Search Behavior of Individual Foragers Involves Neurotransmitter Systems Characteristic for Social Scouting

In honey bees search behavior occurs as social and solitary behavior. In the context of foraging, searching for food sources is performed by behavioral specialized foragers, the scouts. When the scouts have found a new food source, they recruit other foragers (recruits). These recruits never search for a new food source on their own. However, when the food source is experimentally removed, they start searching for that food source. Our study provides a detailed description of this solitary search behavior and the variation of this behavior among individual foragers. Furthermore, mass spectrometric measurement showed that the initiation and performance of this solitary search behavior is associated with changes in glutamate, GABA, histamine, aspartate, and the catecholaminergic system in the optic lobes and central brain area. These findings strikingly correspond with the results of an earlier study that showed that scouts and recruits differ in the expression of glutamate and GABA receptors. Together, the results of both studies provide first clear support for the hypothesis that behavioral specialization in honey bees is based on adjusting modulatory systems involved in solitary behavior to increase the probability or frequency of that behavior.

Improving the Accuracy and Reliability of Parathyroid Hormone Levels Through the Mass-Spectrometric Measurement of Full-length PTH and C-Terminal PTH Fragments

Parathyroid hormone (PTH) is a key biomarker for hypo/hyperparathyrioidism as well as chronic kidney disease, one of the leading health conditions in the USA. As a result, the most recent update of the Kidney Disease Improving Global Outcomes (KDIGO) guideline for CKD-MBD management emphasized the role of PTH as one of the key biomarkers of this disorder. The earlier stages of CKD generate few symptoms and only until the kidney is significantly impaired do patients begin experiencing signs of renal failure. Therefore, the measurement of parathyroid hormone (PTH) in serum and/or plasma is critical not only for the correct detection, diagnosis, and prevention of renal failure, but also calcium, phosphate, and vitamin D disorders. Current laboratory methods for PTH show high variability and inaccuracy, thus creating the need for a reference measurement procedure that can help laboratories and assay manufacturers improve their measurement accuracy and reliability to avoid the misclassification of patients. Due to the short half-life of PTH, this 84 amino-acid polypeptide hormone is produced at low circulating levels in normal conditions ranging from 10 - 65 pg/mL. In addition, N-terminal and C-terminal peptides, which have shown to interfere with clinical analyzer platforms, account for over 80% of all circulating PTH levels. Therefore, a highly specific and sensitive method is needed for the accurate detection of full-length PTH and these PTH fragments. There is clinical relevancy in the ability to measure C-terminal PTH fragments as the ratio of these PTH fragments to full-length PTH has been diagnostic for severe or end-stage renal disease, non-dynamic bone disease, and hyperparathyroid-associated bone loss. Bottom-up proteomics approaches that incorporate enzymatic digestion steps during sample preparation will result in the loss of information for the fragments. Therefore, a new, innovative, top-down proteomics method was developed to measure full-length PTH and its breakdown products (fragments) by mass spectrometry (UHPLC-HRMS). This method enabled, for the first time, the detection of full-length PTH at very low concentrations typically observed in patients with hypoparathyroidism as well as C-terminal fragments that may interfere with regular immunoassays typically used in patient care. The highly specific and sensitive method for PTH and related peptides in CKD patient sera demonstrated no interference from the internal standards and other PTH fragments. Therefore, the optimized method was applied for the screening of normal and CKD serum at various stages of disease progression. Preliminary results demonstrated that certain PTH fragments are correlated with eGFR and different stages of chronic kidney diseases (CKD). This method along with the respective findings from this study will help to improve the diagnosis, treatment, and prevention of CKD-MBD.

