Deconjugation potentials of natural estrogen conjugates in sewage and wastewater treatment plant: New insights from model prediction and on-site investigations.

Natural estrogen conjugates play important roles in municipal wastewater treatment plant (WWTP), but their deconjugation potentials are poorly understood. This work is the first to investigate the relationships between the enzyme activities of arylsulfatase/ß-glucuronidase and deconjugation potentials of natural estrogen conjugates. This work led to three important findings. First, the enzyme activity of ß-glucuronidase in sewage is far higher than that of arylsulfatase, while their corresponding activities in activated sludge were similar. Second, a model based on ß-glucuronidase could successfully predict the deconjugation potentials of natural estrogen glucuronide conjugates in sewage. Third, the enzyme activity of arylsulfatase in sewage was too low to lead to evident deconjugation of sulfate conjugates, which means that the deconjugation rate of estrogen sulfates can be regarded as zero. By comparing their theoretical removal based on enzyme activity and on-site investigation, it is reasonable to conclude that reverse deconjugation of estrogen conjugates (i.e., conjugation of natural estrogens to form conjugated estrogens) likely exist in WWTP, which explains well why natural estrogen conjugates cannot be effectively removed in WWTP. Meanwhile, this work provides new insights how to improve the removal performance of WWTP on natural estrogen conjugates. SYNOPSIS: This work is the first to show how arylsulfatase/ß-glucuronidase could affect deconjugation of natural estrogen conjugates and possible way to enhance their removal in wastewater treatment plant.

Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Transcription level and phylogeny analyses of Chlamydomonas reinhardtii arylsulfatases.

Sulfur is a required macroelement for all organisms, and sulfate deficiency causes growth and developmental defects. Arylsulfatases (ARS) hydrolyze sulfate from sulfate esters and make sulfate bioavailable for plant uptake. These enzymes are found in microorganisms and animals; however, plant genomes do not encode any ARS gene. Our database searches found nineteen ARS genes in the genome of Chlamydomonas reinhardtii. Among these, ARS1 and ARS2 were studied in the literature; however, the remaining seventeen gene models were not studied. Our results show that putative polypeptide sequences of the ARS gene models all have the sulfatase domain and sulfatase motifs found in known ARSs. Phylogenetic analyses show that C. reinhardtii proteins are in close branches with Volvox carterii proteins while they were clustered in a separate group from Homo sapiens and bacterial species (Pseudomonas aeruginosa and Rhodopirellula baltica SH1), except human Sulf1, Sulf2, and GNS are clustered with algal ARSs. RT-PCR analyses showed that transcription of ARS6, ARS7, ARS11, ARS12, ARS13, ARS17, and ARS19 increased under sulfate deficiency. However, this increase was not as high as the increase seen in ARS2. Since plant genomes do not encode any ARS gene, our results highlight the importance of microbial ARS genes.

The lysosomal proteome of senescent cells contributes to the senescence secretome.

Senescent cells accumulate in tissues over time, favoring the onset and progression of multiple age-related diseases. Senescent cells present a remarkable increase in lysosomal mass and elevated autophagic activity. Here, we report that two main autophagic pathways macroautophagy (MA) and chaperone-mediated autophagy (CMA) are constitutively upregulated in senescent cells. Proteomic analyses of the subpopulations of lysosomes preferentially engaged in each of these types of autophagy revealed profound quantitative and qualitative changes in senescent cells, affecting both lysosomal resident proteins and cargo proteins delivered to lysosomes for degradation. These studies have led us to identify resident lysosomal proteins that are highly augmented in senescent cells and can be used as novel markers of senescence, such as arylsulfatase ARSA. The abundant secretome of senescent cells, known as SASP, is considered their main pathological mediator; however, little is known about the mechanisms of SASP secretion. Some secretory cells, including melanocytes, use the small GTPase RAB27A to perform lysosomal secretion. We found that this process is exacerbated in the case of senescent melanoma cells, as revealed by the exposure of lysosomal membrane integral proteins LAMP1 and LAMP2 in their plasma membrane. Interestingly, a subset of SASP components, including cytokines CCL2, CCL3, CXCL12, cathepsin CTSD, or the protease inhibitor SERPINE1, are secreted in a RAB27A-dependent manner in senescent melanoma cells. Finally, proteins previously identified as plasma biomarkers of aging are highly enriched in the lysosomes of senescent cells, including CTSD. We conclude that the lysosomal proteome of senescent cells is profoundly reconfigured, and that some senescent cells can be highly active in lysosomal exocytosis.

