Do trees respond to pollution? A network study of the impact of pollution on spruce growth from Europe.

Tree rings have been reliably used as an environmental proxy over the past decades for environmental reconstructions, simulations and forecasting. In our study, we investigated whether tree-ring chronologies are impacted by pollution. We chose sites in the Krusné hory and the Krkonose Mountains in the Czech Republic which have a known history of pollution. We sampled Norway spruce (Picea abies [L.] Karst) in both ranges and compared their chronologies. We found no significant difference in the overall radial growth in the chronologies from both regions. However, we observed an increased heterogeneity in the growth of trees from the 1970s till the 1990s. Coherently, a severe reduction in tree growth from the late 1970s and a recovery towards the early 1990s was evident. We collected and analysed soil samples for pH and exchangeable element concentrations. All seven sampling sites' soils were strongly acidic (pHCaCl2 = 3.3 ± 0.4). The average soil base saturation at Krusné hory was higher than at Krkonose (39% versus 12%), likely due to more intensive liming. Further, we compared these chronologies to other sites in Europe. Analysing 89 sites, we found that most (9 out of 14) of the sites with significantly reduced radial tree growth were located within the former 'Black Triangle', an area which was subjected to heavy industrialisation and pollution from the 1960s to the 1990s. Atmospheric sulphur deposition was found to negatively affect radial tree-growth, while limited quantities of oxidised nitrogen appeared to have a positive effect. Our results are consistent with previous research, indicating that atmospheric SO2 pollution and subsequent acid fog and rime have led to a reduction in annual radial tree growth across the Black Triangle.

Determination of dabigatran and rivaroxaban by ultra-performance liquid chromatography-tandem mass spectrometry and coagulation assays after major orthopaedic surgery.

Major orthopaedic surgery is associated with an increased risk of venous thromboembolism. Direct oral anticoagulants (DOACs) are recommended as thromboprophylactic agents after orthopaedic surgery. Although routine monitoring of DOACs in general is not required, measuring DOAC concentration may be necessary in clinical settings. The effects of DOACs on routine coagulation assays in spiked material are studied extensively, however, few data are available on DOAC concentration in patients after major orthopaedic surgery. We measured trough and peak DOAC concentrations with UPLC-MS/MS and routine coagulation tests in a prospective study including 40 patients receiving thromboprophylactic treatment with dabigatran 220mg od and 40 patients receiving rivaroxaban 10mg od after major orthopaedic surgery. For rivaroxaban, the median trough concentration with UPLC-MS/MS was 17.1ng/mL and median peak concentration was 149ng/mL. The anti-Xa assay displayed a good correlation, but a positive bias in comparison to the reference method. Furthermore, trough levels were mostly below the LOD of the anti-Xa assay. For dabigatran, the median trough concentration with UPLC-MS/MS was 12.1ng/mL, and median peak level was 80.8ng/mL. A positive bias was found when results from coagulation assays were compared to UPLC-MS/MS data. However, the addition of glucuronidated metabolites to dabigatran concentration UPLC-MS/MS data generally resolved most of this bias. Age was found to have a significant influence on dabigatran pharmacokinetics, irrespective of kidney function, whereas no effect of age was found during rivaroxaban treatment. In both treatment groups, female subjects displayed faster pharmacokinetics in comparison to male subjects, although not reaching significance. We conclude that UPLC-MS/MS is the method of choice to measure trough concentrations of DOACs in patients after orthopaedic surgery. Current coagulation assays are not suited for this purpose. We found large heterogeneity in both peak and trough concentrations of DOACs, and showed that pharmacokinetics of novel oral anticoagulants may be influenced by age and gender. Whether patients with high or low trough concentrations are at increased risk for bleeding or thromboembolic events respectively remains to be established.

Cut-off values to rule out urinary tract infection should be gender-specific.