Improvement of high resolution measurements of neodymium isotope compositions to reconstruct past ocean circulation

<p>Palaeoceanographic studies of ocean circulations are crucial for understanding the ocean´s impact on the Earth´s climate system. Circulation patterns and the provenance of water masses can be detected from temporal variations of the neodymium isotopic composition (εNd) of authigenic neodymium, preserved in deep sea sediment.</p><p>Inductively coupled plasma source mass spectrometry allows for the precise and accurate determination of εNd-values of samples and reference material.</p><p>Here, we reevaluate the mass spectrometric measurement protocol and instrument setting with respect to precision and accuracy defined by neodymium standards.</p><p>The shape of the ion beam plays a crucial role, which is manifested in the result that an optimal adjustment of the beam shaping quadrupoles can increase precision by a factor of 4.</p><p>In addition, the optimal standard neodymium concentration level is roughly 50 ppb yielding uncertainties of the mean of repeated measurements as low as 0.07 ε units whereas 5 times lower concentrations yield 10 times higher uncertainties.</p><p>The statistical nature of precision is further demonstrated through an uncertainty inversely proportional to the square root of N measurements. As a consequence, with an increase from 30 to 80 consecutive measurements precision was improved by a factor of 1.22.</p><p>Taking all evaluated aspects into account, precision and accuracy of standards and thus sediment samples can be strongly improved, hence contributing to a better comprehension of past ocean circulation, where neodymium isotope gradients are small.</p>

A new strategy implementing mass spectrometry in the diagnosis of congenital disorders of N-glycosylation (CDG)

ObjectivesCongenital disorders of N-glycosylation (CDG) are a large group of rare metabolic disorders caused by defects in the most common post-translational modification of proteins. CDGs are often difficult to diagnose as they are manifested with non-specific symptoms and signs. Analysis of serum transferrin (TRF) isoforms, as the classical procedure used to identify a CDG patient, enables to predict pathological steps in the N-linked glycosylation process.MethodsWe devised a new strategy based on liquid chromatography-mass spectrometry (LC-MS) for the analysis of TRF isoforms by combining a simple and fast sample preparation with a specific chromatographic cleanup/separation step followed by mass-spectrometric measurement. Single TRF isoform masses were obtained through reconstruction of multiply charged electrospray data collected by quadrupole-MS technology. Hereby, we report the first analyzed serum samples obtained from 20 CDG patients and 100 controls.ResultsThe ratio of desialylated isoforms to total TRF was calculated for patients and controls. CDG-Type I patients showed higher amounts of bi-sialo isoform (range: 6.7–29.6%) compared to controls (<5.5%, mean percentage 3.9%). CDG-Type II pattern showed an increased peak of tri-sialo isoforms. The mean percentage of tri-sialo-TRF was 9.3% (range: 2.9–12.9%) in controls, which was lower than that obtained from two patients with COG5-CDG and MAN1B1-CDG (18.5 and 24.5%). Intraday and between-day imprecisions were less than 9 and 16%, respectively, for bi-sialo- and less than 3 and 6% for tri-sialo-TRF.ConclusionsThis LC-MS-based approach provides a simple, sensitive and fast analytical tool for characterizing CDG disorders in a routine clinical biochemistry while improving diagnostic accuracy and speeding clinical decision-making.

Pharmacokinetics of Lusutrombopag, a Novel Thrombopoietin Receptor Agonist, in Rats by UPLC-MS/MS

Lusutrombopag is a second oral thrombopoietin (TPO) receptor agonist that selectively acts on human TPO receptors. In the study, UPLC-MS/MS was used to establish a selective and sensitive method to determine lusutrombopag with poziotinib as IS (internal standard) in rat plasma. Samples were prepared by precipitating protein with acetonitrile as a precipitant. Separation of lusutrombopag and poziotinib was performed on a CORTECS UPLC C18 column (2.1 ∗ 50 mm, 1.6 μm). The mobile phase (acetonitrile and water containing 0.1% formic acid) with gradient elution was set at a flow rate of 0.4 ml/min. The mass spectrometric measurement was conducted under positive ion mode using multiple reaction monitoring (MRM) of m/z 592.97 ⟶ 491.02 for lusutrombopag and m/z for poziotinib (IS) 492.06 ⟶ 354.55. The linear calibration curve of the concentration range was 2–2000 ng/ml for lusutrombopag, with a lower limit of quantification (LLOQ) of 2 ng/ml. RSD of interday and intraday precision were both no more than 9.66% with the accuracy ranging from 105.82% to 108.27%. The extraction recovery of lusutrombopag was between 82.15% and 90.34%. The developed and validated method was perfectly used in the pharmacokinetic study of lusutrombopag after oral administration in rats.

