Twenty years of Colombian experience with enzymatic screening in patients with features of mucopolysaccharidosis.

Mucopolysaccharidoses (MPSs) are a group of genetic alterations whose effect is the progressive intralysosomal accumulation of glycosaminoglycans. Affected individuals are deficient in one or more lysosomal enzymes which, depending on the MPS, may cause coarse facial features, short stature, multiple skeletal dysplasia, joint stiffness, or developmental delay. Their diagnosis is mostly performed late or incorrectly, and it represents a challenge since it requires specialized tests only performed in major cities. This makes it difficult for patients to have access to physicians since their geographical location is distant and therefore, the use of samples collected in solid-phase represents an advantage for the study of high-risk populations. In addition, epidemiological information about rare diseases, especially in Latin America, is scarce or inconsistent. Our aim was to report the experience of 20 years of selective screening by assessing enzyme activity and reporting incidence values of MPS in Colombia. This study validated a group of fluorometric endpoint techniques in 8239 patients. The samples were dried blood spots (DBS) collected on filter paper and leukocyte extracts. Reference values in the Colombian population for α-l-iduronidase, iduronate 2-sulfatase, α-N-acetylglucosaminidase, N-acetylglucosamine-6-sulfate sulfatase, ß-galactosidase, arylsulfatase B, and ß-glucuronidase were established in leukocyte extracts, and patients reference ranges were updated in the case of DBS samples. Incidence values were calculated for each MPS and the distribution of cases across the country is also shown. This study offers very useful information for the health system, the scientific community, and it facilitates the diagnosis of these disorders. This is indispensable when seeking to develop new diagnostic or treatment approaches for patients.

Fifteen years of enzyme replacement therapy for mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome): a case report.

BACKGROUND: Mucopolysaccharidosis VI, or Maroteaux-Lamy disease, is an autosomal recessive disease characterized by deficiency of the enzyme arylsulfatase B in the lysosomal catabolism of glycosaminoglycans. Due to reduced (or even null) enzyme activity, glycosaminoglycans (mainly dermatan sulfate) accumulates, leading to a multisystemic disease. Mucopolysaccharidosis VI induces reduced growth, coarse face, audiovisual deficits, osteoarticular deformities, and cardiorespiratory issues, hampering the quality of life of the patient. Enzyme replacement therapy with galsulfase (Naglazyme, BioMarin Pharmaceuticals Inc., USA) is the specific treatment for this condition. Although studies have shown that enzyme replacement therapy slows the progression of the disease, the effects of long-term enzyme replacement therapy remain poorly understood. CASE PRESENTATION: A 29-year-old, Caucasian, male patient diagnosed with mucopolysaccharidosis VI was treated with enzyme replacement therapy for over 15 years. Enzyme replacement therapy was initiated when patient was 13 years old. The patient evolved multiplex dysostosis, carpal tunnel syndrome, thickened mitral valve, and hearing and visual loss. CONCLUSIONS: Although enzyme replacement therapy did not prevent the main signs of mucopolysaccharidosis VI, it slowed their progression. Additionally, enzyme replacement therapy was associated with a longer survival compared with the untreated affected sibling. Taken together, the results indicate that enzyme replacement therapy positively modified the course of the disease.

Soil enzymatic activity under coffee cultivation with different water regimes associated to liming and intercropped brachiaria / Atividade enzimática de solo sob cultivo de café com diferentes regimes hídricos associados a calagem e braquiária nas entrelinhas

This research evaluated the effects of coffee cultivation with two different water regimes associated or not with liming and the presence/absence of brachiaria as intercrop on the activities of the soil enzymes ß-glucosidase, arylsulfatase and acid phosphatase. The study was carried out at the experimental farm of Embrapa Cerrados, using the cultivar IAC 144 (Coffea arabica L.), under a clayey dystrophic Cerrado Oxisol. Two water regimes (WR) were considered, WR1 with irrigation shifts throughout the year and WR3 with controlled water stress, for about 70 days, in the dry season. In each water regime, effects of lime application (with/without) and the presence/absence of brachiaria cultivated between the lines of coffee plants were evaluated. The activities of the enzymes ß-glucosidase, arylsulfatase and acid phosphatase were evaluated during the rainy and dry seasons. Liming and intercropped brachiaria positively affected the activities of the three enzymes assessed in this study at varying degrees, depending on season and/or the WR. Our findings evidenced that intercropped brachiaria in coffee rows was the factor that most positively impacted soil enzymes activities.

