Patients with mucopolysaccharidosis IVA (MPS IVA) can present with systemic skeletal dysplasia, leading to a need for multiple orthopedic surgical procedures, and often become wheelchair bound in their teenage years. Studies on patients with MPS IVA treated by enzyme replacement therapy (ERT) showed a sharp reduction on urinary keratan sulfate, but only modest improvement based on a 6-minute walk test and no significant improvement on a 3-minute climb-up test and lung function test compared with the placebo group, at least in the short-term. Surgical remnants from ERT-treated patients did not show reduction of storage materials in chondrocytes. The impact of ERT on bone lesions in patients with MPS IVA remains limited. ERT seems to be enhanced in a mouse model of MPS IVA by a novel form of the enzyme tagged with a bone-targeting moiety. The tagged enzyme remained in the circulation much longer than untagged native enzyme and was delivered to and retained in bone. Three-month-old MPS IVA mice treated with 23 weekly infusions of tagged enzyme showed marked clearance of the storage materials in bone, bone marrow, and heart valves. When treatment was initiated at birth, reduction of storage materials in tissues was even greater. These findings indicate that specific targeting of the enzyme to bone at an early stage may improve efficacy of ERT for MPS IVA. Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21 (DE3) (erGALNS) and in the methylotrophic yeast Pichia pastoris (prGALNS) has been produced as an alternative to the conventional production in Chinese hamster ovary cells. Recombinant GALNS produced in microorganisms may help to reduce the high cost of ERT and the introduction of modifications to enhance targeting. Although only a limited number of patients with MPS IVA have been treated with hematopoietic stem cell transplantation (HSCT), beneficial effects have been reported. A wheelchair-bound patient with a severe form of MPS IVA was treated with HSCT at 15 years of age and followed up for 10 years. Radiographs showed that the figures of major and minor trochanter appeared. Loud snoring and apnea disappeared. In all, 1 year after bone marrow transplantation, bone mineral density at L2–L4 was increased from 0.372 g/cm2 to 0.548 g/cm2 and was maintained at a level of 0.48±0.054 for the following 9 years. Pulmonary vital capacity increased approximately 20% from a baseline of 1.08 L to around 1.31 L over the first 2 years and was maintained thereafter. Activity of daily living was improved similar to the normal control group. After bilateral osteotomies, a patient can walk over 400 m using hip–knee–ankle–foot orthoses. This long-term observation of a patient shows that this treatment can produce clinical improvements although bone deformity remained unchanged. In conclusion, ERT is a therapeutic option for MPS IVA patients, and there are some indications that HSCT may be an alternative to treat this disease. However, as neither seems to be a curative therapy, at least for the sk...
The acetone-methanol extractive distillation using water as an entrainer was simulated on Aspen Plus software package using rigorous methods. Calculation of the vapor-liquid equilibrium for the ternary system was done by the UNIQUAC model according to the experimental results obtained in a previous work. The effects of the solvent to feed molar ratio, reflux ratio, feed stage, feed solvent stage, and solvent feed temperature were studied to obtain the best design of the extractive distillation column with minimal energy requirements. The most appropriate configuration is 52 theoretical stages. The best binary mixture and entrainer feeding stages were obtained at 48 and 22 respectively with a solvent to feed molar and reflux ratio of 2.0 and 5.0, respectively. The simulation results show the effect of the main variables on the extractive distillation process.
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.
H3K14ac (acetylation of lysine 14 of histone H3) is one of the most important epigentic modifications. Aberrant changes in H3K14ac have been associated with various diseases, including cancers and neurological disorders. Tools that enable detection and quantification of H3K14ac levels in cell extracts and in situ are thus of critical importance to reveal its role in various biological processes. Current detection techniques of specific histone modifications, however, are constrained by tedious sample pretreatments, lack of quantitative accuracy, and reliance on high quality antibodies. To address this issue, we engineered recombinant sensors that are suitable for probing histone acetylation levels using various biological samples. The protein sensor contains recongition domain(s) with sequences derived from the bromodomain of human polybromo-1 (PB1), a natural H3K14ac reader domain. Various sensor designs were tested using nuclear extracts and live cells. The sensor containing dimeric repeats of bromodomain was found most effective in quantifying H3K14ac level in both in vitro and in situ assays. The sensor has a linear detection range of 0.5-50 nM when mixed with nuclear extracts. The sensor colocalizes with H3K14ac antibodies in situ when transfected into human embryonic kidney 293T (HEK293T) cells and is thus capable of providing spatial details of histone modification within the nucleus. Corrected nuclear fluorescence intensity was used to quantify the modification level in situ and found to correlate well with our in vitro assays. Our sensor offers a novel tool to characterize the histone modification level using nuclear extracts and probe histone modification change in live cells.
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