Sample preservation through heat stabilization of proteins: principles and examples.

Very long‐chain acyl‐CoA dehydrogenase deficiency (VLCADD) is a recessive disorder of fatty acid beta‐oxidation with variable phenotype. Patients may present during the neonatal period with lethal multi‐organ failure or during adulthood with a myopathic phenotype. VLCADD is included in the Swedish newborn screening (NBS) program since 2010. The study describes the phenotype and biochemical findings in relation to the genotype, enzyme activity, and screening data in a Swedish cohort of pediatric patients with VLCADD. A total of 22 patients (20 diagnosed via NBS between 2010 and 2019, two diagnosed pre NBS) were included. Parameters analyzed were enzyme activity (palmitoyl CoA oxidation rate); ACADVL genotype; NBS results including Collaborative Laboratory Integrated Reports (CLIR) score; biochemical findings; treatment; clinical outcome. A clinical severity score (CSS) was compiled using treatment interventions and clinical symptoms. A possible correlation between CSS and VLCAD residual enzyme activity and NBS CLIR score was analyzed. The most common ACADVL variant (c.848T>C) was identified in 24/44 alleles. Five novel variants were detected. Clinical manifestations varied from asymptomatic to severe. There was a correlation between CSS, residual enzyme activity, and CLIR scores. Most patients diagnosed via NBS had less severe disease compared to those clinically diagnosed. In conclusion, the identified correlation between the NBS CLIR score, residual enzyme activity, and clinical outcome suggests that information available neonatally may aid in treatment decisions.

Effect of Ultra High Pressure Homogenization on Alkaline Phosphatase and Lactoperoxidase Activity in Raw Skim Milk

<p dir="ltr">Mitochondrial beta-oxidation disorders are a group of recessive diseases characterized by fasting intolerance and symptoms ranging from intermittent myalgia and rhabdomyolysis to cardiomyopathy and severe hypoketotic hypoglycemia. The studies in this thesis have focused on some of the unresolved issues regarding the pathogenesis of these disorders and how to best care for patients: phenotype prediction (study I), mitochondrial function (study II) and fasting metabolism (study III and IV).</p><p dir="ltr">In study I, the phenotype, biochemical data and clinical outcome of 22 Swedish pediatric patients with VLCADD were related to newborn screening data and VLCAD residual enzyme activity. Patients had a wide phenotypic spectrum, varying from asymptomatic to severely affected cases. The identified correlation between newborn screening results, residual enzyme activity analysis, and clinical outcome could significantly aid in treatment decisions and disease severity prediction.</p><p dir="ltr">In study II, mitochondrial respiration and glycolysis in peripheral blood mononuclear cells (PBMCs) from 21 patients with VLCADD, MCADD or CUD were determined using an XFe flux analyzer. PMBCs were suitable for analysis within a 6-hour sampling window, and VLCADD and MCADD cells consistently showed lower mitochondrial respiration rates than CUD cells.</p><p dir="ltr">Study III investigated fasting metabolism during a 9-hour night fast in 12 children with VLCADD or MCADD using subcutaneous microdialysis. The VLCADD group showed an earlier fasting-induced initiation of lipolysis compared to the MCADD group, and all patients maintained euglycemia during the fast. Patients with VLCADD with lower residual enzyme activity had an earlier initiation of lipolysis than those with higher enzyme activity.</p><p dir="ltr">Study IV investigated the fasting and postprandial hormonal and incretin response after a 4-hour daytime fast and after the intake of a standardized meal in 15 children with LCHADD, VLCADD or MCADD compared to 12 age-matched healthy controls. All participants showed a normal glycemic response, and most fasting hormone fluctuations were similar to those observed in controls. Findings of possible increased risk for insulin resistance in patients with LCHADD and a blunted postprandial incretin response in patients compared to controls warrant further investigation.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Olsson, D.</b>, M. Barbaro, C. Haglind, M. Halldin, S. Lajic, S. Tucci, R. H. Zetterström and A. Nordenström (2022). Very long-chain acyl-CoA dehydrogenase deficiency in a Swedish cohort: Clinical symptoms, newborn screening, enzyme activity, and genetics. JIMD Rep 63(2): 181-190. <a href="https://doi.org/10.1111/cts.13133" target="_blank">https://doi.org/10.1111/cts.13133</a></p><p dir="ltr">II. Stenlid, R., D. Olsson, J. Cen, H. Manell, C. Haglind, A. I. Chowdhury, P. Bergsten, A. Nordenström and M. Halldin (2022). Altered mitochondrial metabolism in peripheral blood cells from patients with inborn errors of B-oxidation. Clin Transl Sci 15(1): 182-194. <a href="https://doi.org/10.1111/cts.13133" target="_blank">https://doi.org/10.1111/cts.13133</a></p><p dir="ltr">III. <b>Olsson, D.</b>, C. Haglind, M. Halldin, S. Lajic and A. Nordenström. Assessment of fasting metabolism with microdialysis indicates earlier lipolysis in children with VLCADD compared to MCADD. [Submitted]</p><p dir="ltr">IV. <b>Olsson, D.</b>, M. Halldin, C. Haglind, T. Gustafsson, S. Lajic and A. Nordenstrom. Hormonal responses to a short daytime fast in children with beta-oxidation disorders. [Manuscript]</p>