O objetivo desse trabalho foi avaliar os efeitos do cultivo do café sobre a atividade das enzimas do solo ß-glicosidase, arilsulfatase e fosfatase ácida em função de dois diferentes regimes hídricos associados ou não à calagem e ao cultivo de braquiária nas entrelinhas. Esse estudo foi realizado em um experimento conduzido no campo experimental do Centro de Pesquisa Agropecuária dos Cerrados, utilizando a cultivar IAC 144 (Coffea arabica L.), sob um Latossolo Vermelho distrófico argiloso. Foram considerados dois regimes hídricos (RH), RH1 com irrigação plena em turnos de rega ao longo do ano e RH3 com estresse hídrico controlado, por cerca de 70 dias, na época seca. Em cada regime hídrico foram avaliadas a aplicação ou não de calcário em cobertura e a presença ou ausência de braquiária cultivada nas entrelinhas das plantas do cafezal. As atividades das enzimas ß-glicosidase, arilsulfatase e fosfatase ácida foram avaliadas durante as estações chuvosa e seca. A calagem e a presença de braquiária nas entrelinhas tiveram efeito positivo sobre a atividade das três enzimas avaliadas nesse estudo, em graus variáveis, dependendo da época de coleta das amostras e/ou do RH. Nossos resultados evidenciam que a braquiária nas entrelinhas do café foi o fator de maior impacto positivo sobre a atividade enzimática do solo.

Enzyme activities in a sandy soil of Western Bahia under cotton production systems: short-term effects, temporal variability, and the FERTBIO sample concept.

Enzyme activities (EAs) and the FERTBIO sample concept have been increasingly adopted as a novel approach to estimate the soil quality in Brazil. However, the performance of this strategy in sandy soils of the Cerrado biome remains unclear. During 2 years, in a Cerrado's sandy soil, the short-term effects of ten different cropping systems (conventional tillage or no-tillage associated with monoculture, rotations, and/or successions) on the activities of ß-glucosidase, acid phosphatase, and arylsulfatase were studied. Issues related to annual variability and the feasibility of using the FERTBIO sample concept for soil enzymes activities were also evaluated. Soil samples were collected at three different depths (0-10 cm, 10-20 cm, and 20-40 cm) in March 2017 and February 2018. Five years since the beginning of the experiment, the presence of cover crops and no-till promoted improvements in EAs evidencing the importance of regenerative management practices for the sustainability of agroecosystems in sandy soils. Regardless of the cropping systems and depths evaluated, soil organic carbon and EAs showed low temporal variation during the 2 years of monitoring. Our results also showed that it is possible to use the FERTBIO sample concept for the Quartzipsament soils of Western Bahia, Brazil.

[Oral manifestations of Maroteaux-Lamy syndrome (Mucopolysaccharidosis VI)]. / Manifestaciones orales del síndrome de Maroteaux-Lamy (Mucopolisacaridosis VI).

