Soil microbiological properties and enzyme activity in agroforestry systems compared with monoculture, natural regeneration, and native Caatinga / Propriedades microbiológicas e atividade enzimática do solo sob sistemas agroflorestais comparado com monocultura, regeneração natural e caatinga nativa

The objective of this study was to evaluate the influence of agroforestry systems of different ages (AFS1: one-year old; AFS5: five-years old) on the biological attributes of soil; the following systems were used for comparison: a slash-and-burn (SBF) farming area, Caatinga which has been undergoing regeneration for 6 years (CaR6), and native Caatinga (NCa) in Brazil. Enzyme activity, abundance and composition of arbuscular mycorrhizal fungi (AMF), and production of glomalin-related soil proteins (GRSP) were evaluated at soil depths of 0­0.05 m. AMF species composition in the AFS was more similar to that in the NCa than in the SBF and CaR6 systems. In the rainy season, sporulation was most abundant in the AFS-1, CaR6, and SBF systems, whereas GRSP concentrations were highest in the AFS5 during the dry season. Acid phosphatase and arylsulfatase enzyme activity was lower in the AFS1 soils than in the NCa and SBF soils (rainy period), and levels of ß-glucosidase and fluorescein diacetate hydrolysis in the AFS were equal to or higher than those in the NCa in the dry season but lower in the rainy season. AFS thus appear to promote the maintenance of soil biological quality, and may be more sustainable than SBF farming systems in the Brazilian Caatinga over the long term.

O objetivo do estudo foi avaliar a influência de sistemas agroflorestais (AFS1: um ano de idade; AFS5: cinco anos de idade), nos atributos biológicos do solo usando como referência, uma área de agricultura de corte e queima (SBF), Caatinga em regeneração há 6 anos (CaR6), e Caatinga nativa (NCa), in Brasil. A atividade enzimática, a abundância e composição dos fungos micorrízicos arbusculares (AMF), e a produção de proteína do solo relacionada à glomalina (GRSP) foram avaliados, na profundidade de 0-5 cm do solo. A composição das espécies de AMF nos AFS foi mais semelhante a observada na NCa, do que os sistemas SBF e CaR6. Na estação chuvosa, a esporulação foi mais abundante em AFS-1, CaR6 and SBF quando comparada as outras áreas, enquanto a GRSP apresentou maiores teores no AFS5 no período seco. AFS1 apresentou atividade da fosfatase ácida e arilsulfatase inferiores tanto a NCa quanto a SBF, no período chuvoso. No período seco, a atividade de ß-glicosidase e a hidrólise do diacetato de fluoresceína (FDA) na AFS foram iguais ou superiores a Nca, mas menor no período chuvoso. Verifica-se que os AFS são potenciais para a manutenção da qualidade biológica do solo, podendo, em longo prazo, serem mais sustentáveis que a SBF, em ambiente de Caatinga.

Gene therapy for neurodegenerative disorders: advances, insights and prospects

Gene therapy is rapidly emerging as a powerful therapeutic strategy for a wide range of neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Some early clinical trials have failed to achieve satisfactory therapeutic effects. Efforts to enhance effectiveness are now concentrating on three major fields: identification of new vectors, novel therapeutic targets, and reliable of delivery routes for transgenes. These approaches are being assessed closely in preclinical and clinical trials, which may ultimately provide powerful treatments for patients. Here, we discuss advances and challenges of gene therapy for neurodegenerative disorders, highlighting promising technologies, targets, and future prospects.

Leucodistrofia metacromática infantil tardía: Presentación de un caso / Late infantile metachromatic leukodystrophy: case report

La leucodistrofia metacromática es una enfermedad autosómi-ca recesiva poco común ocasionada por el déficit de la enzima lisosomal arilsulfatasa A, el cual provoca una desmielinización progresiva con manifestaciones neurológicas subsecuentes. Dentro de sus formas de manifestación, la infantil tardía es la de peor pronóstico. La resonancia magnética juega un papel importante en la caracterización de anormalidades subyacentes, lo que permite descartar otras afecciones clínicas y aproximar un diagnóstico, que, posteriormente, es confirmado mediante los análisis moleculares apropiados. Dado el escaso conocimiento de esta enfermedad, sumado a un curso clínico generalmente fatal, se hace fundamental una identificación temprana y precisa con el fin de iniciar un manejo paliativo y asesoría genética. Se presenta a una paciente femenina de 24 meses de edad con historia de retardo psicomotor y hallazgos imagenológicos compatibles con leucodistrofia. Los estudios enzimáticos y moleculares confirmaron el diagnóstico de leu-codistrofia metacromática infantil tardía.

