N-Acetylgalactosamine-4-sulfatase (Arylsulfatase B) Regulates PD-L1 Expression in Melanoma by an HDAC3-Mediated Epigenetic Mechanism.

The effects of the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate, on the expression of PD-L1 were determined, and the underlying mechanism of PD-L1 expression was elucidated. Initial experiments in human melanoma cells (A375) showed that PD-L1 expression increased from 357 ± 31 to 796 ± 50 pg/mg protein (p < 10-11) when ARSB was silenced in A375 cells. In subcutaneous B16F10 murine melanomas, PD-L1 declined from 1227 ± 189 to 583 ± 110 pg/mg protein (p = 1.67 × 10-7), a decline of 52%, following treatment with exogenous, bioactive recombinant ARSB. This decline occurred in association with reduced tumor growth and prolongation of survival, as previously reported. The mechanism of regulation of PD-L1 expression by ARSB is attributed to ARSB-mediated alteration in chondroitin 4-sulfation, leading to changes in free galectin-3, c-Jun nuclear localization, HDAC3 expression, and effects of acetyl-H3 on the PD-L1 promoter. These findings indicate that changes in ARSB contribute to the expression of PD-L1 in melanoma and can thereby affect the immune checkpoint response. Exogenous ARSB acted on melanoma cells and normal melanocytes through the IGF2 receptor. The decline in PD-L1 expression by exogenous ARSB may contribute to the impact of ARSB on melanoma progression.

Biochemical and molecular analysis of pediatric patients with metachromatic leukodystrophy in South China: functional characterization of five novel ARSA variants.

Metachromatic leukodystrophy (MLD) is a rare hereditary neurodegenerative disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). This study described the clinical and molecular characteristics of 24 Chinese children with MLD and investigated functional characterization of five novel ARSA variants. A retrospective analysis was performed in 24 patients diagnosed with MLD at Guangzhou Women and Children's Medical Center in South China. Five novel mutations were further characterized by transient expression studies. We recruited 17 late-infantile, 3 early-juvenile, 4 late-juvenile MLD patients. In late-infantile patients, motor developmental delay and gait disturbance were the most frequent symptoms at onset. In juvenile patients, cognitive regression and gait disturbance were the most frequent chief complaints. Overall, 25 different ARSA mutations were identified with 5 novel mutations.The most frequent alleles were p.W320* and p.G449Rfs. The mutation p.W320*, p.Q155=, p.P91L, p.G156D, p.H208Mfs*46 and p.G449Rfs may link to late-infantile type. The novel missense mutations were predicted damaging in silico. The bioinformatic structural analysis of the novel missense mutations showed that these amino acid replacements would cause severe impairment of protein structure and function. In vitro functional analysis of the six mutants, showing a low ARSA enzyme activity, clearly demonstrated their pathogenic nature. The mutation p.D413N linked to R alleles. In western blotting analysis of the ARSA protein, the examined mutations retained reduced amounts of ARSA protein compared to the wild type. This study expands the spectrum of genotype of MLD. It helps to the future studies of genotype-phenotype correlations to estimate prognosis and develop new therapeutic approach.

Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

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.

A systematic review on the birth prevalence of metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficiency in arylsulfatase A (ASA) activity arising primarily from ASA gene (ARSA) variants. Late-infantile, juvenile and adult clinical subtypes are defined by symptom onset at ≤ 2.5, > 2.5 to < 16 and ≥ 16 years, respectively. Epidemiological data were sought to address knowledge gaps and to inform decisions regarding the clinical development of an investigational drug. METHODS: To synthesize all available estimates of MLD incidence and birth prevalence worldwide and in selected countries, Ovid MEDLINE and Embase were searched systematically (March 11, 2022) using a population, intervention, comparator, outcome, time and setting framework, complemented by pragmatic searching to reduce publication bias. Where possible, results were stratified by clinical subtype. Data were extracted from non-interventional studies (clinical trials, non-clinical studies and case reports were excluded; reviews were used for snowballing only). RESULTS: Of the 31 studies included, 14 reported birth prevalence (13 countries in Asia-Pacific, Europe, the Middle East, North America and South America), one reported prevalence and none reported incidence. Birth prevalence per 100,000 live births ranged from 0.16 (Japan) to 1.85 (Portugal). In the three European studies with estimates stratified by clinical subtypes, birth prevalence was highest for late-infantile cases (0.31-1.12 per 100,000 live births). The distribution of clinical subtypes reported in cases diagnosed over various time periods in 17 studies varied substantially, but late-infantile and juvenile MLD accounted for at least two-thirds of cases in most studies. CONCLUSIONS: This review provides a foundation for further analysis of the regional epidemiology of MLD. Data gaps indicate the need for better global coverage, increased use of epidemiological measures (e.g. prevalence estimates) and more stratification of outcomes by clinical and genetic disease subtype.