Mucopolysaccharidosis type VI, also known as Maroteaux-Lamy syndrome, is an autosomal recessive lysosomal disorder, due to the deficiency of the enzyme arylsulfatase B that leads to the accumulation of dermatan sulfate in the tissues and its urinary excretion. Mucopolysaccharide deposition leads to a progressive disorder affecting multiple organs that often results in death at a young age. This disease has several oral manifestations, among which dental complications can be serious and include follicles similar to dentigerous cysts, malocclusions, condylar defects and gingival hyperplasia, in addition to a short neck, corneal opacity, macroglossia, skull enlargement, anteroposterior dimension long, claw hand is some of the clinical features. A case of a 14-month-old patient is presented, who attended a pediatric dentistry consultation for episodes of fever, low weight, severe gingival hyperplasia. Physical examination revealed coarse facies, short neck, pectus excavatus, hands with decreased grip, and neurodevelopmental delay. On intraoral examination, dental eruption delayed, generalized gingival hyperplasia, palate with little transverse growth. On radiographic examination, dental organs included and poor position in the anterior sector, upper molars within the maxillary sinus, rotated lower canines. He is referred to medicine for biochemical tests and genetics for diagnosis. Detailed biochemistry MPS type VI, confirmed by molecular testing. The clinical manifestations in this case correspond to the clinical form of rapid progression reported in these patients. They report: short stature, skeletal malformations and alterations at the oral level. Children with severe MPS VI start early and progress rapidly, bone radiographs and urine GAG measurement are helpful for diagnosis with genetic and ARSB enzyme activity. It is necessary to strengthen the knowledge in dentistry and the general population about the clinical characteristics of type VI mucopolysaccharides in order to have an early diagnosis and management of pathologies in these patients.

Arylsulfatase A pseudodeficiency in Mexico: Enzymatic activity and haplotype analysis.

BACKGROUND: Metachromatic Leukodystrophy (MLD, OMIM 250100) is a neurodegenerative disease caused by mutations in the ARSA gene (OMIM 607574) that lead to deficiency in Arylsulfatase A (ASA). ASA pseudodeficiency (PD-ASA) is a biochemical condition that substantially diminishes ASA activity but is not associated with clinical manifestations. PD-ASA is associated with the c.1055A>G (p.Asn352Ser) (rs2071421) and c.*96A>G (rs6151429) variants, which have an estimated frequency of 2% in the population. OBJECTIVE: To determine the activity of Arylsulfatase A and to identify variants and haplotypes in the ARSA gene in Mexican individuals with pseudodeficiency. METHODS: Two-hundred apparently healthy individuals were included to determine the enzymatic activity of ASA in leukocytes by spectrophotometric analysis, and identification of the PD-ASA alleles was performed by PCR-RFLP assays. Genotypes were confirmed by semi-automated Sanger sequencing. Haplotypes were constructed using Arlequin v.10.04, and linkage disequilibrium analysis was performed with Cube X. RESULTS: The enzymatic activity of ASA was determined to be 1.74-2.09 nmol/mg protein/min and later correlated with genotypes and haplotypes. For the (p.Asn352Ser) variant, we found 126 (0.63) individuals with the AA genotype, 62 with AG (0.31) and 12 with GG (0.06); the frequency of the polymorphic allele was 0.215 (86 alleles, 21.5%), and the variant was in HWE (p = .2484). The variant c.*96A>G was also in HWE (p = .2105): 185 individuals (0.925) with the AA genotype, 14 (0.07) with AG, and 1 (0.005) with (GG), with a frequency of 0.04 (4%) for the polymorphic allele. The inference of haplotypes resulted in 312 (0.78) AA, 72 (0.18) GA, and 16 (0.04) GG haplotypes. The AG haplotype was not found. The variants were found to be in linkage disequilibrium (D' = 1). Of the nine possible diplotypes, AA/AG, AA/GG, and AG/GG were not found, in concordance with the hypothesis that the G allele of c.*96A>G does not occur in the absence of the G allele of c.1055A>G. We found a slight correlation between ASA biochemical activity and variants, mainly due to the G allele of c.*96A>G in either genotypes or haplotypes. CONCLUSIONS: In Northwestern Mexico, the presence of PD-ASA alleles was biochemically and molecularly determined, and the frequencies were found to be in HWE. The frequency of PD-ASA for the North Western Mexican mestizo is 8%.

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.

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.

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

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.

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.

