, GBA3, and its relationship with hepatocellular carcinoma (HCC). The expression of GBA3 mRNA in HCC was evaluated first using the TCGA database, and then by immunohistochemistry using tissue microarrays of 328 clinically characterized HCC samples and 151 non-tumor liver controls. Moreover, the presence of a correlation between GBA3 expression and clinicopathological characteristics of patients was examined. The obtained results indicated that the expression of GBA3 mRNA was significantly lower in HCC than in the adjacent non-tumor liver tissues. The expression of GBA3 was inversely related to the number of tumors (p=0.041), tumor size (p<0.001), Edmondson grade (p=0.007), microvascular invasion (p=0.049), patient status (p<0.001), and α-fetoprotein level (p<0.001). Patients exhibiting low GBA3 expression had a shorter survival time than those with high expression (p<0.001). In conclusion, the decreased GBA3 expression is strongly associated with a poor prognosis in HCC patients, and GBA3 may be a potential therapeutic target for HCC.

Plant diversity in integrated crop-livestock systems increases the soil enzymatic activity in the short term

ABSTRACT Integrated crop-livestock systems (ICLS) intercropped in the pasture phase promote alterations in the dynamics of soil attributes, among them the enzymatic activity. This study aimed to evaluate soil enzymatic and microbial activities, following the implementation of ICLS with increased plant diversity during the pasture phase. The study consisted of soybean cultivation at season and pasture with different diversity levels in the off-season: very low - monocropped Urochloa brizantha cv. BRS Paiaguás with no animal entry; low - monocropped Paiaguás; medium - Paiaguás and cowpea bean (Vigna unguiculata); high - Paiaguás, cowpea bean and niger (Guizotia abyssinica); very high - Paiaguás, cowpea bean, niger, buckwheat (Fogopyrum esculentum) and forage turnip (Raphanus sativus). The introduction of plants from different families, associated to grazing, positively affected the enzymatic activities of acid phosphatase, β-glucosidase and urease. In relation to microbial biomass carbon and basal respiration, there were no effects from the treatments. Therefore, among the evaluated biochemical soil attributes, the activities of some soil enzymes were more sensitive in demonstrating, in the short term, the positive effects of the increased plant variety in the ICLS pasture phase.

Shifts in soil traits and arbuscular mycorrhizal symbiosis represent the conservation status of Araucaria araucana forests and the effects after fire events

Araucaria araucana is a long-lived native conifer from the sub-Antarctic Andean forests of South America. Currently, the species is classified as endangered due to fragmentation, deforestation and increasing fire events affecting southern Chile. Although fire is a key factor shaping the dynamics of A. araucana forests, the impact of fire on the chemical and microbiological soil properties remains elusive. Here, we aimed to characterize the (bio)chemical and microbiological soil traits in different sites of the Andes and the Coastal range of southern Chile, as a way to describe the conservation status of A. araucana forests in contrasting environments with different fire history. The study was conducted in old-growth A. araucana forests within protected areas affected by different fire events during the last 18 years. Roots were analyzed for arbuscular mycorrhizal (AM) colonization and soil samples were processed for (bio)chemical and microbiological analyses. Our results revealed that AM colonization was strongly affected by fire, being closely related with the acid phosphatase and β-glucosidase enzymatic activities, which also showed lower values in fire-affected sites. Glomalin-related protein in the soil was also a good indicator of soil stability associated to environments non-affected by fire. Based on our results, the conservation status benchmark for A. araucana is in the Coastal range, showing high rates of AM colonization, fungal structures and biochemical activities in environments subjected to minor geological risks and no fire events. This research provides helpful information for finding efficient biological inoculants, including indigenous AM fungal species, which can help to enhance A. araucana plantlets with an optimized rhizosphere oriented to the restoration of burned A. araucana forests in south-central Chile.

Preliminary Results of a Prospective Observational Study to Assess the Prevalence of Gaucher Disease in an Adult Population Affected By MGUS