Geomedical application of copper isotope ratios: change of δ65Cu in xenograft model of human cancers

&lt;p&gt;In geosciences, high precision isotope ratio determination provides essential information about processes in geological systems. Novel ambitions evolve closer to biological applications. Copper is an essential metal for human body taking part of several cellular processes (e.g. respiratory chain, enzyme function, iron metabolism, elimination of reactive oxygen species, cell signalling pathways etc). However, the disorder of copper homeostasis causes serious diseases like Wilson disease (Cu accumulation in liver caused by genetical disorder) and it could also promote tumour growth by supporting angiogenesis and metastasis formation [Denoyer et al., 2015]. Despite numerous experiments, focusing on copper concentration determination in different tumour tissues (e.g. breast, lung cancer, etc.) hoping to assist in tumour diagnosis, the results are not convincing enough. However, previous studies on hepatocellular cancer and oral squamous cell carcinoma showed that tumour tissue appears to be relatively enriched in &lt;sup&gt;65&lt;/sup&gt;Cu compared to normal tissue whereas the &amp;#948;&lt;sup&gt;65&lt;/sup&gt;Cu in blood of tumorous patient decreased according to data obtained from control population [Balter et al., 2015, Lobo et al., 2017]. Our main aim is to elaborate a method to understand better the change in &lt;sup&gt;63&lt;/sup&gt;Cu/&lt;sup&gt;65&lt;/sup&gt;Cu stable isotope ratio during tumour growth. In this approach, we present our first results on copper isotope ratio determination in a xenograft mouse model. Our model was established in SCID (severe combined immunodeficiency disease) mice by injecting human cancer cells (1x10&lt;sup&gt;7&lt;/sup&gt; cells) subcutaneously. After the tumour reached approximately 2-3 cm diameter, the tumour mass was cut it in small, equal pieces and transplanted further into 10 mice increasing the experimental set-up homogeneity. All the animals were sacrificed by cardiac puncture under deep terminal anaesthesia within four weeks. Tumour and organs were removed by ceramic knife then were frozen with liquid nitrogen and stored at -80&amp;#176;C. We measured the copper concentration and &amp;#948;&lt;sup&gt;65&lt;/sup&gt;Cu in the tumour tissue, blood, liver, kidney and brain. A clean laboratory ambience was chosen to perform the sample preparation processes decreasing the environmental contamination. Separation of copper from other biologically essential element (Na, Mg, Fe, Zn) interfering the copper isotope measurement is a serious condition of the preparation [Lauwens et al., 2017]. Effects of sodium (&lt;sup&gt;23&lt;/sup&gt;Na&lt;sup&gt;40&lt;/sup&gt;Ar&lt;sup&gt;+&lt;/sup&gt;) and magnesium (&lt;sup&gt;25&lt;/sup&gt;Mg&lt;sup&gt;40&lt;/sup&gt;Ar&lt;sup&gt;+&lt;/sup&gt;) on copper isotope ratio were solved by choosing not the peak center but the interference-free plateau. Our measurements have been carried out on a Thermo Neptune PLUS multicollector mass spectrometer equipped with 9 moveable Faraday detectors, 3 amplifiers with a resistance of 10&lt;sup&gt;13&lt;/sup&gt; Ohm, and 6 amplifiers with a resistance of 10&lt;sup&gt;11&lt;/sup&gt; Ohm, in wet plasma conditions. The mass spectrometric measurement of the copper isotope ratio is doped either with Ni or Ga reference material which have a well-known isotope ratios.&lt;/p&gt;&lt;p&gt;&amp;#160;&lt;/p&gt;&lt;p&gt;References:&lt;/p&gt;&lt;p&gt;Balter V. et al. PNAS 2015; 112: 982&amp;#8722;985.&lt;/p&gt;&lt;p&gt;Denoyer D. et al. Metallomics 2015; 7: 1459&amp;#8722;76.&lt;/p&gt;&lt;p&gt;Lauwens S. et al. J. Anal. At. Spectrom. 2017; 32: 597&amp;#8722;608.&lt;/p&gt;&lt;p&gt;Lobo L. et al. Talanta 2017; 165: 92&amp;#8722;97.&lt;/p&gt;