Atividade enzimática do solo em pastagens de Urochloa manejados sob ofertas de forragem / Soil enzymatic activity of Urochloa pastures managed under different forage allowances

A atividade enzimática do solo reflete a atividade dos microrganismos que decompõem a matéria orgânica e liberam nutrientes às plantas. O objetivo foi avaliar a atividade enzimática no solo em pastagens de cultivares de Urochloa manejados sob as ofertas de forragem de 4, 7, 10 e 13 kg de matéria seca (MS)/100 kg de peso corporal (PC). O método de pastejo adotado foi mob-stocking, com quatro ciclos de pastejo (entre dezembro de 2008 e fevereiro de 2009). Também foi realizada uma amostragem em junho de 2009 para melhor caracterização, pois a mineralização da matéria orgânica no solo ocorre em longo prazo. Assim, as datas de coleta de solo para análise foram descritas como dias de avaliações e não como pastejos: 1 (de 17 a 20 de dezembro de 2008), 21 (de 07 a 10 de janeiro de 2009), 42 (de 28 a 31 de janeiro de 2009), 63 (de 18 a 21 de fevereiro de 2009), 183 (de 18 a 20 de junho de 2009). Foi avaliada a atividade enzimática no solo da desidrogenase, protease, arilsulfatase e celulase. O delineamento experimental foi inteiramente casualizado, com três repetições por oferta. Foi realizada a análise de variância e teste de Tukey ao nível de 5% de probabilidade, para comparação de médias, utilizando-se o procedimento o programa estatístico R. As análises foram realizadas de maneira individual para cada cultivar. De modo geral, nas pastagens das três cultivares estudadas, a atividade da desidrogenase foi maior (P<0,05) aos 63 dias e da protease no último dia de avaliação. O decorrer do período experimental implicou em mudanças na atividade da arilsulfatase e da celulase em relação às ofertas de forragem, mas sem um padrão de comportamento definido. As ofertas de forragem mostraram efeito sobre a atividade das enzimas ao longo do período de avaliação, porém sem padrão de comportamento definido, sendo observadas oscilações cíclicas da atividade dessas enzimas. As ofertas de forragem estudadas afetaram a atividade enzimática da desidrogenase, protease, arilsulfatase e...(AU)

Soil enzymatic activity reflects the activity microorganisms that decompose the organic matter and release nutrients into the soil that can be used by plants. The objective of this study was to evaluate soil enzymatic activity in Urochloa cultivar pastures managed under forage allowances of 4, 7, 10 and 13 kg dry matter/100 kg body weight. The grazing method adopted was mob-stocking, with four grazing cycles (between December 2008 and February 2009). Soil sampling was performed in June 2009 for better characterization since the mineralization of soil organic matter is a long-term process. Thus, the dates of soil sampling for analysis are reported as evaluation days and not as grazing: 1 (December 17 to 20, 2008), 21 (January 7 to 10, 2009), 42 (January 28 to 31, 2009), 63 (February 18 to 21, 2009), 183 (June 18 to 20, 2009). The soil enzymatic activity of dehydrogenase, protease, arylsulfatase, and cellulase was evaluated. The experimental design was completely randomized, with three replications per forage allowance. Analysis of variance and Tukeys test at the 5% probability level were applied to compare means using the R statistical program. The analyses were performed individually for each cultivar. Overall, in pastures of the three cultivars studied, dehydrogenase activity was higher (P<0.05) on day 63 and protease on the last day of evaluation. The experimental period implied changes in the activity of arylsulfatase and cellulase according to forage allowance, but without a defined pattern. Forage allowances exerted an effect on the activity of the enzymes throughout the evaluation period, but there was no defined pattern. Cyclic oscillations in the activity of these enzymes were observed. The forage allowances studied affected the soil enzymatic activity of dehydrogenase, protease, arylsulfatase and cellulase in Urochloa pastures of the Marandu, Xaraes and Mulato varieties, but no pattern was found in...(AU)

Atividade enzimática do solo em pastagens de Urochloa manejados sob ofertas de forragem / Soil enzymatic activity of Urochloa pastures managed under different forage allowances