Complex genotypes in family with metachromatic leukodystrophy: Effect of trans and cis mutations distribution on the phenotype variability.

Metachromatic leukodystrophy (MLD) is a severe metabolic disorder caused by the deficient activity of arylsulfatase A due to ARSA gene mutations. According to the age of onset, MLD is classified into three forms: infantile, juvenile, and adult. In our study, we aimed to perform a genetic analysis for two siblings with juvenile MLD for a better characterization of the molecular mechanisms behind the disease. A consanguineous family including two MLD patients (PII.1 and PII.2) was enrolled in our study. The diagnosis was made based on the clinical and neuroimaging investigations. The sequencing of ARSA gene was performed followed by in silico analysis. Besides, the cis/trans distribution of the variants was verified through a PCR-RFLP. The ARSA gene sequencing revealed three known variants, two exonic c.1055A > G and c.1178C > G and an intronic one (c.1524 + 95A > G) in the 3'UTR region. All variants were present at heterozygous state in the two siblings and their mother. The assessment of the cis/trans distribution showed the presence of these variants in cis within the mother, while PII.2 and PII.2 present the c.1055A > G/c.1524 + 95A > G and the c.1178C > G in trans. Additionally, PII.1 harbored a de novo novel missense variant c.1119G > T, whose pathogenicity was supported by our predictive results. Our genetic findings, supported by a clinical examination, confirmed the affection of the mother by the adult MLD. Our results proved the implication of the variable distribution of the found variants in the age of MLD onset. Besides, we described a variable severity between the two siblings due to the de novo pathogenic variant. In conclusion, we identified a complex genotype of ARSA variants within two MLD siblings with a variable severity due to a de novo variant present in one of them. Our results allowed the establishment of an adult MLD diagnosis and highlighted the importance of an assessment of the trans/cis distribution in the cases of complex genotypes.

A radiation of Psylliodes flea beetles on Brassicaceae is associated with the evolution of specific detoxification enzymes.

Flea beetles of the genus Psylliodes have evolved specialized interactions with plant species belonging to several distantly related families, mainly Brassicaceae, Solanaceae, and Fagaceae. This diverse host use indicates that Psylliodes flea beetles are able to cope with different chemical defense metabolites, including glucosinolates, the characteristic defense metabolites of Brassicaceae. Here we investigated the evolution of host use and the emergence of a glucosinolate-specific detoxification mechanism in Psylliodes flea beetles. In phylogenetic analyses, Psylliodes species clustered into four major clades, three of which contained mainly species specialized on either Brassicaceae, Solanaceae, or Fagaceae. Most members of the fourth clade have broader host use, including Brassicaceae and Poaceae as major host plant families. Ancestral state reconstructions suggest that Psylliodes flea beetles were initially associated with Brassicaceae and then either shifted to Solanaceae or Fagaceae, or expanded their host repertoire to Poaceae. Despite a putative ancestral association with Brassicaceae, we found evidence that the evolution of glucosinolate-specific detoxification enzymes coincides with the radiation of Psylliodes on Brassicaceae, suggesting that these are not required for using Brassicaceae as hosts but could improve the efficiency of host use by specialized Psylliodes species.