INTRODUCTION: Gaucher disease (GD) is a rare, autosomal recessive genetic disorder. It is due to a deficiency of the lysosomal enzyme, glucocerebrosidase, which leads to an accumulation of its substrate, glucosylceramide, in tissue macrophage with damage to hematological, visceral, and skeletal organ systems. Although GD has a continuous spectrum of severity, it is traditionally classified into three forms: type 1 (chronic; lacking early onset neuronopathy), type 2 (acute; with early onset neuronopathy),and type 3 (chronic; with early onsetneuronopathy). Type 1GD accounts for more than 90% all GD patients. Its prevalence world-wide is 1 in 50,000-100,000 but it is as high as ~1 in 850 in individuals of Ashkenazi heritage. Type 1 GD is frequently associated to monoclonal gammopathies; despite the emergence of theories advanced to explain these observations, the cause remains unknown. OBJECTIVE: Aim of the ongoing observational study is to determine the prevalence of unrecognized type I GD in a selected Italian population with MGUS. MATERIALS AND METHODS: From January 2018, dried blood spots (DBS) sample from patients with laboratory evidence of MGUS coming from five hematology units of Sicily and Calabria were collected and tested for the acid β-glucosidase enzyme activity. The study was approved by the local institutional review board. All patients provided informed consent for the prospective collection of their data. In case of DBS positive result, a confirmatory test was carried over and, if GD was confirmed, the patient was referred to one of the Regional Reference Centers for Metabolic Disease, as for current clinical practice in Italy. RESULTS: To date, 308 patients with MGUS were enrolled; acid β-glucosidase enzyme activity was low in 22 patients (7%). Sequence analysis of GBA gene was performed in these selected patients, but we have found only 4 patients with heterozygous mutation in the GBA1 gene, 1 homozygous(c.1226A&gt;G -N370S) and 1 compound heterozygous (c.1226A&gt;G -N370S and c.1448T&gt;C -L444P); the last 2 patients had signs of GD (hepato-splenomegaly and mild thrombocytopenia). CONCLUSIONS: Type 1 GD remains a rare lysosomal storage disorder but preliminary results of our observational study show that it should be considered in the diagnostic framework of patients with MGUS, particularly when other GD symptoms are present. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Di Raimondo:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding.

Biomarker Response to Oral Eliglustat in Adults with Gaucher Disease Type 1: Results from 4 Completed Clinical Trials

INTRODUCTION: Gaucher disease type 1 (GD1) is caused by hereditary deficiency in activity of lysosomal acid β-glucosidase and lysosomal dysfunction due to abnormal recycling and storage of glycosphingolipids especially in tissue macrophages. Clinical manifestations can include anemia, thrombocytopenia, hepatosplenomegaly, and debilitating bone disease. Hematologists care for most patients with GD1 but may be unaware of the importance of plasma biomarkers not only as tissue response surrogates that are useful for monitoring treatment efficacy but also as pathogenic molecules that can directly contribute to disease manifestations. These biomarkers include chitotriosidase, macrophage inflammatory protein 1β (MIP-1β), glucosylceramide, glucosylsphingosine, and monosialodihexosyl ganglioside (GM3). Here we show differing changes in biomarker levels after inception of eliglustat therapy in treatment-naïve GD1 patients and in patients previously treated with enzyme replacement therapy (ERT). Eliglustat is an oral inhibitor of glucosylceramide biosynthesis approved by FDA as a first-line treatment of adults with GD1 who have extensive, intermediate, or poor CYP2D6 metabolizer phenotypes (&gt;90% of patients). METHODS: We evaluated percent change from baseline in biomarker levels during time on eliglustat in the 4 completed clinical trials of oral eliglustat for GD1 adults (Sanofi Genzyme): Phase 2/NCT00358150 (treatment-naïve patients with moderate to severe baseline disease, N=26); Phase 3 ENGAGE/NCT00891202 (treatment-naïve patients with mild to moderate baseline disease, N=40); Phase 3 ENCORE/NCT00943111 (switch patients at prespecified therapeutic goals stabilized after a mean of 10 years of ERT], N=159); Phase 3 EDGE/NCT01074944 (mixture of treatment-naïve [N=22] and ERT-switch patients [N=148] with mild to moderate baseline disease and variable prior time on ERT). All percentages were calculated in the subset of patients with biomarkers measured at baseline and the reported timepoint. The attrition of patients with data at the longer-term time points is attributable primarily to a combination of 2-year enrollment periods, trial design, and protocol-mandated switches to commercial therapy for US patients. Most patients (81%) continued until eliglustat became commercially available or completion of their trial; 9 patients (2.3% overall) withdrew due to adverse events reported as drug-related. For Phase 2, ENGAGE, and ENCORE, chitotriosidase genotypes were obtained and values were normalized; patients homozygous for the CHIT null mutation were excluded and values for heterozygous patients were doubled. For EDGE patients, chitotriosidase genotype data were not obtained but patients with ≥4 zero values (n=12) were excluded. RESULTS: In treatment-naïve patients, all biomarkers were elevated at baseline. Median levels of glucosylceramide, GM3, and MIP-1β normalized within 1 year of starting eliglustat. Chitotriosidase and glucosylsphingosine levels markedly decreased but did not normalize. After 3-8 years on treatment, median percent reduction in chitotriosidase and glucosylsphingosine (Phase 2 and ENGAGE) was 85-92% (Table) but values did not normalize. In patients switching from ERT, median chitotriosidase was still modestly elevated at baseline. Median values for other biomarkers were normal or near normal. After 1 year of treatment, chitotriosidase decreased (Table). Median glucosylceramide, GM3, and MIP-1β remained normal or normalized. After 3-4 years, GM3 and MIP-1β remained normal. Median percent reduction in chitotriosidase from baseline was 60-63% but values did not normalize. Eliglustat was generally well-tolerated in all 4 trials; 97% of adverse events were mild/moderate and 86% considered unrelated to eliglustat by the investigator. CONCLUSIONS: Gaucher disease biomarkers decreased substantially in treatment-naïve patients after eliglustat treatment. In switch patients, including very stable patients after a mean of 10 years on ERT, chitotriosidase and MIP-1β also decreased substantively after eliglustat treatment. Future longitudinal follow-up of eliglustat-treated patients should indicate whether the biomarker and plasma glycosphingolipid responses reported here in stable patients at long-term therapeutic goals are associated with immunological and clinical improvements. Disclosures Weinreb: Shire HGT: Consultancy, Honoraria, Speakers Bureau; Sanofi Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cox:Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement, Research Funding. Mistry:Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement, Research Funding; NIH NIMS: Research Funding; Shire: Honoraria, Other: Travel Reimbursement, Research Funding; Pfizer: Honoraria, Other: Travel Reimbursement; Synageva: Honoraria, Other: Travel Reimbursement. Charrow:BioMarin: Research Funding; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amicus: Research Funding. Lukina:JSC GENERIUM: Research Funding; Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement, Research Funding; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement. Foster:Sanofi Genzyme: Employment. Peterschmitt:Sanofi Genzyme: Employment.