A atividade enzimática do solo reflete a atividade dos microrganismos que decompõem a matéria orgânica e liberam nutrientes às plantas. O objetivo foi avaliar a atividade enzimática no solo em pastagens de cultivares de Urochloa manejados sob as ofertas de forragem de 4, 7, 10 e 13 kg de matéria seca (MS)/100 kg de peso corporal (PC). O método de pastejo adotado foi mob-stocking, com quatro ciclos de pastejo (entre dezembro de 2008 e fevereiro de 2009). Também foi realizada uma amostragem em junho de 2009 para melhor caracterização, pois a mineralização da matéria orgânica no solo ocorre em longo prazo. Assim, as datas de coleta de solo para análise foram descritas como dias de avaliações e não como pastejos: 1 (de 17 a 20 de dezembro de 2008), 21 (de 07 a 10 de janeiro de 2009), 42 (de 28 a 31 de janeiro de 2009), 63 (de 18 a 21 de fevereiro de 2009), 183 (de 18 a 20 de junho de 2009). Foi avaliada a atividade enzimática no solo da desidrogenase, protease, arilsulfatase e celulase. O delineamento experimental foi inteiramente casualizado, com três repetições por oferta. Foi realizada a análise de variância e teste de Tukey ao nível de 5% de probabilidade, para comparação de médias, utilizando-se o procedimento o programa estatístico R. As análises foram realizadas de maneira individual para cada cultivar. De modo geral, nas pastagens das três cultivares estudadas, a atividade da desidrogenase foi maior (P<0,05) aos 63 dias e da protease no último dia de avaliação. O decorrer do período experimental implicou em mudanças na atividade da arilsulfatase e da celulase em relação às ofertas de forragem, mas sem um padrão de comportamento definido. As ofertas de forragem mostraram efeito sobre a atividade das enzimas ao longo do período de avaliação, porém sem padrão de comportamento definido, sendo observadas oscilações cíclicas da atividade dessas enzimas. As ofertas de forragem estudadas afetaram a atividade enzimática da desidrogenase, protease, arilsulfatase e...

Soil enzymatic activity reflects the activity microorganisms that decompose the organic matter and release nutrients into the soil that can be used by plants. The objective of this study was to evaluate soil enzymatic activity in Urochloa cultivar pastures managed under forage allowances of 4, 7, 10 and 13 kg dry matter/100 kg body weight. The grazing method adopted was mob-stocking, with four grazing cycles (between December 2008 and February 2009). Soil sampling was performed in June 2009 for better characterization since the mineralization of soil organic matter is a long-term process. Thus, the dates of soil sampling for analysis are reported as evaluation days and not as grazing: 1 (December 17 to 20, 2008), 21 (January 7 to 10, 2009), 42 (January 28 to 31, 2009), 63 (February 18 to 21, 2009), 183 (June 18 to 20, 2009). The soil enzymatic activity of dehydrogenase, protease, arylsulfatase, and cellulase was evaluated. The experimental design was completely randomized, with three replications per forage allowance. Analysis of variance and Tukey’s test at the 5% probability level were applied to compare means using the R statistical program. The analyses were performed individually for each cultivar. Overall, in pastures of the three cultivars studied, dehydrogenase activity was higher (P<0.05) on day 63 and protease on the last day of evaluation. The experimental period implied changes in the activity of arylsulfatase and cellulase according to forage allowance, but without a defined pattern. Forage allowances exerted an effect on the activity of the enzymes throughout the evaluation period, but there was no defined pattern. Cyclic oscillations in the activity of these enzymes were observed. The forage allowances studied affected the soil enzymatic activity of dehydrogenase, protease, arylsulfatase and cellulase in Urochloa pastures of the Marandu, Xaraes and Mulato varieties, but no pattern was found in...

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.

Diagnostic and treatment strategies in mucopolysaccharidosis VI.