Exogenous recombinant N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) inhibits progression of B16F10 cutaneous melanomas and modulates cell signaling.

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p < 0.0001) and improved the probability of survival up to 21 days (p = 0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), carbohydrate sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed ARSB siRNA. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression.

Predicting the pathogenicity of missense variants based on protein instability to support diagnosis of patients with novel variants of ARSL.

Rare diseases are estimated to affect 3.5%-5.9% of the population worldwide and are difficult to diagnose. Genome analysis is useful for diagnosis. However, since some variants, especially missense variants, are also difficult to interpret, tools to accurately predict the effect of missense variants are very important and needed. Here we developed a method, "VarMeter", to predict whether a missense variant is damaging based on Gibbs free energy and solvent-accessible surface area calculated from the AlphaFold 3D protein model. We applied this method to the whole-exome sequencing data of 900 individuals with rare or undiagnosed disease in our in-house database, and identified four who were hemizygous for missense variants of arylsulfatase L (ARSL; known as the genetic cause of chondrodysplasia punctata 1, CPDX1). Two individuals had a novel Ser89 to Asn (Ser89Asn) or Arg469 to Trp (Arg469Trp) substitution, respectively predicted as "damaging" or "benign"; the other two had an Arg111 to His (Arg111His) or Gly117 to Arg (Gly117Arg) substitution, respectively predicted as "damaging" or "possibly damaging" and previously reported in patients showing clinical manifestations of CDPX1. Expression and analysis of the missense variant proteins showed that the predicted pathogenic variants (Ser89Asn, Arg111His, and Gly117Arg) had complete loss of sulfatase activity and reduced protease resistance due to destabilization of protein structure, while the predicted benign variant (Arg469Trp) had activity and protease resistance comparable to those of wild-type ARSL. The individual with the novel pathogenic Ser89Asn variant exhibited characteristics of CDPX1, while the individual with the benign Arg469Trp variant exhibited no such characteristics. These findings demonstrate that VarMeter may be used to predict the deleteriousness of variants found in genome sequencing data and thereby support disease diagnosis.

Engineered arylsulfatase A with increased activity, stability and brain delivery for therapy of metachromatic leukodystrophy.

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme's bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.

Detection, production, modification, and application of arylsulfatases.

Arylsulfatase is a subset of sulfatase which catalyzes the hydrolysis of aryl sulfate ester. Arylsulfatase is widely distributed among microorganisms, mammals and green algae, but the arylsulfatase-encoding gene has not yet been found in the genomes of higher plants so far. Arylsulfatase plays an important role in the sulfur flows between nature and organisms. In this review, we present the maturation and catalytic mechanism of arylsulfatase, and the recent literature on the expression and production of arylsulfatase in wild-type and engineered microorganisms, as well as the modification of arylsulfatase by genetic engineering are summarized. We focus on arylsulfatases from microbial origin and give an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the researches about arylsulfatase application on the field of agar desulfation, soil sulfur cycle and soil evaluation are also discussed. Finally, the perspectives concerning the future research on arylsulfatase are prospected.

The arylsulfatase- and phospholipase-rich venom of the plutoniumid centipede Theatops posticus.

Research on centipede venoms has led to the discovery of a diverse array of novel proteins and peptides, including those with homology to previously discovered toxin families (e.g., phospholipase A2s and pM12a metalloproteases) and novel toxin families not previously detected in venoms (e.g., ß-pore forming toxins and scoloptoxins). Most of this research has focused on centipedes in the order Scolopendromorpha, particularly those in the families Scolopendridae, Cryptopidae, and Scolopocryptopidae. To generate the first high-throughput venom characterization for a centipede in the scolopendromorph family Plutoniumidae, we performed venom-gland transcriptomics and venom proteomics on two Theatops posticus. We identified a total of 64 venom toxins, 60 of which were detected in both the venom-gland transcriptome and venom proteome and four of which were only detected transcriptomically. We detected a single highly abundant arylsulfatase B (ARSB) toxin, the first ARSB toxin identified from centipede venoms. As ARSBs have been detected in other venomous species (e.g., scorpions), ARSBs in T. posticus highlights a new case of convergent evolution across venoms. Theatops posticus venom also contained a much higher abundance and diversity of phospholipase A2 toxins compared to other characterized centipede venoms. Conversely, we detected other common centipedes toxins, such as CAPs and scoloptoxins, at relatively low abundances and diversities. Our observation of a diverse set of toxins from T. posticus venom, including those from novel toxin families, emphasizes the importance of studying unexplored centipede taxonomic groups and the continued potential of centipede venoms for novel toxin discovery and unraveling the molecular mechanisms underlying trait evolution.

Evaluating meaningful changes in physical functioning and cognitive declines in metachromatic leukodystrophy: a caregiver interview study.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disease caused by deficient activity of arylsulfatase A (ASA). Treatment options for patients are limited; gene therapy based on haematopoietic stem cell transplantation is the only approved treatment for some subtypes of MLD. Any therapeutic benefit of treatments must be meaningful for patients and their families. We evaluated the clinical meaningfulness of slowing the decline in gross motor function as measured by the Gross Motor Function Classification in MLD (GMFC-MLD) from the caregiver perspective via semi-structured telephone interviews with caregivers of children with late-infantile MLD. We also evaluated the perceived significance of declines in communication abilities measured by the Expressive Language Function Classification in MLD (ELFC-MLD). This work could help to inform the endpoints of a phase 2 clinical trial (NCT03771898) assessing the efficacy of intrathecal recombinant human ASA in MLD. RESULTS: Twelve caregivers were recruited, reporting on 12 children with MLD. Children had a mean age of 6.1 years; mean age at symptom onset was 17.6 months. Most children (10/12) progressed from walking without support (categories 0-1) to a loss of locomotion (categories 5-6) in ≤ 2 years. Caregivers felt that GMFC-MLD and ELFC-MLD accurately described motor and language declines in their children, respectively. Most caregivers (10/12) reported that the idea of delaying disease progression would be meaningful. Further, a slowing of motor function decline in GMFC-MLD, from category 1 to category 3 or from category 2 to category 4 over 2 years, was seen as meaningful by all caregivers asked; however, only 3/12 caregivers reported that delayed decline would be meaningful if baseline category was ≥ 3. Caregivers also reported that delaying expressive language decline at any level that did not indicate a complete loss of expressive language (indicated by categories 1-3) would be meaningful. CONCLUSIONS: Caregivers of children with MLD felt that a delayed decline in gross motor function, as assessed by the GMFC-MLD, would be meaningful, supporting the selection of primary and secondary endpoints for the phase 2 clinical trial. Communication abilities were another area of significance for consideration in future clinical trial design.

Increased Cerebral Serum Amyloid A2 and Parameters of Oxidation in Arylsulfatase B (N-Acetylgalactosamine-4-Sulfatase)-Null Mice.

Background: Chondroitin sulfate and chondroitin sulfate proteoglycans have been associated with Alzheimer's disease (AD), and the impact of modified chondroitin sulfates is being investigated in several animal and cell-based models of AD. Published reports have shown the role of accumulation of chondroitin 4-sulfate and decline in Arylsulfatase B (ARSB; B-acetylgalactosamine-4-sulfatase) in other pathology, including nerve injury, traumatic brain injury, and spinal cord injury. However, the impact of ARSB deficiency on AD pathobiology has not been reported, although changes in ARSB were associated with AD in two prior reports. The enzyme ARSB removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate and dermatan sulfate and is required for their degradation. When ARSB activity declines, these sulfated glycosaminoglycans accumulate, as in the inherited disorder Mucopolysaccharidosis VI. Objective: Reports about chondroitin sulfate, chondroitin sulfate proteoglycans, and chondroitin sulfatases in AD were reviewed. Methods: Measurements of SAA2, iNOS, lipid peroxidation, chondroitin sulfate proteoglycan 4 (CSPG4), and other parameters were performed in cortex and hippocampus from ARSB-null mice and controls by QRT-PCR, ELISA, and other standard assays. Results: SAA2 mRNA expression and protein, CSPG4 mRNA, chondroitin 4-sulfate, and iNOS were increased significantly in ARSB-null mice. Measures of lipid peroxidation and redox state were significantly modified. Conclusion: Findings indicate that decline in ARSB leads to changes in expression of parameters associated with AD in the hippocampus and cortex of the ARSB-deficient mouse. Further investigation of the impact of decline in ARSB on the development of AD may provide a new approach to prevent and treat AD.