Metachromatic leukodystrophy is an uncommon autosomal recessive disease caused by the deficiency of the arylsulfatase A lysosomal enzyme, which causes a progressive demyelin-ation with subsequent neurological manifestations. Between its manifestation forms, the one presenting in late childhood has the worst prognosis. Magnetic resonance plays an important role in the characterization of underlying abnormalities, which makes it possible to rule out other clinical conditions and approximate a diagnosis that is later confirmed by the appropriate molecular studies. Given the limited knowledge of the condition, coupled with a generally fatal clinical course, an early and accurate identification is fundamental in order to start palliative management and genetic counseling. A 24 months old female patient with psychomotor retardation history and imaging findings compatible with leukodystrophy is presented. Enzymatic and molecular studies confirmed a diagnosis of late childhood metachromatic leukodystrophy.

Comparison of approaches for site-directed saturation mutagenesis of anarylsulfatase / 西安交通大学学报(医学版)

Objective: To compare the overall efficiency and cost of the construction and screening of libraries of Pseudomonas aeruginosa arylsulfatase (PAAS) through site-directed saturation mutagenesis with random primers (NNN) and precise primers for PCR. Methods: Site-directed mutagenesis libraries were constructed using the random primers (NNN) of the selected site, an equal-mole mixture of precise primers and a purified precise primer each time for the amplification of the fusion vector of Escherichia coli alkaline phosphatase (ECAP) and PAAS through PCR. The libraries were screened in a high-throughput mode through the assay of activity ratios of PAAS/mutants to ECAP after alkaline lysis of host cells. Three approaches for site-directed saturation mutagenesis were compared for their number of monoclones screened, the number of their expected mutants discovered, overall time consumed, overall cost and other pertinent factors. Results: The site-directed saturation mutagenesis of M72 with random primers for PCR resulted in only 10 among 20 expected mutants after the screening of over 600 monoclones, besides obvious codon bias. In contrast, no obvious codon bias was observed and there were already 18 among 20 expected mutants after the screening of less than 150 monoclones for site-directed saturation mutagenesis of G138 with similar random primers. The site-directed saturation mutagenesis of M72 with an equal-mole mixture of precise primers yielded all of the 19 expected mutants after the screening of less than 190 monoclones; the site-directed saturation mutagenesis of M72 with the purified precise primers for PCR one-by-one gave all of the 19 expected mutants after the screening of just 2 monoclones for every expected mutant. The use of the purified precise primers for PCR one-by-one was thus more favorable for the construction of libraries of site-directed saturation mutagenesis for the minimum number of monoclones screened, the least overall time and cost. In comparison to the use of the purified precise primers for PCR one-by-one, the use of random primers or the equal-mole mixture of precise primers for PCR to generate the libraries tolerated much greater overall cost and longer time. Conclusion: The generation and screening of the site-directed saturation mutagenesis libraries with precise primers for PCR one-by-one were more practical in elucidating the sequence-activity relationship while the use of equal-mole mixture of precise primers for PCR was preferable for the screening of positive mutants during site-directed saturation mutagenesis.

Long-Term Galsulfase Treatment Associated With Improved Survival of Patients With Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome): 15-Year Follow-Up From the Survey Study

Abstract Mucopolysaccharidosis VI (MPS VI) is a progressive lysosomal storage disorder with multiorgan and multisystemic pathology. Currently, galsulfase enzyme replacement therapy (ERT) is the only approved treatment for MPS VI. A crosssectional survey study of 121 patients with MPS VI conducted in 2001 to 2002 and a 10-year follow-up study of the same patients (resurvey study; ClinicalTrials.gov NCT01387854) found that those receiving galsulfase at any time showed physical improvements and a lower mortality rate (16.5%) versus treatment-naive patients (50%). After *15 years, galsulfasetreated patients (n » 104) continue to have a survival advantage over treatment-naive patients (n » 14), as demonstrated by a 24% versus 57% mortality rate. This survival advantage is further supported by data from the commercial use of galsulfase (2005-2016), which show a 5-year mortality rate for galsulfase-treated patients of 12.5%. Together, these findings suggest that galsulfase ERT can increase life expectancies for patients with MPS VI over a period of at least 15 years.

Identification of a novel mutation in ARSA gene in three patients of an Iranian family with metachromatic leukodystrophy disorder

Abstract Metachromatic leukodystrophy disorder (MLD) is an autosomal recessive and lysosomal storage disease. The disease is caused by the deficiency of the enzyme arylsulfatase A (ARSA) which is encoded by the ARSA gene. Different mutations have been reported in different populations. The present study was aimed to detect the mutation type of the ARSA gene in three relative Iranian patients. We found a novel homozygous missense mutation c.1070 G > T (p.Gly357Val) in exon 6 of these patients. The mutation was found to be reported for the first time in MLD patients. The data can update the mutation profile and contribute toward improved clinical management and counseling of MLD patients.