Microbial phytases are enzymes with biotechnological interest for the feed industry. In this article, the effect of spray-drying conditions on the stability and activity of extracellular phytase produced by R. microsporus var. microsporus biofilm is described. The phytase was spray-dried in the presence of starch, corn meal (>150 μm), soy bean meal (SB), corn meal (<150 μm) (CM), and maltodextrin as drying adjuvants. The residual enzyme activity after drying ranged from 10.7% to 60.4%, with SB and CM standing out as stabilizing agents. Water concentration and residual enzyme activity were determined in obtained powders as a function of the drying condition. When exposed to different pH values, the SB and CM products were stable, with residual activity above 50% in the pH range from 4.5 to 8.5 for 60 min. The use of CM as drying adjuvant promoted the best retention of enzymatic activity compared with SB. Spray drying of the R. microsporus var. microsporus phytase using different drying adjuvants showed interesting results, being quite feasible with regards their biotechnological applications, especially for poultry diets.

Carnitine-acylcarnitine translocase deficiency is a rare and life-threatening mitochondrial fatty acid beta-oxidation disorder. We describe a patient who, despite a severe clinical course and an extremely low carnitine-acylcarnitine translocase activity, is currently alive and in good health. We performed an extensive analysis of all previously published cases in order to evaluate the clinical features and prognostic factors. Reports on 21 patients with carnitine-acylcarnitine translocase deficiency were obtained. Only 5 out of the 21 patients survived early childhood. At least 20 siblings are reported to have died of sudden unexplained death in the neonatal period. Although phenotype and residual enzyme activity have been suggested to be related to outcome, we were not able to establish such a relationship. Phenotype and residual enzyme activity do not appear to be major prognostic factors. Vigorous work-up in order to reach an expedite diagnosis and prompt medical intervention during acute episodes, especially in the neonatal period, may prevent fatal complications.

ABSTRACTCongenital adrenal hyperplasia (CAH) is a genetic disorder characterized by impaired cortisol production and consequent elevated adrenocorticotropic hormone (ACTH): CAH patients often require lifelong hydrocortisone therapy. Disease severity reflects residual 21‐hydroxylase enzyme activity, crucial for cortisol synthesis. Accurate assessment of residual enzymatic activity is key to developing individualized dosing. This study aimed to estimate enzymatic activity using a previously developed healthy adult ACTH‐cortisol model and to evaluate the potential for individualized therapy. Leveraging ACTH (n = 62) and cortisol (n = 66) concentrations from 51 (20 pediatric, 31 adult) untreated CAH patients, and assuming maximal cortisol production (Emax) = 100% in healthy individuals, residual enzymatic activity was estimated as an Emax scaling factor. To assess proof‐of‐concept feasibility of individualized therapy, simulations of individual untreated 24‐h ACTH and cortisol profiles were performed, and for one patient hydrocortisone dosing regimens (15–25 mg/day in 3 doses, q4h or q6h) were compared to simulated untreated and healthy profiles. The original model failed to capture elevated ACTH in severe CAH and was refined to predict observed data across all patients. Using the refined model, estimated enzymatic activity was higher than in vitro values for adults, while children under 13 years old showed 31.6% of adult enzymatic activity. Shortening dosing intervals had a greater impact on reducing the patient's ACTH overexposure than increasing the daily dose. This model‐based approach captured in vivo endogenous cortisol production and enabled simulation‐based evaluation of individualized therapy in adults. In children, further validation of the ACTH‐cortisol dynamics model and enzymatic activity estimates is needed to evaluate individualized therapy.

