A novel method for the measurement of plasma metanephrines using online solid phase extraction-liquid chromatography tandem mass spectrometry

Background: Measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine is useful in the diagnosis of phaeochromocytomas, but many assays require a large volume of plasma due to poor assay sensitivity, and often require lengthy sample preparation. Our aim was to develop a method for measurement of plasma metanephrines using a small sample volume with minimal hands-on preparation. Methods: Samples were deproteinised using 10 K spin filters prior to online solid phase extraction using a Waters Acquity UPLC Online SPE Manager (Waters, Manchester, UK) coupled to a Waters Xevo TQ-S mass spectrometer (Waters, Manchester, UK). The assay was validated and results compared to a previously published method. Results: We achieved a limit of quantification of 37.5 pmol/L for metanephrine and 3-methoxytyramine and 75 pmol/L for normetanephrine using only 150 mL of sample. The assay was linear up to 30,000 pmol/L for all analytes and in a method comparison study results showed good agreement with a previously published LC-MS/MS assay. Conclusions: We have developed a simple method for measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine using only 150 mL of sample. There is minimal hands-on sample preparation required and the assay is suitable for routine use in a clinical laboratory

Local topography increasingly influences the mass balance of a retreating cirque glacier

Local topographically driven processes – such as wind drifting, avalanching, and shading – are known to alter the relationship between the mass balance of small cirque glaciers and regional climate. Yet partitioning such local effects from regional climate influence has proven difficult, creating uncertainty in the climate representativeness of some glaciers. We address this problem for Sperry Glacier in Glacier National Park, USA, using field-measured surface mass balance, geodetic constraints on mass balance, and regional climate data recorded at a network of meteorological and snow stations. Geodetically derived mass changes during 1950–1960, 1960–2005, and 2005–2014 document average mass change rates during each period at −0.22 ± 0.12, −0.18 ± 0.05, and −0.10 ± 0.03 m w.e. yr−1, respectively. A correlation of field-measured mass balance and regional climate variables closely (i.e., within 0.08 m w.e. yr−1) predicts the geodetically measured mass loss from 2005 to 2014. However, this correlation overestimates glacier mass balance for 1950–1960 by +1.20 ± 0.95 m w.e. yr−1. Our analysis suggests that local effects, not represented in regional climate variables, have become a more dominant driver of the net mass balance as the glacier lost 0.50 km2 and retreated further into its cirque

Assessing the seasonal evolution of snow depth spatial variability and scaling in complex mountain terrain

Dynamic natural processes govern snow distribution in mountainous environments throughout the world. Interactions between these different processes create spatially variable patterns of snow depth across a landscape. Variations in accumulation and redistribution occur at a variety of spatial scales, which are well established for moderate mountain terrain. However, spatial patterns of snow depth variability in steep, complex mountain terrain have not been fully explored due to insufficient spatial resolutions of snow depth measurement. Recent advances in uncrewed aerial systems (UASs) and structure from motion (SfM) photogrammetry provide an opportunity to map spatially continuous snow depths at high resolutions in these environments. Using UASs and SfM photogrammetry, we produced 11 snow depth maps at a steep couloir site in the Bridger Range of Montana, USA, during the 2019–2020 winter. We quantified the spatial scales of snow depth variability in this complex mountain terrain at a variety of resolutions over 2 orders of magnitude (0.02 to 20 m) and time steps (4 to 58 d) using variogram analysis in a high-performance computing environment. We found that spatial resolutions greater than 0.5 m do not capture the complete patterns of snow depth spatial variability within complex mountain terrain and that snow depths are autocorrelated within horizontal distances of 15 m at our study site. The results of this research have the potential to reduce uncertainty currently associated with snowpack and snow water resource analysis by documenting and quantifying snow depth variability and snowpack evolution on relatively inaccessible slopes in complex terrain at high spatial and temporal resolutions.</p

Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization

Prognostic impact of MGMT promoter methylation and MGMT and CD133 expression in colorectal adenocarcinoma

Background: New biomarkers are needed for the prognosis of advanced colorectal cancer, which remains incurable by conventional treatments. O6-methylguanine DNA methyltransferase (MGMT) methylation and protein expression have been related to colorectal cancer treatment failure and tumor progression. Moreover, the presence in these tumors of cancer stem cells, which are characterized by CD133 expression, has been associated with chemoresistance, radioresistance, metastasis, and local recurrence. The objective of this study was to determine the prognostic value of CD133 and MGMT and their possible interaction in colorectal cancer patients. Methods: MGMT and CD133 expression was analyzed by immunohistochemistry in 123 paraffin-embedded colorectal adenocarcinoma samples, obtaining the percentage staining and intensity. MGMT promoter methylation status was obtained by using bisulfite modification and methylation-specific PCR (MSP). These values were correlated with clinical data, including overall survival (OS), disease-free survival (DFS), tumor stage, and differentiation grade. Results: Low MGMT expression intensity was significantly correlated with shorter OS and was a prognostic factor independently of treatment and histopathological variables. High percentage of CD133 expression was significantly correlated with shorter DFS but was not an independent factor. Patients with low-intensity MGMT expression and ≥50% CD133 expression had the poorest DFS and OS outcomes. Conclusions: Our results support the hypothesis that MGMT expression may be an OS biomarker as useful as tumor stage or differentiation grade and that CD133 expression may be a predictive biomarker of DFS. Thus, MGMT and CD133 may both be useful for determining the prognosis of colorectal cancer patients and to identify those requiring more aggressive adjuvant therapies. Future studies will be necessary to determine its clinical utility.This study was supported by FEDER, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I + D + I), Instituto de Salud Carlos III (FIS) through Project no. PI11/01862 and by the Consejería de Salud de la Junta de Andalucía through Project no. PI-0338. The authors are grateful to the Biobank of the Andalusian Public Healthcare System (Granada, Spain) for invaluable assistance

Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair

<p>Abstract</p> <p>Background</p> <p>Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.</p> <p>Methods</p> <p>A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured <it>in vitro </it>and <it>in vivo </it>in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific <it>in situ </it>hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.</p> <p>Results</p> <p>The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. <it>In vitro </it>and <it>in vivo </it>(subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels <it>in vitro </it>and <it>in vivo</it>, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the <it>in vitro </it>cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific <it>in situ </it>hybridization.</p> <p>Conclusions</p> <p>The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.</p

Metabolomics, machine learning and immunohistochemistry to predict succinate dehydrogenase mutational status in phaeochromocytomas and paragangliomas

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background inover one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and severalother tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlight-ing the importance of identifying SDHx mutations for patient management. Genetic variants of unknown signi-cance, where implications for the patient and family members are unclear, are a problem for interpretation. Forsuch cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB(SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatogra-phy–mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions providean alternative method. Here, we compare SDHB-IHC with metabolite proling in 189 tumours from 187 PPGLpatients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to estab-lish predictive models for interpreting metabolite data. Metabolite proling showed higher diagnostic specicitycompared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite proles improved predictive ability over that of the SFR, in particular forhard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, thecombination of metabolite proling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classied allbut one of the false negatives from metabolite proling strategies, while metabolite proling correctly classied allbut one of the false negatives/positives from SDHB-IHC. From 186 tumours with conrmed status of SDHx variantpathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benets ofboth strategies for patient management

G-protein signaling: back to the future

Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Gα·GDP/Gβγ heterotrimers to promote GDP release and GTP binding, resulting in liberation of Gα from Gβγ. Gα·GTP and Gβγ target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation — a cycle accelerated by ‘regulators of G-protein signaling’ (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) β is activated by Gαq and Gβγ, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Gα nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways