To Örebro University

BACKGROUND: Procollagen III, aminoterminal peptide (PIIINP) is a degradation product of collagen type III-synthesis. Collagen type III is distributed in many tissues, and an increase in serum PIIINP could reflect an increase in collagen turnover and pro-fibrotic activity. In this study, on a population of younger, healthy adults, we examined whether serum PIIINP correlates with early markers of vascular health, to evaluate its potential as a biomarker for early screening of preclinical cardiovascular risk.

METHODS: PIIINP levels, pulse wave velocity (PWV) and Carotid-intima media thickness (cIMT) was measured in 834 healthy, non-smoking, individuals aged 18-26. In univariable and multivariable linear regression models, we examined the association between PIIINP and vascular measurements, PWV and cIMT with adjustment for serum lipids, liver enzymes and systolic blood pressure.

RESULTS: The average of PIIINP, PWV and cIMT measurements in this population, were low (7.1 and 7.3 µg/L, 5.5 and 5.2 m/s, and 0.50 and 0.49 mm for men and women, respectively). In univariable analyses, PIIINP correlated positively with cIMT (p = 0.0061) and negatively with PWV (p = 0.0069). In multivariable analyses, a statistically significant association remained between PIIINP and cIMT (p < 0.001), but not with PWV.

CONCLUSION: Serum PIIINP correlates with cIMT in a healthy population, indicating its potential as a biomarker of cardiovascular risk at a preclinical stage. PIIINP measurement being easier to perform and less examiner dependent than the more time consuming and cumbersome cIMT, are suggestive of its possible merits as an early screening tool for cardiovascular disease.

Background and aim: Evaluate the diagnostic performance of automated, quantitative bilirubin measurement, modified to extend its lower measurement ranges, in cerebrospinal fluid (CSF) using the Siemens analyzer Advia XPT. Results were compared with the gold standard spectrophotometry for diagnosis of subarachnoid haemorrhage (SAH).

Method: Eighty clinical samples were analyzed on an Advia XPT, and results were compared to spectrophotometric results using the Agilent Cary 100 bio system. Method performance at low concentrations were evaluated using diluted control material and patient plasma and CSF samples. ROC curve analysis determined a suitable cutoff.

Result: Evaluation of low-concentration performance, below 2 mu mol/L on Advia XPT, showed a measurement bias of-1.0 %, and a linear regression equation of y = 0.843x + 0.0351 (R2 of 0.975), describing the relationship between measured and expected concentrations of diluted samples. The coefficient of variation, (CV), was 2.92 % at 0.598 mu mol/L and 26.6 % at 0.161 mu mol/L. Using the outcome of the analysis on Agilent Cary 100 as reference, sensitivity was 100 % and specificity 96 %, employing a cutoff of 0.41 mu mol/L.

Conclusion: Quantitative measurement of bilirubin in CSF using the bilirubin oxidase method on the automated Advia XPT platform perform well, with the analysis of low concentrations of bilirubin displaying a high precision and a high concordance with the results of spectrophotometry. These preliminary findings are indicative of the merits of quantitative measurement, that warrants further study of its diagnostic potential as an alternative to the more cumbersome spectrophotometry for diagnosing SAH.

Oxidative stress plays a vital role for the adaptive responses to physical training. However, excessive oxidative stress can precipitate cellular damage, necessitating protective mechanisms to mitigate this effect. Glucosinolates, found predominantly in cruciferous vegetables, can be converted into isothiocyanates, known for their antioxidative properties. These compounds activate crucial antioxidant defence pathways and support mitochondrial function and protein integrity under oxidative stress, in both Nrf2-dependent and independent manners. We here administered glucosinolate-rich broccoli sprouts (GRS), in a randomized double-blinded cross-over fashion to 9 healthy subjects in combination with daily intense exercise training for 7 days. We found that exercise in combination with GRS significantly decreased the levels of carbonylated proteins in skeletal muscle and the release of myeloperoxidase into blood. Moreover, it lowered lactate accumulation during submaximal exercise, and attenuated the severe nocturnal hypoglycaemic episodes seen during the placebo condition. Furthermore, GRS in combination with exercise improved physical performance, which was unchanged in the placebo condition.

OBJECTIVE: The amino-terminal peptide of type III procollagen (PIIINP) is a byproduct of type III collagen synthesis that exhibits promise as a biomarker of fibrosis, specifically in monitoring hepatic fibrosis in methotrexate treated patients. The Advia Centaur^® PIIINP assay is developed for track-based automated laboratory systems and is suitable for large volume analysis. Reference intervals in children and younger adults have been published previously. Here we measured PIIINP to determine reference ranges, specifically including elderly patients, for whom such are currently lacking.

METHODS: Samples were collected from subjects ranging from 20 to 98 years of age. Blood donors and clinical samples from primary care patients were used for reference interval calculation. Samples were analysed using the Advia Centaur^® PIIINP assay. After exclusion of samples high in alanine transaminase (AST), aspartate transaminase (ALT), and C-reactive protein (CRP) 386 samples were used in the reference interval calculation.

RESULTS AND CONCLUSION: We determined the following reference interval for the Advia Centaur^® PIIINP assay: the lower limit of the reference interval (2.5% percentile with 95% CI) was 4.42 (4.20-4.65) µg/L and the upper limit of the reference interval (97.5% percentile 95% CI) 16.0 (15.04-17.02) µg/L.No significant differences in mean PIIINP concentrations were found between men and women. While differing mean PIIINP concentrations were seen among subjects in different age groups, the differences were small and partitioning of reference range was determined not to be necessary.

Endocytosis is a fundamental process underlying diverse eukaryotic physiology. The terminal stage of this process is membrane fusion between the perimeter membrane of a late endosome filled with intraluminal vesicles, or multivesicular body (MVB), and the lysosome membrane to facilitate catabolism of internalized biomaterials or surface polytopic proteins. To comprehensively understand the mechanisms underlying MVB-lysosome membrane fusion, we developed a quantitative, cell-free assay to study this SNARE-mediated event in molecular detail using Saccharomyces cerevisiae and its vacuolar lysosome, or vacuole, as models. This involves separately isolating organelles from two yeast strains each expressing a different complementary fusion probe targeted to the lumen of either MVBs or vacuoles. Isolated organelles are mixed in vitro under fusogenic conditions. Upon MVB-vacuole membrane fusion, luminal contents mix to facilitate probe interaction, reconstituting β-lactamase activity recorded by a colorimetric enzyme activity assay. This method accommodates a multitude of approaches (e.g., genetics, addition of purified protein reagents) to study this process in isolation, and in theory could be repurposed to study other SNARE-mediated fusion events within cells.

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion.

Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity.

Inorganic ions such as phosphate and sulfate are essential macronutrients required for a broad spectrum of cellular functions and their regulation. In a constantly fluctuating environment microorganisms have for their survival developed specific nutrient sensing and transport systems ensuring that the cellular nutrient needs are met. This chapter focuses on the S. cerevisiae plasma membrane localized transporters, of which some are strongly induced under conditions of nutrient scarcity and facilitate the active uptake of inorganic phosphate and sulfate. Recent advances in studying the properties of the high-affinity phosphate and sulfate transporters by means of site-directed mutagenesis have provided further insight into the molecular mechanisms contributing to substrate selectivity and transporter functionality of this important class of membrane transporters. © Springer International Publishing Switzerland 2016.

Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity P_i transceptor in Saccharomyces cerevisiae Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by P_i:H⁺ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of P_i A comparison of this conformation with the model for P_i release in PiPT revealed that Tyr¹⁷⁹ in Pho84 (Tyr¹⁵⁰ in PiPT) is not part of the P_i binding site. This difference may be due to a lack of detailed information on the P_i release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr¹⁷⁹, Asp¹⁷⁸, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr¹⁷⁹ then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr¹⁷⁹ in regulating P_i release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of P_i is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled P_i transport cycle of a transceptor.