Property of arylsulfatase and ß-glucuronidase extracted from digestive tracts of Cipangopaludina chinensis and their cleavage performance on conjugated natural estrogens.

Arylsulfatase and ß-glucuronidase are the two substantial enzymes having a significant role in the cleavage of conjugated natural estrogens (C-NEs). The present study reports that arylsulfatase and ß-glucuronidase have been abundantly found in the digestive tracts of Cipangopaludina chinensis; in which, their corresponding activities were 60 and 5 U/g wet waste, respectively. The arylsulfatase from Cipangopaludina chinensis could show high activity at low temperatures. Hence, its activity still remained at 53.2% of maximal activity even at an extremely low temperature of 4 ℃; while the corresponding activities of arylsulfatase from Helix pomatia or activated sludge were less than 20% and 10%, respectively. The arylsulfatase and ß-glucuronidase from Cipangopaludina chinensis could efficiently cleave C-NEs suggesting that they could be alternative enzymes derived from Helix pomatia that are used for cleavage of conjugated compounds in environmental or biological sample analysis. Meanwhile, they might also be used to enhance the cleavage of C-NEs in municipal wastewater.

Arylsulfatase I is a prognostic biomarker for head and neck squamous cell carcinoma and Pan-cancer.

BACKGROUND: Sulfatase gene family members mediate various biological functions in tumor stroma and tumor cell environments. However, the expressions and prognostic value of Arylsulfatase I (ARSI), a sulfatase gene family member, in head and neck squamous cell carcinoma (HNSC) have not been fully established. METHODS: Arylsulfatase I expressions in pan-cancer were profiled using publicly available databases. Then, univariate Cox regression, Kaplan-Meier, and the Pearson's correlation analyses were performed to determine correlations between ARSI expressions and cancer prognosis, immune cell status, and drug sensitivity. Gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA) were used to assess the potential mechanisms underlying ARSI functions in HNSC. RESULTS: Arylsulfatase I was highly expressed in 15 cancer types, with significant expressions in HNSC. Elevated ARSI levels were associated with worse prognostic outcomes in HNSC patients. In addition, GSVA and GSEA showed that ARSI was highly involved in tumor cell escape and inflammatory responses. Expressions of ARSI negatively correlated with tumor mutation burden or microsatellite instability and positively correlated with immune-related genes. Elevated ARSI expressions conferred poor tolerance to daporinad and sinularin, but increased cell sensitivity to dasatinib and XAV939. CONCLUSION: Arylsulfatase I is a promising prognostic and therapeutic target for HNSC.

Sulfite may disrupt estrogen homeostasis in human via inhibition of steroid arylsulfatase.

Steroid arylsulfatase is an important enzyme in human, which plays an important role in dynamic equilibrium of natural estrogens. On the other hand, sulfite can be endogenously produced as a consequence of human body's metabolism of sulfur-containing amino acids, while its main sources to human are mainly derived from food as it is a widely used additive. Sulfite-sensitivity is a well-known phenomenon to a small proportion of populations. However, its potential adverse effects on healthy individuals have been hardly reported. It was for the first time reported in this study that sulfite could effectively inhibit arylsulfatase, and its IC50 values for the snail- and human urine-derived arylsulfatase were determined to be 71.9 and 142.8 µM, which were lower than the concentration of sulfite in some healthy population. Consequently, it appears that sulfite might disrupt estrogen homeostasis in human, and this deserves further investigation.

ARSD, a novel ERα downstream target gene, inhibits proliferation and migration of breast cancer cells via activating Hippo/YAP pathway.

Advanced breast cancer (BC), especially basal like triple-negative BC (TNBC), is a highly malignant tumor without viable treatment option, highlighting the urgent need to seek novel therapeutic targets. Arylsulfatase D (ARSD), localized at Xp22.3, is a female-biased gene due to its escaping from X chromosome inactivation (XCI). Unfortunately, no systematic investigation of ARSD on BC has been reported. In this study, we observed that ARSD expression was positively related to ERα status either in BC cells or tissue specimens, which were associated with good prognosis. Furthermore, we found a set of hormone-responsive lineage-specific transcription factors, FOXA1, GATA3, ERα, directly drove high expression of ARSD through chromatin looping in luminal subtype BC cells. Opposingly, ARSD still subjected to XCI in TNBC cells mediated by Xist, CpG islands methylation, and inhibitory histone modification. Unexpectedly, we also found that ectopic ARSD overexpression could inhibit proliferation and migration of TNBC cells by activating Hippo/YAP pathway, indicating that ARSD may be a molecule brake on ERα signaling pathway, which restricted ERα to be an uncontrolled active status. Combined with other peoples' researches that Hippo signaling maintained ER expression and ER + BC growth, we believed that there should exist a regulative feedback loop formation among ERα, ARSD, and Hippo/YAP pathway. Collectively, our findings will help filling the knowledge gap about the influence of ARSD on BC and providing evidence that ARSD may serve as a potential marker to predict prognosis and as a therapeutic target.