A Korean Patient with Early Juvenile Form of Metachromatic Leukodystrophy: Biochemical and Molecular Genetic Investigation

Metachromatic leukodystrophy is an inherited lysosomal storage disorder caused by the deficiency of arylsulfatase A activity. The patient in this study, a 5-yr-old girl, presented with progressive psychomotor regression. An MRI image of her brain showed bilateral symmetrical demyelination. The arylsulfatase A activity in her leukocytes was decreased to 8.0 nmol/hr/mg protein (reference range, 25-80 nmol/hr/mg protein). Mutation analysis of ARSA, using PCR and direct sequencing, showed two heterozygote pathogenic variations of c.449C>T (p.Pro150Leu) and c.640G>A (p.Ala214Thr). In summary, we report a Korean patient with an early juvenile form of metachromatic leukodystrophy, who was diagnosed based on her clinical symptoms as well as by using biochemical, radiological, and molecular genetic investigations.

Identification of a Novel Nonsense Mutation in the ARSE Gene of a Patient with X-Linked Recessive Chondrodysplasia Punctata

X-linked recessive chondrodysplasia punctata (CDPX1) is caused by a hemizygous mutation in the arylsulfatase E (ARSE) gene located on chromosome Xp22.3. It is a rare congenital disorder of punctate calcifications in cartilages, leading to short stature and facial and limb anomalies. These clinical features are frequently observed in all types of chondrodysplasia punctata and have also been seen in other cartilage developmental disorders. Because of the phenotypical similarities, specific testing for only one gene is inefficient and time consuming. The advent of next-generation sequencing has provided an opportunity to improve diagnostic accuracy as well as save on time and cost. Here, we report on a patient diagnosed with CDPX1, who was identified via diagnostic exome sequencing to have a novel nonsense mutation in the ARSE gene, that was inherited from the mother.

Biochemical and Genetic Analysis of Seven Korean Individuals With Suspected Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by a deficiency in arylsulfatase A (ARSA). However, decreased ARSA activity is also observed in pseudodeficiency (PD). To distinguish between MLD and PD, we performed gene mutation and sulfatide analyses by using dried blood spots (DBSs) from seven Korean individuals who underwent an analysis of ARSA activity. DNA was extracted from DBSs, and PCR-direct sequencing of ARSA was performed. The cDNA obtained was analyzed to confirm a novel mutation. Of the seven subjects, three were confirmed as having MLD, one was confirmed as having MLD-PD, one was confirmed as having PD, and the remaining two were obligate heterozygotes. We verified the novel pathogenic variant c.1107+1delG by performing familial and cDNA analyses. Sulfatide concentrations in DBSs were analyzed and were quantified by using ultra-performance liquid chromatography and tandem mass spectrometry, respectively. Total sulfatide concentration was inversely correlated with ARSA activity (Spearman's coefficient of rank correlation, P=0.929, P=0.0025). The results of this mutational and biochemical study on MLD will increase our understanding of the genetic characteristics of MLD in Koreans.

Significance of arylsulfatase A gene detection for the diagnosis of metachromatic leukodystrophy / 中华实用儿科临床杂志

Objective To detect genetic causes of seizures and developmental retardation in 60 patients with abnormal head magnetic resonance imaging(MRI) ,and to analyze the clinical manifestations and head MRI manifestations in carriers of arylsulfatase A (ARSA) gene mutation.Methods The blood samples of children and genomic DNA were collected.Sixty cases of children with suspected metachromatic leukodystrophy were tested (MLD) by using the second generation sequencing technology.The genotype and phenotype and head MRI findings were analyzed.Results Of the 60 cases of children, 15 cases with gene mutations.There were 7 kinds of ARSA gene mutations, and 3 of them, c.1178C ＞ G, c.1055A ＞ G and c.883 G ＞ A were pathogenic.The others were single nucleotide polymorphism(SNP), which had no relationship with this disease.One of the patients carried only SNP, and 14 of them were carrying pathogenic mutation, c.1055A ＞ G (53.33％) ,c.1178C ＞ G (40.00％) were more common,and c.1055A ＞ G mutation was in 8 cases, of which 5 cases were late-onset type.One case of the 3 patients who were late infantile type was carrying c.1178C ＞ G mutation at the same time.All the eight cases had retardation.One case had hydrocephalus, and 5 cases had epilepsy.All of the 6 patients with c.1178C ＞ G were late-infantile type, and had retardation, including 4 cases of epilepsy, c.883G ＞ A mutation in 1 case,was late-infantile type,and the first symptom was binaural deafness and mental retardation.Three different types of mutations showed no significant difference in brain MRI.Conclusions There are 14 patients who were diagnosed as MLD.c.1178C ＞ G and c.883G ＞ A were late infantile type,and c.1055A ＞G was mostly late-onset type.The changes in head MRI caused by different types of ARSA gene mutations were of no significant differences in performance.