Determination of sulfur isotopic ratio by HR-ICP-MS method

&lt;p&gt;Sulfur is an important geochemical element that is part of many natural compounds. Sulfur takes part in most natural processes. In this case, a change in the valency of this element can occur. The change in sulfur valency is accompanied by isotope fractionation, which gives rise to noticeable shifts in the isotopic composition of this element. Thus, sulfur is one of the important geochemical indicators that can be used to reconstruct the redox conditions of various geological processes, including sedimentation processes in marine basins. For many decades, the traditional method of analyzing the isotopic composition of sulfur in sulfides remains the method involving the oxidation of sulfur to SO2 and the subsequent measurement of the 34S/32S ratio in gas using a mass spectrometer (Giesemann et al., 1994; Grassineau et al., 2001; Studley et al., 2002, etc.). However, with the advent of the inductively coupled plasma method of multi-collector mass spectrometry, new in isotope geochemistry, the corresponding methodological direction began to develop in which this method is used to analyze the sulfur isotopic composition (Krupp et al., 2004; You and Li, 2005; Clough et al ., 2006; Mason et al., 2006; Craddock et al., 2008 and others). The results presented in these works suggest that the MC-ICP-MS method can be used to solve certain problems of studying the natural variations in the sulfur isotopic composition. Therefore, we created optimized methodic for measuring isotope ratio of sulfur. The experimental work was carried out on the instrument base of the laboratory of SEC &quot;Geothermohronology&quot; of the Institute of Geology and Oil and Gas Technologies at Kazan Federal University (IGiNGT KFU, Kazan) and was aimed at creating a set of analytical procedures for chemical sample preparation and mass spectrometric analysis methods. The chemical preparation of pyrite samples included the stage of their acid decomposition using concentrated inorganic acids, which ensured the quantitative transfer of the sulfur of the sample into solution, as well as the stage of obtaining pure sulfur preparations based on the use of ion exchange chromatography. The optimal methodological scheme of mass spectrometric measurement of the 34S / 32S ratio was determined on a Neptune Plus multi-collector mass spectrometer. It takes into account the influence of factors unfavorable for analysis of the isotopic composition of sulfur, such as interference overlays of ions of oxygen, carbon, and nitrogen compounds, as well as the effect of instrumental mass discrimination. The work was supported by the Ministry of Science and High Education of the Russian Federation contract No. 14.Y26.31.0029 in the framework of the Resolution No.220 of the Government of the Russian Federation.&lt;/p&gt;

Functional genetic discovery of enzymes using full-scan mass spectrometry metabolomics

Our understanding of metabolic networks is incomplete, and new enzymatic activities await discovery in well-studied organisms. Mass spectrometric measurement of cellular metabolites reveals compounds inside cells that are unexplained by current maps of metabolic reactions, and existing computational models are unable to account for all activities observed within cells. Additional large-scale genetic and biochemical approaches are required to elucidate metabolic gene function. We have used full-scan mass spectrometry metabolomics of polar small molecules to examine deletion mutants of candidate enzymes in the model yeast Saccharomyces cerevisiae. We report the identification of 25 genes whose deletion results in focal metabolic changes consistent with loss of enzymatic activity and describe the informatic approaches used to enrich for candidate enzymes from uncharacterized open reading frames. Triumphs and pitfalls of metabolic phenotyping screens are discussed, including estimates of the frequency of uncharacterized eukaryotic genes that affect metabolism and key issues to consider when searching for new enzymatic functions in other organisms.