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).

The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wild-type and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate.

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

La mucopolisacaridosis tipo VI, también conocida como síndrome de Maroteaux-Lamy, es un trastorno lisosómico autosómico recesivo, causado por la deficiencia de la enzima arilsulfatasa B, lo que conduce a la acumulación de dermatán sulfato en los tejidos y su excreción urinaria. La deposición de mucopolisacáridos genera un trastorno progresivo que afecta a múltiples órganos y que, a menudo, resulta en la muerte a temprana edad. Esta enfermedad tiene varias manifestaciones orales, entre las que destacan las complicaciones dentales, que pueden ser graves e incluir folículos similares a quistes dentígeros, maloclusiones, defectos condilares e hiperplasia gingival, además de características clínicas como cuello corto, opacidad corneal, macroglosia y agrandamiento del cráneo, dimensión anteroposterior larga y mano en garra. Se presenta el caso de un paciente de 14 meses de edad que acudió a consulta de odontopediatría por episodios de fiebre, bajo peso e hiperplasia gingival severa. El examen físico evidenció facies tosca, cuello corto, pectus excavatus, manos con disminución en agarre y retardo en el neurodesarrollo. El examen intraoral halló retardo de la erupción dental, hiperplasia gingival generalizada y paladar con poco crecimiento transversal. El examen radiográfico detectó órganos dentarios incluidos y mala posición en el sector anterior, molares superiores dentro del seno maxilar y caninos inferiores rotados. El paciente fue remitido a medicina para exámenes bioquímicos y genéticos para definir el diagnóstico. La bioquímica reveló MPS tipo VI, lo que fue confirmado mediante prueba molecular. Las manifestaciones clínicas en este caso corresponden a la forma clínica de progresión rápida reportada en estos pacientes: talla baja, malformaciones esqueléticas y alteraciones a nivel oral. Los niños con MPS VI grave comienzan temprano y progresan rápidamente, las radiografías óseas y la medición de GAG en orina son útiles para el diagnóstico con actividad de la enzima ARSB y genética. Es necesario fortalecer el conocimiento en odontología y la población en general sobre las características clínicas de mucopolisacáridos tipo VI para tener un diagnóstico temprano y un mejor manejo de patologías en estos pacientes. (AU)

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. (AU)

Multimodal imaging and genetic findings in a case of ARSG-related atypical Usher syndrome.

Background: Atypical Usher syndrome has recently been associated with arylsulfatase G (ARSG) variants. In these cases, characteristic findings include progressive sensorineural hearing loss (SNHL) without vestibular involvement and ring-shaped late-onset retinitis pigmentosa (RP).Materials and Methods: One patient with atypical Usher syndrome and a novel homozygous ARSG variant was included in this study. The patient underwent a comprehensive ophthalmic examination, including multimodal imaging and genetic testing.Results: A 60-year-old male of Persian decent presented to our clinic with a history of 20 years of progressive SNHL, and 10 years of progressive peripheral vision loss and pigmentary retinopathy. Consistent with previous reports of ARSG-related atypical Usher syndrome, fundus examination revealed ring-shaped retinal hyperpigmentation and fundus autofluorescence (FAF) demonstrated a six-zone pattern of autofluorescence. Optical coherence tomography (OCT) showed extensive cystoid spaces concentrated in the ganglion cell layer. Widefield OCT angiography at the level of the choriocapillaris showed signs of atrophy that corresponded to the FAF hypofluorescent zone. The patient was homozygous for a novel ARSG variant c. 1270 C > T, p. Arg424Cys.Conclusion: We report a novel ARSG variant in a case of atypical Usher syndrome and describe multimodal imaging findings that further characterize the effect of ARSG in the pathogenesis of atypical Usher syndrome.

Evaluation of Arylsulfatase D (ARSD) and long noncoding RNA ARSD-AS1 gene expression in breast cancer patients and their association with oncogenic transcription factors.