The Application of Sulfur Influences Microbiome of Soybean Rhizosphere and Nutrient-Mobilizing Bacteria in Andosol.

This study aimed to determine the effect of sulfur (S) application on a root-associated microbial community resulting in a rhizosphere microbiome with better nutrient mobilizing capacity. Soybean plants were cultivated with or without S application, the organic acids secreted from the roots were compared. High-throughput sequencing of 16S rRNA was used to analyze the effect of S on microbial community structure of the soybean rhizosphere. Several plant growth-promoting bacteria (PGPB) isolated from the rhizosphere were identified that can be harnessed for crop productivity. The amount of malic acid secreted from the soybean roots was significantly induced by S application. According to the microbiota analysis, the relative abundance of Polaromonas, identified to have positive association with malic acid, and arylsulfatase-producing Pseudomonas, were increased in S-applied soil. Burkholderia sp. JSA5, obtained from S-applied soil, showed multiple nutrient-mobilizing traits among the isolates. In this study, S application affected the soybean rhizosphere bacterial community structure, suggesting the contribution of changing plant conditions such as in the increase in organic acid secretion. Not only the shift of the microbiota but also isolated strains from S-fertilized soil showed PGPB activity, as well as isolated bacteria that have the potential to be harnessed for crop productivity.

Safety and Efficacy of Intravenous and Intrathecal Delivery of AAV9-Mediated ARSA in Minipigs.

Metachromatic leukodystrophy (MLD) is a hereditary neurodegenerative disease characterized by demyelination and motor and cognitive impairments due to deficiencies of the lysosomal enzyme arylsulfatase A (ARSA) or the saposin B activator protein (SapB). Current treatments are limited; however, gene therapy using adeno-associated virus (AAV) vectors for ARSA delivery has shown promising results. The main challenges for MLD gene therapy include optimizing the AAV dosage, selecting the most effective serotype, and determining the best route of administration for ARSA delivery into the central nervous system. This study aims to evaluate the safety and efficacy of AAV serotype 9 encoding ARSA (AAV9-ARSA) gene therapy when administered intravenously or intrathecally in minipigs, a large animal model with anatomical and physiological similarities to humans. By comparing these two administration methods, this study contributes to the understanding of how to improve the effectiveness of MLD gene therapy and offers valuable insights for future clinical applications.

Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in Chlamydomonas reinhardtii.

Anti-lipopolysaccharide factor 3 (ALFPm3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALFPm3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALFPm3 in Chlamydomonas reinhardtii. In this study, signal peptides ARS1 and CAH1 were fused with ALFPm3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALFPm3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALFPm3 could be detected in algal cells and culture medium by immunoblot, meaning that ALFPm3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALFPm3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi, V. alginolyticus, V. anguillarum, and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALFPm3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALFPm3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALFPm3 peptide. Our results provided a new strategy for the secretory production of ALFPm3 with high antibacterial activity in C. reinhardtii, which could improve the application potentiality of ALFPm3 in the aquaculture industry.

The remarkably enzyme-rich venom of the Big Bend Scorpion (Diplocentrus whitei).