Mucopolysaccharidosis VI (MPS VI) is a very rare autosomal recessive disorder caused by mutations in the ARSB gene, which lead to deficient activity of the lysosomal enzyme ASB. This enzyme is important for the breakdown of the glycosaminoglycans (GAGs) dermatan sulfate and chondroitin sulfate, which accumulate in body tissues and organs of MPS VI patients. The storage of GAGs (especially dermatan sulfate) causes bone dysplasia, joint restriction, organomegaly, heart disease, and corneal clouding, among several other problems, and reduced life span. Despite the fact that most cases are severe, there is a spectrum of severity and some cases are so attenuated that diagnosis is made late in life. Although the analysis of urinary GAGs and/or the measurement of enzyme activity in dried blood spots are useful screening methods, the diagnosis is based in the demonstration of the enzyme deficiency in leucocytes or fibroblasts, and/or in the identification of pathogenic mutations in the ARSB gene. Specific treatment with enzyme replacement has been available since 2005. It is safe and effective, bringing measurable benefits and increased survival to patients. As several evidences indicate that early initiation of therapy may lead to a better outcome, newborn screening is being considered for this condition, and it is already in place in selected areas where the incidence of MPS VI is increased. However, as enzyme replacement therapy is not curative, associated therapies should be considered, and research on innovative therapies continues. The management of affected patients by a multidisciplinary team with experience in MPS diseases is highly recommended.

Genotypic characterization of Brazilian patients with infantile and juvenile forms of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder inherited as an autosomal recessive trait. MLD is caused by the deficiency of arylsulfatase A (ARSA), a lysosomal enzyme that catalyzes the first step in the degradation of sulfated glycolipids, which are essential components of the myelin sheet. Notably, between 7% and 15% of healthy individuals show in vitro deficiency of ARSA, a condition called ARSA pseudodeficiency (ARSA-PD). To date, 151 ARSA-MLD mutations have been reported in the gene encoding ARSA (ARSA), among which IVS2+1G>A and P426L occur at high frequencies in most of the studied populations. The aim of this work was to identify ARSA mutant alleles in a cohort of 27 unrelated Brazilian MLD patients. The most frequent ARSA-MLD mutation, IVS2+1G>A, and the ARSA-PD polymorphisms, N350S and 1524+95A>G, were detected using real-time PCR, while the remaining mutations were detected using direct sequencing of ARSA. In concordance with previous reports, IVS2+1G>A and P426L were the most common ARSA-MLD mutations in our cohort of MLD patients, found at frequencies of 0.05 and 0.08, respectively. Interestingly, two mutations previously reported as rare, 103_110del8 and 1190_1191insC, were found at higher frequencies in our cohort of MLD patients, 0.08 and 0.06, respectively. Additionally, 11 other rare ARSA-MLD mutations were found at lower frequencies in our cohort of MLD patients. To our knowledge, this is the first systematic genotypic characterization of MLD patients from Latin America. This work highlights the genetic heterogeneity of MLD, and supports genotype-phenotype associations, which become more important as specific treatments are being developed for this devastating disorder.

Improving arylsulfatase activity determination in dried blood spots: Screening and diagnostic approaches for Maroteaux-Lamy syndrome (MPS VI).

BACKGROUND: Mucopolysaccharidosis type VI can be screened by measuring the lysosomal arylsulfatase B (ARSB) residual enzyme activity in dried blood spots (DBS) using synthetic substrates. However, we have found experimental obstacles when determining ARSB activity with the fluorescent method due to the significant quenching effect rendered by DBS components. METHODS: We adapted the methods originally described by Chamoles et al. [1] and Civallero et al. [2] and put forward 2 distinct approaches for ARSB activity quantification from DBS samples by measuring the 4-methylumbelliferone (ß-MU) fluorescence generated from the ARSB 4-methylumbelliferone sulfate (ß-MUS) substrate. RESULTS: We demonstrate the high throughput feasibility of a novel approach for measuring ARSB activities by incorporating tailor-made calibration curves according to each patient's DBS sample quenching properties. The second method is used to calculate ARSB activities by measuring the fluorescence and absorbance parameters in each reaction sample with a single DBS-free calibration curve. CONCLUSIONS: The quantitative correlation between the DBS sample absorbance and its quenching effect can be used to calculate predictive ARSB activities and would serve as an affordable first tier screening test. The method described herein demonstrates the critical importance of adapting the ß-MU calibration curves to each patient's unique DBS sample matrix and its positive impact on the accuracy and reliability of ARSB activity measurements.