PURPOSE: Long non-coding RNAs (LncRNAs) are thought as tumorigenic factors in cancer progression. We investigated the clinical significance of arylsulfatase D (ARSD) and ARSD antisense in breast cancer patients. METHODS: Eighty breast cancer tumors were obtained from the Tumor Bank of Cancer Institute, Imam Khomeini Hospital. The expression level of ARSD and ARSD-AS1 were examined in breast tumors in comparison to the margin of normal tissues using quantitative real-time PCR. Demographic information and the clinicopathologic characteristics including tumor grade, presence of cell receptors, lymph node and vascular invasion were also evaluated. Bioinformatics databases were used for identification of ARSD and ARSD-AS1 molecular targets and their association with cancer. RESULTS: Significant up-regulation of ARSD was observed in tumor tissues in comparison with its antisense (p<0.05). Both ARSD and ARSD-AS1 expression in tumor specimens were notably lower than those in adjacent normal tissue. High expression of ARSD was associated to lower tumor grade (p<0.05). Bioinformatics results revealed the interaction of ARSD with STS and SUMF1 proteins was attributed to the inhibiting of sulfates activity. Also, ARSD co-expressed genes were associated with oncogenic transcription factors, MAF and GATA. TP53 transcription factor site was identified as a target of ARSD-AS1 mRNA. The interaction of this antisense with microRNA (miR-618) could explain its participation in tumor cell proliferation. CONCLUSION: Low expression of ARSD was associated with higher tumor grade. The evidence from this study enhance our understanding of ARSD and ARSD-AS1 function in cancer gene therapy. Accordingly, they could be introduced as great potential targets for breast cancer treatment.

Discovery of the First Human Arylsulfatase A Reversible Inhibitor Impairing Mouse Oocyte Fertilization.

Arylsulfatase A (ARSA) plays a crucial role in the reproduction of mammals due to its involvement in the specific gamete interaction preceding sperm and egg fusion leading to fertilization. Recently, it has been shown that zona pellucida (ZP) sperm binding and in vivo fertilization in mice are markedly hampered by using a specific anti-ARSA antibody. Herein, the design and discovery of the first ARSA small molecule inhibitor based on a coumarin-containing polycycle are presented. Through a structure-based approach applied on our in-house library, compound 1r was identified as an ARSA reversible inhibitor (ARSAi); then its activity was validated through both surface plasmon resonance and biochemical inhibition experiments, the first providing a KD value of 21 µM and the latter an IC50 value of 13.2 µM. Further investigations highlighted that compound 1r induced 20% sperm death at 25 µM and also impaired sperm motility; nevertheless both the effects were mediated by ROS production, since they were rescued by the cotreatment of 1r and N-acetyl cysteine (NAC). Interestingly, while 1r was not able to hamper the ZP/sperm binding, it markedly decreased the in vitro oocyte fertilization by mouse sperm up to 60%. Notably, this effect was not hampered by 1r/NAC coadministration, hence allowing the ruling out of an ROS-dependent mechanism. In conclusion, herein is reported the first ever hit of ARSAi as a chemical tool that will enable better exploration of ARSA's biological role in fertilization as well as provide a starting point for developing 1r structure optimization aimed at increasing enzyme inhibition potency but also providing a deeper understanding of the involvement of ARSA in the fertilization pathway mechanism.

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.

Proteoglycan desulfation: a critical step in oncogenesis?

Proteoglycans are essential constituents of tissue- and organ microenvironments, modulating both the structural scaffolds that surround cells as well as signaling cues that determine cellular phenotype. An important modification of proteoglycans is the sulfation of the monosaccharides that comprise them. Sulfates are added by sulfotransferases and desulfation occurs through the action of sulfatases. In this essay, we examine the biochemistry of a conserved family of desulfating enzymes known as arylsulfatases. A subset of these enzymes mediates the desulfation of proteoglycans. We review the consequences of their aberrant expression in the light of carcinogenesis and carcinomatosis: the dissemination of cancer cells. A closer understanding of their cellular-molecular roles reveals their promise for future strategies for cancer therapy.

Interactive effect of salinity and cadmium toxicity on soil microbial properties and enzyme activities.

