Characterization of Early Disease Status in Treatment-Naive Male Paediatric Patients with Fabry Disease Enrolled in a Randomized Clinical Trial

Trial Design

This analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.

Methods

Males aged 5–18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13–17 years), renal function, and glycolipid levels (plasma, urine).

Results

Plasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m² (range 90.4–161.0 mL/min/1.73 m²) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0–27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.

Conclusions

These data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.

Trial Registration

ClinicalTrials.gov NCT00701415     

Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2

Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.     

Molecular Cloning,Functional Characterization,and Evolutionary Analysis of Vitamin D Receptors Isolated from Basal Vertebrates

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC₅₀ values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D₃ acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D₃ throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.     

Modeling changes in probabilistic reinforcement learning during adolescence

In the real world, many relationships between events are uncertain and probabilistic. Uncertainty is also likely to be a more common feature of daily experience for youth because they have less experience to draw from than adults. Some studies suggest probabilistic learning may be inefficient in youths compared to adults, while others suggest it may be more efficient in youths in mid adolescence. Here we used a probabilistic reinforcement learning task to test how youth age 8-17 (N = 187) and adults age 18-30 (N = 110) learn about stable probabilistic contingencies. Performance increased with age through early-twenties, then stabilized. Using hierarchical Bayesian methods to fit computational reinforcement learning models, we show that all participants’ performance was better explained by models in which negative outcomes had minimal to no impact on learning. The performance increase over age was driven by 1) an increase in learning rate (i.e. decrease in integration time scale); 2) a decrease in noisy/exploratory choices. In mid-adolescence age 13-15, salivary testosterone and learning rate were positively related. We discuss our findings in the context of other studies and hypotheses about adolescent brain development.     

Autophagy and the integrated stress response

Autophagy is a tightly regulated pathway involving the lysosomal degradation of cytoplasmic organelles or cytosolic components. This pathway can be stimulated by multiple forms of cellular stress, including nutrient or growth factor deprivation, hypoxia, reactive oxygen species, DNA damage, protein aggregates, damaged organelles, or intracellular pathogens. Both specific, stimulus-dependent and more general, stimulus-independent signaling pathways are activated to coordinate different phases of autophagy. Autophagy can be integrated with other cellular stress responses through parallel stimulation of autophagy and other stress responses by specific stress stimuli, through dual regulation of autophagy and other stress responses by multifunctional stress signaling molecules, and/or through mutual control of autophagy and other stress responses. Thus, autophagy is a cell biological process that is a central component of the integrated stress response.     

Anaerobic digestion of harvested aquatic weeds: water hyacinth (Eichhornia crassipes), cabomba (Cabomba Caroliniana) and salvinia (Salvinia molesta)

The aim of this study was to explore the potential of three aquatic weeds, water hyacinth, cabomba, and salvinia, as substrates for anaerobic digestion. A set of four pilot-scale, batch digestions were undertaken to assess the yield and quality (% methane) of biogas from each plant species, and the rate of degradation. A set of 56 small-scale (100 mL) biological methane potential (BMP) tests were designed to test the repeatability of the digestions, and the impact of drying and nutrient addition.The results of the pilot-scale digestions show that both water hyacinth and cabomba are readily degradable, yielding 267 L biogas kg^?1 VS and 221 L biogas kg^?1 VS, respectively, with methane content of approximately 50%. There is evidence that the cabomba fed reactor leaked midway through the digestion therefore the biogas yield is potentially higher than measured in this case. Salvinia proved to be less readily degradable with a yield of 155 L biogas kg^?1 VS at a quality of 50% methane.The BMPs showed that the variability was low for water hyacinth and cabomba but high for salvinia. They also showed that the addition of nutrient solution and manure did not significantly increase the biogas yields and that drying was detrimental to the anaerobic degradability of all three substrates.Based on these results treatment of both water hyacinth and cabomba by anaerobic digestion can be recommended. Anaerobic digestion of Salvinia cannot be recommended due to the low biogas yields and high variability for this substrate.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Thymus-associated parathyroid hormone has two cellular origins with distinct endocrine and immunological functions

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2 ^−/− mutants. However, the developmental ontogeny and cellular identity of these “thymic” PTH–expressing cells is unknown. We found that the lethality of aparathyroid Gcm2 ^−/− mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2 ^−/− mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant “thymic PTH.”