Factors Influencing the Measurement of Lysosomal Enzymes Activity in Human Cerebrospinal Fluid

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson''s disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson''s disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson''s disease and other neurodegenerative disorders.     

Juvenile recruitment in loggerhead sea turtles linked to decadal changes in ocean circulation

Given the threats of climate change, understanding the relationship of climate with long‐term population dynamics is critical for wildlife conservation. Previous studies have linked decadal climate oscillations to indices of juvenile recruitment in loggerhead sea turtles (Caretta caretta), but without a clear understanding of mechanisms. Here, we explore the underlying processes that may explain these relationships. Using the eddy‐resolving Ocean General Circulation Model for the Earth Simulator, we generate hatch‐year trajectories for loggerhead turtles emanating from Japan over six decades (1950–2010). We find that the proximity of the high‐velocity Kuroshio Current to the primary nesting areas in southern Japan is remarkably stable and that hatchling dispersal to oceanic habitats itself does not vary on decadal timescales. However, we observe a shift in latitudes of trajectories, consistent with the Pacific Decadal Oscillation (PDO). In a negative PDO phase, the Kuroshio Extension Current (KEC) is strong and acts as a physical barrier to the northward transport of neonates. As a result, hatch‐year trajectories remain mostly below 35°N in the warm, unproductive region south of the Transition Zone Chlorophyll Front (TZCF). During a positive PDO phase, however, the KEC weakens facilitating the neonates to swim north of the TZCF into cooler and more productive waters. As a result, annual cohorts from negative PDO years may face a lack of resources, whereas cohorts from positive PDO years may find sufficient resources during their pivotal first year. These model outputs indicate that the ocean circulation dynamics, combined with navigational swimming behavior, may be a key factor in the observed decadal variability of sea turtle populations.     

Stable Single α-Helices Are Constant Force Springs in Proteins

Single α-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (<30 pN) without a characteristic unfolding peak. Molecular dynamics simulations showed that the SAH domain unfolds progressively as the length is increased and refolds progressively as the length is reduced. This enables the SAH domain to act as a constant force spring in the mechanically dynamic environment of the cell.     

High resolution melting (HRM) analysis for the detection of ER22/23EK, BclI, and N363S polymorphisms of the glucocorticoid receptor gene

Polymorphisms in the glucocorticoid receptor (GR) gene have been associated with altered sensitivity to glucocorticoids. We designed a high-resolution melting (HRM) assay to detect, simultaneously, the three most intriguing GR polymorphisms, selected on the bases of clinical relevance and frequencies in caucasian population as described in literature. HRM enables the detection of ER22/23EK and N363S genotypes but fails to discriminate homozygous mutant for the BclI polymorphism from wild-type samples, however a simple spike experiment leads to a clear discrimination between these genotypes. The analyses were performed on a cohort of 70 healthy Caucasian subjects. The method was validated by restriction fragment length polymorphisms; HRM results were found to be in 100% concordance with those observed with the restriction enzymes. We also employed this method on a population of 40 Crohn Disease patients; the analysis demonstrated a significantly higher frequency of the BclI polymorphism in patients than in healthy volunteers.This is, at now, the less expensive and time-and work-saving method to detect GR mutations, providing precision, fast screening and high throughput capabilities.     

An open-label trial of L-5-hydroxytryptophan in subjects with romantic stress

This open-label trial assessed the clinical efficacy of L-5-hydroxytryptophan (5-HTP), a natural serotonin precursor, in nondepressed young subjects with high levels of romantic stress. Since both neurotrophins and serotonin have been linked to human romantic attachment, we sought to investigate the changes in serum brain-derived neurotrophic factor (BDNF) levels and platelet serotonin content in relation to the changes in romantic stress throughout the study. A total of 15 healthy subjects (11 females and 4 males, mean age: 23.3 ± 2.1 years) who experienced a recent romantic break-up or reported recent romantic problems took part in the study. The participants were treated openly for 6 weeks with L-5-hydroxytryptophan (60 mg Griffonia simplicifolia extract containing 12.8 mg 5-HTP b.i.d., Amorex, Coropharm, Villach, Austria). The subjects were evaluated at baseline, at 3 weeks and at the end of the 6-week trial using an adapted version of the Seiffge-Krenke's Problem Questionnaire. BDNF and platelet serotonin content were determined at baseline, at 3 weeks, and after the completion of the 6-week trial. We observed significant improvements in romantic stress scores from weeks 0 through 3 (p=0.007) but no further significant improvement was evident from weeks 3 through 6 (p=0.19). At 6 weeks, subjects had a significant increase from baseline in both BDNF and platelet serotonin values. Our data suggest that direct modulation of the serotonergic system may have use for the treatment of psychological suffering associated with unreciprocated romantic love.     

On the catalytic role of the active site residue E121 of E. colil-aspartate oxidase

l-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD⁺. The enzyme oxidizes l-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor.     

Functional properties and evolutionary splicing constraints on a composite exonic regulatory element of splicing in CFTR exon 12

In general, splicing regulatory elements are defined as Enhancers or Silencers depending on their positive or negative effect upon exon inclusion. Often, these sequences are usually present separate from each other in exonic/intronic sequences. The Composite Exonic Splicing Regulatory Elements (CERES) represent an extreme physical overlap of enhancer/silencer activity. As a result, when CERES elements are mutated the consequences on the splicing process are difficult to predict. Here, we show that the functional activity of the CERES2 sequence in CFTR exon 12 is regulated by the binding, in very close proximity to each other, of several SR and hnRNP proteins. Moreover, our results show that practically the entire exon 12 sequence context participate in its definition. The consequences of this situation can be observed at the evolutionary level by comparing changes in conservation of different splicing elements in different species. In conclusion, our study highlights how it is increasingly difficult to define many exonic sequences by simply breaking them down in isolated enhancer/silencer or even neutral elements. The real picture is close to one of continuous competition between positive and negative factors where affinity for the target sequences and other dynamic factors decide the inclusion or exclusion of the exon.     

Synthesis of the Glc3Man N-glycan tetrasaccharide by iterative allyl IAD

The synthesis of the tetrasaccharide alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-alpha-D-Manp-OMe, corresponding to the terminal tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved with complete stereocontrol by the use of iterative allyl protecting group mediated intramolecular aglycon delivery (allyl IAD) demonstrating the utility of intramolecular glycosylation for the stereocontrolled construction of multiple glycosidic linkages during the synthesis of an oligosaccharide.