The diagnosis of urinary tract infection (UTI) by urine culture is an expensive and time-consuming procedure. Using a screening method, to identify negative samples, would improve the procedure and reduce costs. In this study, urine flow cytometry, of over 7000 urine samples, was assessed by retrospective analysis. With a cut-off value of >200bacteria/µl, we obtained a sensitivity of 93.0%, a specificity of 63.5%, and a negative predictive value (NPV) of 96.2%. As a result the culturing of 49% of all samples could be avoided. In addition, the data was retrospectively analyzed to determine if the introduction of gender-specific cut-off values could improve screening results. The obtained receiver operator curves are indeed significantly different when gender specific cut-offs were used. When a NPV of 95% is considered acceptable the unisex cut-off value of >200bacteria/µl can be used for women (NPV 94.9%), but the cut-off value for men could be raised to >400bacteria/µl without diminishing the NPV (NPV 95.0%).

Urine flow cytometry can rule out urinary tract infection, but cannot identify bacterial morphologies correctly.

The diagnosis of urinary tract infection (UTI) by urine culture is a time-consuming and costly procedure. Usage of a screening method, to identify negative samples, would therefore affect time-to-diagnosis and laboratory cost positively. Urine flow cytometers are able to identify particles in urine. Together with the introduction of a cut-off value, which determines if a urine sample is subsequently cultured or not, the number of cultures can be reduced, while maintaining a low level of false negatives and a high negative predictive value. Recently, Sysmex developed additional software for their urine flow cytometers. Besides measuring the number of bacteria present in urine, information is given on bacterial morphology, which may guide the physician in the choice of antibiotic. In this study, we evaluated this software update. The UF1000i classifies bacteria into two categories: 'rods' and 'cocci/mixed'. Compared to the actual morphology of the bacterial pathogen found, the 'rods' category scores reasonably well with 91% chance of classifying rod-shaped bacteria correctly. The 'cocci/mixed' category underperforms, with only 29% of spherical-shaped bacteria (cocci) classified as such. In its current version, the bacterial morphology software does not classify bacteria, according to their morphology, well enough to be of clinical use in this study population.

It takes acid, rather than ice, to freeze glucose.

Plasma glucose levels provide the cornerstone of diabetes evaluation. Unfortunately, glucose levels drop in vitro due to glycolysis. Guidelines provide suitable conditions which minimize glycolysis, such as immediate centrifugation or the use of ice/water slurry storage containers. For obvious practical reasons, most laboratories use blood collection tubes containing glycolysis inhibitors. We describe the effect of a variety of commonly used blood collection tubes on in vitro stability of glucose. Furthermore, we looked at the validity of the assumption that glycolytic activity is minimal when blood is kept in an ice/water slurry. Sodium fluoride alone does not reduce in vitro glycolysis in the first 120 minutes after phlebotomy. Addition of citrate almost completely prevented in vitro glycolysis, but showed a positive bias (0.2 mmol/l) compared to control. This is partly due to a small drop in glucose level in control blood, drawn according to the current guidelines. This drop occurs within 15 minutes, in which glycolysis has been described to be minimal and acceptable. NaF-EDTA-citrate based test tubes provide the best pre-analytical condition available. Furthermore, glucose levels are not stable in heparinized blood placed in an ice/water slurry. We strongly advise the use of NaF-EDTA-citrate based test tubes in diabetes research.

Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants.

BACKGROUND: Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. OBJECTIVE: To develop and validate a reference ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. METHODS: The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. RESULTS: The UPLC-MS/MS method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay. CONCLUSIONS: Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal.

Urine flow cytometry as a primary screening method to exclude urinary tract infections.