A patient presenting a 22q13 deletion associated with an apparently balanced translocation t(16;22): An illustrative case in the investigation of patients with low ARSA activity.

A 10-year-old speechless, mentally deficient male, with low arylsulfatase A (ARSA) activity, and presumably, methachromatic leukodystrophy, underwent genetic evaluation. As the clinical picture was not compatible with this diagnosisan ARSA gene and chromosome analysis were performed, showing the presence of a pseudodeficiency ARSA allele and a de novo apparently balanced t(16;22)(p11.2;q13) translocation. A deletion on the long arm of chromosome 22 encompassing the ARSA gene, as shown by FISH and array-CGH, indicated a 22q13 deletion syndrome. This case illustrates the importance of detailed cytogenetic investigation in patients presenting low arylsulfatase A activity and atypical/unspecific clinical features.

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.

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.

ATIVIDADE MICROBIANA E PROPRIEDADES BIOQUÍMICAS DO SOLO RESULTANTES DA APLICAÇÃO DE GESSO AGRÍCOLA NA CULTURA DO REPOLHO

The present study investigated the impact of the application of gypsum and of a sulfur-rich source on the microbial and biochemical characteristics of a dark red latossol used for cabbage cultivation. Gypsum was applied in amounts of 134, 268, and 402 kg S/ha. The following controls were included: a treatment with no sulfur; fertilization with ammonium sulfate (402 kg S/ha); and organic matter (20 t/ha lab-lab, dry weight). Application of increasing sulfur doses to the soil led to an increase in total number of bacteria and a decrease in Gram-negative bacteria. The remaining groups of microorganisms, Bacillus spp, actinomycetes and fungi, did not vary significantly or did not present a satisfatory response. The increase in total number of bacteria compared to control was 29 to 60% in the rhizosphere and 15 to 107% in the non-rhizosphere, with the number of microorganisms in the rhizosphere exceeding the number of microorganisms in the non-rhizosphere by 5 to 251%. The microbial biomass of the rhizosphere increased in the treatments with ammonium sulfate and organic matter and was unchanged in the non-rhizosphere. In agreement with microbial count data, the microbial biomass also increased in the rhizosphere compared to the non-rhizosphere. Dehydrogenase and urease activity increased in comparison to the control, although no effect was observed on arylsulfatase activity. Cabbage production on treated soil was 52.2 to 81.1% higher as compared to the control. Increasing sulfur concentrations were observed in the soil and in the cabbage aerial portion.

Este estudo investigou o impacto da aplicação de gesso agrícola e de uma fonte rica em enxofre sobre as características microbianas e bioquímicas de um latossolo vermelho escuro cultivado com repolho. As aplicações de gesso foram de 134, 268 e 402 kg de S/ha. Foram incluídos um tratamento controle, sem enxofre, um contendo a adubação normal de sulfato de amônio (402kg de S/ha) e outro com adubo verde (20t/ha de lab-lab, peso seco). Em resposta a adições crescentes de enxofre no solo verificou-se que enquanto o número de bactérias totais aumentou, o número de bactérias Gram-negativas diminuiu. Os demais grupos de microrganismos, Bacillus spp, actinomicetos e fungos, não variaram significativamente ou não apresentaram uma resposta satisfatória. O aumento do número de bactérias totais, em relação ao controle, foi de 29 a 60% na rizosfera e de 15 a 107% na não-rizosfera, respectivamente. O número de microrganismos na rizosfera foi de 5 a 251 % superior ao da não-rizosfera. A biomassa microbiana da rizosfera aumentou nos tratamentos com sulfato de amônio e adubo verde, permanecendo invariável na não-rizosfera. Confirmando os resultados da contagem microbiana, a biomassa microbiana também aumentou na rizosfera em relação à da não-rizosfera. A atividade da desidrogenase e da urease aumentou em relação ao controle, não se constatando, no entanto, efeito na atividade da arilsulfatase. A produção de repolho foi superior ao controle de 52,5 a 81,1 %. Concentrações crescentes de enxofre foram observadas no solo e na parte aérea do repolho.