Evaluation of Organic Substrates and Microorganisms as Bio-Fertilisation Tool in Container Crop Production

Microorganisms are only effective when adequate conditions for their survival and development are provided. Among the factors that influence its effectiveness includes the type of soil or culture substrate, which works as an energy source reserve. Therefore, a tomato and a melon crop were established in different cycles to assess the effect of the physicochemical properties of organic substrates based on coconut fibre and vermicompost in three proportions, 0:100, 40:60 and 60:40 (% v:v), on the microbial activity in the rhizosphere when the bacteria Azotobacter vinelandii, Bacillus megaterium and Frateuria aurantia were applied. Concentrations of NO3−, H2PO4−, K+ and Ca2+ in the petiole cellular extract (PCE) were quantified at 60, 90 and 120 days after transplantation (DAT) for tomato and 45 and 65 DAT for melon. We analysed dehydrogenase activity (DHA), acid phosphatase activity (FTA) and β-glucosidase activity (β-GLU). In order to maintain optimal volumetric moisture for the survival of microorganisms, automatic control was used to manage the irrigation frequency between 22%–28%. The results showed that physicochemical substrate properties, by incorporating 40% vermicompost into the coconut fibre mixture, increased enzymatic activity. Plants that were inoculated with Azotobacter vinelandii and Frateuria aurantia showed an improvement in NO3− and K+ assimilation achieving highest yields.

Toxicity comparison of three imidazolium bromide ionic liquids to soil microorganisms

Ionic liquids (ILs) are extensively used in several chemistry fields. And research about the effects of ILs on soil microbes is needed. In this study, brown soil was exposed to 1-butyl-3-methylimidazolium bromide ([C4mim]Br), 1-hexyl-3-methylimidazolium bromide ([C6mim]Br) and 1-decyl-3-methylimidazolium bromide ([C10mim]Br). The toxicities of the three ILs are evaluated by measuring the soil culturable microbial number, enzyme activity, microbial diversity and, abundance of the ammonia monooxygenase (amoA) genes of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Results showed that all tested ILs caused a decrease in culturable microbial abundance. Tested ILs exposure inhibit urease activity and promote acid phosphatase and β-glucosidase activities. Tested ILs reduced soil microbial diversity and the abundances of AOB-amoA and AOA-amoA genes significantly. After a comparison of the integrated biomarker response (IBR) index, the toxicities of tested ILs to soil microorganisms were as follows: [C10mim]Br > [C6mim]Br > [C4mim]Br. Among all collected biomarkers, the abundance of the AOA-amoA gene was the most sensitive one and was easily affected after ILs exposure.