Scorpion venoms have long been studied for their peptide discovery potential, with modern high-throughput venom-characterization techniques paving the way for the discovery of thousands of novel putative toxins. Research into these toxins has provided insight into the pathology and treatment of human diseases, even resulting in the development of one compound with Food and Drug Administration (FDA) approval. Although most of this research has focused on the toxins of scorpion species considered medically significant to humans, the venom of harmless scorpion species possess toxins that are homologous to those from medically significant species, indicating that harmless scorpion venoms may also serve as valuable sources of novel peptide variants. Furthermore, as harmless scorpions represent a vast majority of scorpion species diversity, and therefore venom toxin diversity, venoms from these species likely contain entirely new toxin classes. We sequenced the venom-gland transcriptome and venom proteome of two male Big Bend scorpions (Diplocentrus whitei), providing the first high-throughput venom characterization for a member of this genus. We identified a total of 82 toxins in the venom of D. whitei, 25 of which were identified in both the transcriptome and proteome, and 57 of which were only identified in the transcriptome. Furthermore, we identified a unique, enzyme-rich venom dominated by serine proteases and the first arylsulfatase B toxins identified in scorpions.

Atidarsagene autotemcel for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare autosomal recessive disorder of sphingolipid metabolism, due to a deficiency of the enzyme arylsulfatase A (ARSA). The main clinical signs of the disease are secondary to central and peripheral nervous system demyelination. MLD is subdivided into early- and late-onset subtypes based upon the onset of neurological disease. The early-onset subtype is associated with a more rapid progression of the disease that leads to death within the first decade of life. Until recently, no effective treatment was available for MLD. The blood-brain barrier (BBB) prevents systemically administered enzyme replacement therapy from reaching target cells in MLD. The evidence for the efficacy of hematopoietic stem cell transplantation is limited to the late-onset MLD subtype. Here, we review the preclinical and clinical studies that facilitated the approval of the ex vivo gene therapy atidarsagene autotemcel for early-onset MLD by the European Medicines Agency (EMA) in December 2020. This approach was studied in an animal model first and then in a clinical trial, eventually proving its efficacy in preventing disease manifestations in presymptomatic patients and stabilizing its progression in paucisymptomatic subjects. This new therapeutic consists of patients' CD34+ hematopoietic stem/progenitor cells (HSPCs) transduced with a lentiviral vector encoding functional ARSA cDNA. The gene-corrected cells get reinfused into the patients after a cycle of chemotherapy conditioning.

Respecting catalytic efficiency of soil arylsulfatase as soil Sb contamination bio-indicator by enzyme kinetic strategy.

Antimony (Sb), a toxic metalloid, is ubiquitous in the environment and threatens human and ecological health. Soil arylsulfatase (ARS) activity indicates heavy metal pollution. However, the enzyme's substrate concentration can affect the toxicity evaluation of heavy metals using enzyme activity. Enzyme kinetic parameters directly reflect the potency of heavy metals, and the magnitude of these parameters does not change with the substrate concentration of soil enzyme. In this work, seventeen soils were exposed to Sb contamination to investigate the change of kinetic parameters of soil arylsulfatase under Sb stress. Results showed that Sb inhibited soil arylsulfatase activity. The maximum reaction rate (Vmax) of soil arylsulfatase was reduced by 11.58-46.72% in 16 tested soils and unchanged in S15 when exposed to Sb. The Michaelis constant (Km) presented three trends: unchanged, increased by 28.46-41.27%, and decreased by 19.71-29.91% under Sb stress. The catalytic efficiency (Ka as the ratio of Vmax to Km) decreased by 12.56-55.17% in all soils except for S12 and S16. Antimony acted as a non-competitive and linear mixed inhibitor by decreasing ARS activity in S1-S12, S14, and S17-S18 soils, as an uncompetitive inhibitor in S13 and S16 soils and as a competitive inhibitor in S15. The competitive and uncompetitive inhibition constants (Kic and Kiu) were 0.058-0.142 mM and 0.075-0.503 mM. The ecological dose values of Sb to catalytic efficiency (Ka) of ARS (ED10-Ka) ranged from 50 to 1315 mg kg-1. Soil pH and total phosphorus (TP) contents were the dominant factors responsible for Sb toxicity on Ka by affecting the interaction of inhibitor (Sb) with enzyme-substrate (ES) complex. The findings of this study advance the current knowledge on Sb toxicity to soil enzymes and have significant implications for the risk assessment of Sb in soils.