PURPOSE: To exclude urinary tract infections, culture is the gold standard method, although it is time consuming and costly. Current strategies using dipstick analysis are unsatisfactory as screening methods, because of inadequate sensitivity/specificity. Urine flow cytometry is an attractive alternative. To exclude urinary tract infections, a cutoff value to screen for negative cultures was determined. METHODS: 281 outpatients (51 % male) of a general population visiting the urology department were included. Urine samples were measured by flow cytometry and compared with culture results and dipstick analysis. ROC analysis was performed to evaluate the screening performance of flow cytometry and dipstick analysis compared to culture. RESULTS: 18 % of cultures were positive, defined as >10(4) colony forming units/mL. Bacterial count by flow cytometry alone provides the best sensitivity and specificity to exclude a urinary tract infection. A cutoff value of 60 bacteria/µL urine leads to a sensitivity of 100 % and a specificity of 60 %. Retrospectively, with a cutoff value of 60 bacteria/µL urine, 49 % of the cultures would have been redundant. 20 % of patients receiving antibiotics possibly had received those unnecessarily. The calculated percentage of false negatives was 0 % (95 % confidence interval 0-3.3 %). CONCLUSIONS: Urine flow cytometry is a reliable screening method to exclude urinary tract infections. With a cutoff value of 60 bacteria/µL urine, negative predictive value is 100 % and the calculated percentage of false negatives is 0 % (95 % confidence interval 0-3.3 %). Using flow cytometry as a screening method could lead to a reduction in cultures and antibiotics.

Linking mass spectrometric imaging and traditional peptidomics: a validation in the obese mouse model.

MALDI mass spectrometry imaging (MSI) is a promising technique in the field of molecular (immuno)histology but is confronted with the problematic large-scale identification of peptides from thin tissue sections. In this study we present a workflow that significantly increased the number of identified peptides in a given MALDI-MSI data set and we evaluated its power concerning relative peptide quantifications. Fourier transform mass spectrometry (FTMS) profiling on matrix-coated thin tissue sections allowed us to align spectra of different MS sources, matching identical peaks in the process, thus linking MSI data to tandem mass spectrometry (MS/MS) on one hand and semiquantitative liquid chromatography (LC)/MS data on the other. Bonanza clustering was applied in order to group MS/MS spectra of structurally related peptides, making it possible to infer the identity of MSI-detected compounds based on identified members within the same cluster, effectively increasing the number of identifications in a single MSI data set. Out of 136 detected peptides with MALDI-MSI, we were able to identify 46 peptides. For 31 of these, a LC/quadrupole time-of-flight (QTOF) counterpart was detected, and we observed similar obese (ob/ob) to wild-type (wt) peak intensity ratios for 18 peptides. This workflow significantly increased the number of identifications of peptide masses detected with MALDI-MSI and evaluated the power of this imaging method for relative quantification of peptide levels between experimental conditions.

Interaction between electrical stimulation, protein coating and matrix elasticity: a complex effect on muscle fibre maturation.

In skeletal muscle tissue engineering, it remains a challenge to produce mature, functional muscle tissue. Mimicking the in vivo niche in in vitro culture might overcome this problem. Niche components include, for example, extracellular matrix proteins, neighbouring cells, growth factors and physical factors such as the elasticity of the matrix. Previously, we showed the effects of matrix stiffness and protein coating on proliferation and differentiation of muscle progenitor cells in a two-dimensional (2D) situation. In the present study we have investigated the additional effect of electrical stimulation. More precisely, we investigated the effect of electrical stimulation on primary myoblast maturation when cultured on top of Matrigel™ - or laminin-coated substrates with varying elasticities. The effect of electrical stimulation on differentiation and maturation was found to be dependent on coating and stiffness. Although electrical stimulation enhanced myoblast maturation, the effect was mild. We therefore conclude that, with the current regimen, electrical stimulation is not essential to create functional, mature muscle tissue.

Essential environmental cues from the satellite cell niche: optimizing proliferation and differentiation.

The use of muscle progenitor cells (MPCs) for regenerative medicine has been severely compromised by their decreased proliferative and differentiative capacity after being cultured in vitro. We hypothesized the loss of pivotal niche factors to be the cause. Therefore, we investigated the proliferative and differentiative response of passage 0 murine MPCs to varying substrate elasticities and protein coatings and found that proliferation was influenced only by elasticity, whereas differentiation was influenced by both elasticity and protein coating. A stiffness of 21 kPa optimally increased the proliferation of MPCs. Regarding differentiation, we demonstrated that fusion of MPCs into myotubes takes place regardless of elasticity. However, ongoing maturation with cross-striations and contractions occurred only on elasticities higher than 3 kPa. Furthermore, maturation was fastest on poly-d-lysine and laminin coatings.