Effect of tillage systems and permanent groundcover intercropped with orange trees on soil enzyme activities

The objective of this study was to evaluate the effect of different soil tillage systems and groundcover crops intercropped with orange trees on soil enzyme activities. The experiment was performed in an Ultisol soil in northwestern Paraná State. Two soil tillage systems were evaluated [conventional tillage (CT) across the entire area and strip tillage (ST) with a 2-m strip width] in combination with various groundcover vegetation management systems. Soil samples were collected after five years of experimental management at a depth of 0-15 cm under the tree canopy and in the inter-row space in the following treatments: (1) CT-Calopogonium mucunoides; (2) CT-Arachis pintoi; (3) CT-Bahiagrass; (4) CT-Brachiaria humidicola; and (5) ST-B. humidicola. The soil tillage systems and groundcover crops influenced the soil enzyme activities both under the tree canopy and in the inter-row space. The cultivation of B. humidicola provided higher amylase, arylsulfatase, acid phosphatase and alkaline phosphatase than other groundcover species. Strip tillage increased enzyme activities compared to the conventional tillage system.

A Case of Metachromatic Leukodystrophy Confirmed by Molecular Genetic Analysis / 대한소아신경학회지

Metachromatic leukodystrophy (MLD) is the rare neurometabolic disease caused by the deficiency of the enzyme arylsulfatase A resulting in a deficiency of sulfatide degradation and the target gene is ARSA gene. We report a case of the late infantile form of MLD that was confirmed by means of enzyme assay and gene analysis with typical brain MRI and MR spectroscopy finding.

Estudio de las enzimas lisosomales N-acetil-a-D-glucosaminidasa y arilsulfatasa A en adultos cubanos / Assay of lysosomal N-acetyl-a-D-glucosaminidase and arylsulfatase A activities in Cuban adults

El objetivo de este estudio fue determinar, en una muestra de individuos cubanos, un rango preliminar de valores normales de actividad específica de N-acetil- a-D-glucosaminidasa y arilsulfatasa A, enzimas deficientes en la mucopolisacaridosis tipo III B y la leucodistrofia metacromática, respectivamente. Se realizó una investigación de corte transversal, en muestras de sangre de 38 individuos adultos. Se realizó la extracción de los leucocitos y se determinó la actividad específica de las enzimas por métodos espectrofotométricos. Todos los participantes fueron caracterizados desde el punto de vista clínico como sanos para ambas enfermedades. Se obtuvieron valores medios de actividad específica de N-acetil-a-D-glucosaminidasa y arilsulfatasa A de 1,52±0,30 y 121,37±20,14 nmol/mg/h, respectivamente. No se encontraron diferencias significativas en relación a las características étnicas para ninguna de las dos enzimas (p<0,05). Este constituye el primer estudio cubano en el cual se publican rangos de actividad específica para estas enzimas en individuos cuya condición de sanos y no relacionados familiarmente con la enfermedad ha sido clínicamente demostrada. El análisis de un mayor número de muestras permitirá establecer los puntos de corte que dotarán al diagnóstico bioquímico de estas enfermedades de una mayor confiabilidad.

The aim of this study was to determine, in a sample of Cuban individuals, a preliminary range of normal values of N-acetyl-a-D-glucosaminidase and arylsulfatase A activity, enzymes that are deficient in mucopolysaccharidosis type III B and metachromatic leukodystrophy, respectively. A cross-sectional research was conducted. Blood samples were obtained out of 38 adult individuals. The leucocytes were extracted and the specific enzymatic activity was assayed by spectrophotometric methods. All participants were characterized from the clinical point of view as healthy for both diseases. Average values of N-acetyl-a-D-glucosaminidase and arylsulfatase A specific activities of 1.52 ± 0.30 and 121.37 ± 20.14 nmol/mg/h, respectively were obtained. There were no significant differences related to ethnicity for any of the two enzymes (p <0.05). This is the first Cuban study in which ranges of activity for these enzymes have been reported in healthy individuals whose healthy and non-familiarly related with the disease status have been clinically demonstrated. The analysis of more samples will establish the cutoff points that will increase the reliability of the biochemical diagnosis of these diseases.