ABSTRACTWe describe 14 patients with 12 novel missense mutations in ASPA, the gene causing Canavan disease (CD). We developed a method to study the effect of these 12 variants on the function of aspartoacylase—the hydrolysis of N‐acetyl‐l‐aspartic acid (NAA) to aspartate and acetate. The wild‐type ASPA open reading frame (ORF) and the ORFs containing each of the variants were transfected into HEK293 cells. Enzyme activity was determined by incubating cell lysates with NAA and measuring the released aspartic acid by LC–MS/MS. Clinical data were obtained for 11 patients by means of questionnaires. Four patients presented with a non‐typical clinical picture or with the milder form of CD, whereas seven presented with severe CD. The mutations found in the mild patients corresponded to the variants with the highest residual enzyme activities, suggesting that this assay can help evaluate unknown variants found in patients with atypical presentation. We have detected a correlation between clinical presentation, enzyme activity, and genotype for CD.

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.

Pompe disease is caused by the accumulation of glycogen in the lysosomes due to a deficiency of the lysosomal acid-α-glucosidase (GAA) enzyme. Depending on residual enzyme activity, the disease manifests two distinct phenotypes. In this study, we assess an enzymatic and genetic analysis of Hungarian patients with Pompe disease. Twenty-four patients diagnosed with Pompe disease were included. Enzyme activity of acid-α-glucosidase was measured by mass spectrometry. Sanger sequencing and an MLPA of the GAA gene were performed in all patients. Twenty (83.33%) patients were classified as having late-onset Pompe disease and four (16.66%) had infantile-onset Pompe disease. Fifteen different pathogenic GAA variants were detected. The most common finding was the c.-32-13 T > G splice site alteration. Comparing the α-glucosidase enzyme activity of homozygous cases to the compound heterozygous cases of the c.-32-13 T > G disease-causing variant, the mean GAA activity in homozygous cases was significantly higher. The lowest enzyme activity was found in cases where the c.-32-13 T > G variant was not present. The localization of the identified sequence variations in regions encoding the crucial protein domains of GAA correlates with severe effects on enzyme activity. A better understanding of the impact of pathogenic gene variations may help earlier initiation of enzyme replacement therapy (ERT) if subtle symptoms occur. Further information on the effect of GAA gene variation on the efficacy of treatment and the extent of immune response to ERT would be of importance for optimal disease management and designing effective treatment plans.

Predicting clinical phenotypes of metachromatic leukodystrophy based on the arylsulfatase A activity and the ARSA genotype? - Chances and challenges.

The residual enzyme activity in tissues of a 6-year-old boy with myoglobinuria and carnitine palmitoyltransferase (CPT) deficiency was studied with malonyl-CoA, a specific inhibitor of CPT-I in rat tissues. In this patient the enzyme deficiency was limited to the CPT fraction insensitive to malonyl-CoA, since the residual activity was an increased amount of CPT sensitive to the inhibitor. CPT sensitivity to malonyl-CoA was also assayed in human liver mitochondria, and inhibition was similar to that found in rat liver. Moreover, comparative data on human liver mitochondria and biopsy specimens showed that, after freeze-thawing and homogenization, CPT sensitivity to malonyl-CoA was decreased in both these preparations, indicating that studies of CPT inhibition by malonyl-CoA in homogenates of frozen tissues may be equated to those in homogenates of frozen mitochondria.

Photoperiod/thermo-sensitive genic male sterility (P/TGMS) is critical for rice two-line hybrid system. Previous studies showed that slow development of pollen is a general mechanism for sterility-to-fertility conversion of TGMS in Arabidopsis. However, whether this mechanism still exists in rice is unknown. Here, we identified a novel rice TGMS line, ostms16, which exhibits abnormal pollen exine under high temperature and fertility restoration under low temperature. In mutant, a single base mutation of OsTMS16, a fatty acyl-CoA reductase (FAR), reduced its enzyme activity, leading to defective pollen wall. Under high temperature, the mOsTMS16M549I couldn't provide sufficient protection for the microspores. Under low temperature, the enzyme activity of mOsTMS16M549I is closer to that of OsTMS16, so that the imperfect exine could still protect microspore development. These results indicated whether the residual enzyme activity in mutant could meet the requirement in different temperature is a determinant factor for fertility conversion of P/TGMS lines. Additionally, we previously found that res2, the mutant of a polygalacturonase for tetrad pectin wall degradation, restored multiple TGMS lines in Arabidopsis. In this study, we proved that the osres2 in rice restored the fertility of ostms16, indicating the slow development is also suitable for the fertility restoration in rice.

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A 2 by modification of carboxylate groups

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions

Functional characterization of novel variants found in patients with suspected Fabry disease