Salinity has been proposed to increase the mobility and availability of heavy metals, with a potentially significant consequence for greater metal toxicity. However, the interactive effect of salinity and metal pollution on soil microbial properties and functions is still unknown. This investigation was performed to examine the response of several soil microbial properties and processes to the combined salinity and cadmium (Cd) toxicity in a clay loam soil amended with plant residue. The NaCl salt (0, 32.5 and 78.3 mM NaCl kg-1 soil), Cd (0 and 30 mg kg-1 soil) and alfalfa residue (0 and 1%) were added to the soil and the mixtures were incubated for 90 days under standard laboratory conditions (25 ±â€¯1 °C and 70% of water holding capacity). Similar treatments without residue addition were also included in the experimental arrangement. Salinity increased soil Cd availability and toxicity, and subsequently decreased soil microbial respiration rate, microbial biomass and enzyme activity. The negative effect of increasing salinity on soil microbial and biochemical properties was stronger in Cd-polluted than unpolluted soils and at high than low salinity levels. The declines in soil microbial attributes and enzyme activity were linearly related to the concentration of soil available Cd. Nevertheless, the negative effect of salinity was reduced with addition of alfalfa residue in Cd-polluted soils. The interactive effect of Cd and NaCl was synergistic in residue-unamended soils, but antagonistic in residue-amended soils. It is concluded that (i) the multiple stresses induced by salinity and Cd pollution may synergistically affect soil microbial processes and attributes and (ii) application of organic residues has a high potential for lowering the synergistic effect of salinity in Cd-polluted environments and improving the important microbial indicators of soil quality.

SULFATION PATHWAYS: Steroid sulphatase inhibition via aryl sulphamates: clinical progress, mechanism and future prospects.

Steroid sulphatase is an emerging drug target for the endocrine therapy of hormone-dependent diseases, catalysing oestrogen sulphate hydrolysis to oestrogen. Drug discovery, developing the core aryl O-sulphamate pharmacophore, has led to steroidal and non-steroidal drugs entering numerous clinical trials, with promising results in oncology and women's health. Steroidal oestrogen sulphamate derivatives were the first irreversible active-site-directed inhibitors and one was developed clinically as an oral oestradiol pro-drug and for endometriosis applications. This review summarizes work leading to the therapeutic concept of sulphatase inhibition, clinical trials executed to date and new insights into the mechanism of inhibition of steroid sulphatase. To date, the non-steroidal sulphatase inhibitor Irosustat has been evaluated clinically in breast cancer, alone and in combination, in endometrial cancer and in prostate cancer. The versatile core pharmacophore both imbues attractive pharmaceutical properties and functions via three distinct mechanisms of action, as a pro-drug, an enzyme active-site-modifying motif, likely through direct sulphamoyl group transfer, and as a structural component augmenting activity, for example by enhancing interactions at the colchicine binding site of tubulin. Preliminary new structural data on the Pseudomonas aeruginosa arylsulphatase enzyme suggest two possible sulphamate-based adducts with the active site formylglycine as candidates for the inhibition end product via sulphamoyl or sulphonylamine transfer, and a speculative choice is suggested. The clinical status of sulphatase inhibition is surveyed and how it might develop in the future. Also discussed are dual-targeting approaches, development of 2-substituted steroidal sulphamates and non-steroidal derivatives as multi-targeting agents for hormone-independent tumours, with other emerging directions.

A homozygous founder missense variant in arylsulfatase G abolishes its enzymatic activity causing atypical Usher syndrome in humans.

PURPOSE: We aimed to identify the cause of disease in patients suffering from a distinctive, atypical form of Usher syndrome. METHODS: Whole-exome and genome sequencing were performed in five patients from three families of Yemenite Jewish origin, suffering from distinctive retinal degeneration phenotype and sensorineural hearing loss. Functional analysis of the wild-type and mutant proteins was performed in human fibrosarcoma cells. RESULTS: We identified a homozygous founder missense variant, c.133G>T (p.D45Y) in arylsulfatase G (ARSG). All patients shared a distinctive retinal phenotype with ring-shaped atrophy along the arcades engirdling the fovea, resulting in ring scotoma. In addition, patients developed moderate to severe sensorineural hearing loss. Both vision and hearing loss appeared around the age of 40 years. The identified variant affected a fully conserved amino acid that is part of the catalytic site of the enzyme. Functional analysis of the wild-type and mutant proteins showed no basal activity of p.D45Y. CONCLUSION: Homozygosity for ARSG-p.D45Y in humans leads to protein dysfunction, causing an atypical combination of late-onset Usher syndrome. Although there is no evidence for generalized clinical manifestations of lysosomal storage diseases in this set of patients, we cannot rule out the possibility that mild and late-onset symptoms may appear.

Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase.

To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t 0.5) at 37 °C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t 0.5 of ~6.0 days in 50 mM HEPES, but ~42 days in 10 mM sodium borate with >85 % activity after 15 days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t 0.5 of PAAS was ~14 days in 50 mM Tris-HCl, and ~21 days in 10 mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14 days at 37 °C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.

Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example.

Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution.