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Using e(+)e(-) annihilation data corresponding to an integrated luminosity of 6.32 fb(-1) collected at center-of-mass energies between 4.178 and 4.226 GeV with the BESIII detector, we perform the first amplitude analysis of the decay D-s(+) -> (KSK+)-K-0 pi(0) and determine the relative branching fractions and phases for intermediate processes. We observe an a(0)-like state with mass of 1.817 GeV in its decay to (KSK+)-K-0 for the first time. In addition, we measure the ratio {B[D-s(+) -> (K*) over bar (892)K-0(+)]/B[D-s(+) -> (K-0) over bar K*(892)(+)]} to be 2.35(-0.23stat)(+0.42) +/- 0.10(syst). Finally, we provide a precision measurement of the absolute branching fraction B(D-s(+) -> (KSK+)-K-0 pi(0))=(1.46 +/- 0.06(stat) +/- 0.05(syst))%.

The main cause of trigeminal neuralgia (TN) is compression of a blood vessel at the root entry zone of the trigeminal nerve. However, a neurovascular conflict does not seem to be the only etiology and other mechanisms are implicated in the development of the disease. We hypothesized that TN patients may have distinct protein expression in the CSF. In this study, lumbar CSF from TN patients (n = 17), scheduled to undergo microvascular decompression, and from controls (n = 20) was analyzed and compared with in depth mass spectrometry TMTbased quantitative proteomics. We identified 2552 unique proteins, of which 46 were significantly altered (26 increased, and 20 decreased, q-value < .05) in TN patients compared with controls. An over-representation analysis showed proteins involved in high-density lipoprotein, such as Apolipoprotein A4, Apolipoprotein M, and Apolipoprotein A1, and the extracellular region, including proteins involved in the complement cascade to be over-represented. We conclude that TN patients have distinct protein expression in the CSF compared to controls. The pathophysiological background of the protein alterations found in this study warrants further investigation in future studies. Perspective: In this article, cerebrospinal fluid from patients with trigeminal neuralgia was analyzed using in depth shotgun proteomics, revealing 46 differentially expressed proteins compared to controls. Among these, apolipoproteins and proteins involved in the complement system were elevated and signif-icantly over-represented, implying an inflammatory component in the pathophysiology of the disease.

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Myalgic encephalitis/chronic fatigue syndrome (ME/CFS) is a severe systemic disorder characterized by neurological, gastrointestinal, inflammatory symptoms and fatigue. Disregulation in tryptophan (TRP) metabolism and excessive kynurenine pathway activation may cause these symptoms. Thus, this thesis investigates TRP in ME/CFS. TRP, a key amino acid, regulates nervous system, immune system, endocrine system, and energy metabolism. The main pathway of TRP metabolism is kynurenine, with a minor percentage shuttled towards serotonin biosynthesis, a brain-essential neurotransmitter. Kynurenine metabolism generates kynurenic acid (neuroprotective) and quinolinic acid (neurotoxic).

Our current knowledge of TRP metabolism in ME/CFS is insufficient. Few studies have quantified TRP in ME/CFS, and even fewer have employed high-resolution mass spectrometry, essential for accurate measurements and comprehensive metabolomics. Additionally, many studies disregarded factors like age and sex, which influence TRP metabolite levels. Lastly, preclinical research on the neuroprotective effects of KYN as a potential treatment is notably lacking

To address these research questions, we developed an accurate and comprehensive analytical method using liquid chromatography coupled with high-resolution mass spectrometry. This method quantifies TRP and its metabolites, along with the vitamins B2 and B6, essential for the enzymes in this pathway. Additionally, we measured the oxidative marker hypoxanthine and the amino acids tyrosine and phenylalanine, which compete with TRP to cross the blood-brain barrier, and limit its availability in the brain. We then employed the, TRP method and untargeted metabolomics, to compare the metabolic profiles of ME/CFS patients with those of healthy individuals, considering age and sex. Moreover, the effects of the menstrual cycle on TRP levels were examined by correlating 11 steroids with TRP metabolites. Additionally, the tissue distribution of kynurenine was investigated following both acute and chronic administration in a preclinical model.

The untargeted study found alterations in the vitamin B3, arginine-proline, aspartate-asparagine, L-Adrenaline and S-Adenosyl-L-homocysteine pathways . While, the targeted approach revealed decreased levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in ME/CFS patients. In addition, hypoxanthine and phenylalanine was elevated in ME/CFS patients, suggesting hypoxia and altered amino acid metabolism. The study found strong relationships between TRP metabolites and steroids during the menstrual cycle, suggesting hormones affect this pathway. Preclinical findings showed that kynurenine administration resulted in region-specific effects, with a potential neuroprotective effect in the hippocampus. These studies open avenues for further exploration of TRP metabolism, particularly in relation to ME/CFS and the impact of steroid hormones on this metabolic pathway.

This study explores the metabolic differences between human and murine plasma in addition to differences between murine subcutaneous and visceral white adipose tissue. A quantitative and semi-quantitative targeted method was developed and validated for this purpose. The quantitative method includes tryptophan and its metabolites in addition to tyrosine, phenylalanine, taurine, B vitamins, neopterin, cystathionine and hypoxanthine. While the semi-quantitative method includes; 3-indoleacetic acid, 5-hydroxyindoleacetic acid, acetylcholine, asymmetric dimethylarginine, citrulline and methionine. Sample preparation was based on protein precipitation, while quantification was conducted using ultrahigh-performance liquid chromatography coupled to a quadrupole Orbitrap tandem mass spectrometer with electrospray ionization in the parallel reaction monitoring (PRM) mode. The low limit of quantification for all metabolites ranged from 1 to 200 ng mL^-1. Matrix effects and recoveries for stable isotope labelled internal standards were evaluated, with most having a coefficient of variation (CV) of less than 15%. Results showed that a majority of the analytes passed both the intra- and interday precision and accuracy criteria. The comparative analysis of human and murine plasma metabolites reveals species-specific variations within the tryptophan metabolic pathway. Notably, murine plasma generally exhibits elevated concentrations of most compounds in this pathway, with the exceptions of kynurenine and quinolinic acid. Moreover, the investigation uncovers noteworthy metabolic disparities between murine visceral and subcutaneous white adipose tissues, with the subcutaneous tissue demonstrating significantly higher concentrations of tryptophan, phenylalanine, tyrosine, and serotonin. The findings also show that even a semi-quantitative method can provide comparable results to quantitative methods from other studies and be effective for assessing metabolites in a complex sample. Overall, this study provides a robust platform to compare human and murine metabolism, providing a valuable insight to future investigations. A validated HRMS method for measuring tryptophan metabolites and related compounds has been developed, with simple sample preparation, successfully applied in human and murine plasma, as well as murine white adipose tissue.

Mechanical ventilation (MV), however brief results in the loss of diaphragm muscle mass and strength, termed ventilator induced diaphragm dysfunction (VIDD). VIDD increases dependence, complicates and prolongs weaning and significantly increases discharge mortality rate and health care costs worldwide. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway was recently identified as an important signalling pathway implicated in VIDD, upregulated in the diaphragm following MV and limb muslces during critical care. Regulation of STAT3 is imperritve to skeletal muscle mass and function, as STAT3 is required in proper muscle growth and regeneration, while chronic activation of STAT3 is implicated in muscle dysfunction. As JAK/STAT pathway inhibition can restrict the development of chronic muscle wasting conditons, this study aimed to explore the therapeutic potential of Ruxolitinib, an approved JAK1/2 inhibitor for myelofibrosis, for treatment of CIM. We hypothesised Ruxolitinib would reduce loss of muscle mass and function associated with VIDD. Here, rats were subjected to five days controlled MV (CMV) with and without daily Ruxolitinib gavage. Five-days CMV significantly reduced diaphragm muscle size and impaired specific force, which was associated with 2-fold upregulation of P-STAT3, disrupted mitochondrial structure and respiratory function. Expression of the motor protein myosin was not affected, however CMV may alter myosin function through deamidation post translational modification. Ruxolitinib increases five-day survival rate, restored P-STAT3 expression and preserved diaphragm muscle size and specific force. These functional improvements were associated with improved mitochondrial structure, augmented mitochondrial respiratory function and reversal or augmentation of myosin deamidations. These results provide evidence of the preclinical potential of repurposing Ruxolitinib for the treatment of VIDD.

Background Critical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a by-product of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood; however, loss of mechanical stimuli contributes to critical illness-associated muscle atrophy and weakness. Passive mechanical loading and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care. Methods Rats were subjected to 8 days of critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM. Results Following 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, myosin : actin ratio, and single muscle fibre maximum force normalized to cross-sectional area (CSA; specific force) were reduced by 40-50% (P < 0.0001). ES significantly reduced the loss of soleus muscle fibre CSA and myosin : actin ratio by approximately 30% (P < 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signalling in the soleus muscle following critical care, and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (P < 0.01). ES promoted phosphofructokinase and insulin signalling pathways to control levels (P < 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signalling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (P = 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately two-fold (P = 0.06). Reduction of muscle protein degradation rather than increased synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (P = 0.006) and MuRF1 (P = 0.08) by approximately 50%, downstream of AMPK-FoxO3. Conclusions ES maintained GLUT4 translocation through increased AMPK-TBC1D4 signalling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signalling, and reduced muscle protein degradation in CIM.

Aim: Mechanical ventilation (MV) results in diminished diaphragm size and strength, termed ventilator-induced diaphragm dysfunction (VIDD). VID increases dependence, prolongs weaning, and increases discharge mortality rates. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway is implicated in VIDD, upregulated following MV. JAK/STAT inhibition alleviates chronic muscle wasting conditions. This study aimed to explore the therapeutic potential of Ruxolitinib, an FDA approved JAK1/2 inhibitor (JI) for the treatment of VIDD. Methods: Rats were subjected to 5 days controlled MV (CMV) with and without daily Ruxolitinib gavage. Muscle fiber size and function were assessed. RNAseq, mitochondrial morphology, respirometry, and mass spectrometry were determined. Results: CMV significantly reduced diaphragm size and specific force by 45% (p < 0.01), associated with a two-fold P-STAT3 upregulation (p < 0.001). CMV disrupted mitochondrial content and reduced the oxygen consumption rate (p < 0.01). Expression of the motor protein myosin was unaffected, however CMV alters myosin function via post-translational modifications (PTMs). Daily administration of JI increased animal survival (40% vs. 87%; p < 0.05), restricted P-STAT3 (p < 0.001), and preserved diaphragm size and specific force. JI was associated with preserved mitochondrial content and respiratory function (p < 0.01), and the reversal or augmentation of myosin deamidation PTMs of the rod and head region. Conclusion: JI preserved diaphragm function, leading to increased survival in an experimental model of VIDD. Functional enhancement was associated with maintenance of mitochondrial content and respiration and the reversal of ventilator-induced PTMs of myosin. These results demonstrate the potential of repurposing Ruxolitinib for treatment of VIDD.

The effect of encapsulating oils on the phase behaviour and the microstructure of wormlike micelles formed by polyoxyethylene cholesteryl ether (ChEO10) and triethylene glycol monododecyl ether co-surfactant (C12EO3) was investigated using rheology, Cryo-TEM and small-angle neutron scattering measurements. Six alkyl ester oils bearing small, systematic variations in their molecular structure were encapsulated: ethyl butyrate (EB24), ethyl caproate (ECO26), ethyl caprylate (EC28), methyl enanthate (ME17), methyl caprylate (MC18) and butyl butyrate (BB44), where the subscripts refer to the length of the alkyl chain and fatty acid chain, respectively, on either sides of the ester link. The addition of alkyl ester oils to ChEO10/C12EO3 solutions promotes the longitudinal growth of the surfactant aggregates into wormlike micelles possessing an elliptical cross-section, with rminor 31&#xa0;±&#xa0;2&#xa0;Å and rmajor varying from 45 to 70&#xa0;Å. At fixed alkyl chain length, oils with longer fatty acid chains were found to be more efficient in inducing wormlike micelle formation or their elongation, following the order: EC28&#xa0;&gt;&#xa0;ECO26&#xa0;&gt;&#xa0;EB24. Instead, at fixed fatty acid chain length, increasing the alkyl chain has a negative effect on the longitudinal micellar growth (MC18&#xa0;&gt;&#xa0;EC28 and EB24&#xa0;&gt;&#xa0;BB44). At high co-surfactant concentrations and in the presence of EB24, an unusual phase of ring-like micelles was detected. Overall, the orientation of the oil molecules within the micelles enables them to act as co-surfactants with a small head-group, decreasing the average cross-section area and promoting longitudinal growth of the micelles into worms.

The need for alternative cognitive enhancers has risen due to the fact that clinical trial results of the drugs currently approved for treating these disorders have not been satisfactory.

IRAP has become a possible drug target for treating cognitive impairment brought about by Alzheimer’s disease, head trauma or cerebral ischemia, among others. This came after the revelation that Angiotensin IV enhances memory and learning. Angiotensin IV, the endogenous ligand of IRAP has been structurally modified with the aim of producing potent IRAP inhibitors. However, the peptidic nature of these inhibitors restricts their use; they are not likely to cross the blood brain barrier.

Other strategies for generating IRAP inhibitors have been through structure-based design and receptor based virtual screening. These drug-like molecules have exhibited positive results in animal studies.

IRAP inhibitors have been identified via a HTS of 10500 low-molecular weight compounds to give the hit based on a spirooxindole dihydroquinazolinone scaffold, with an IC₅₀ value of 1.5 µM. In this project, some analogues to this hit compound have successfully been synthesized using a known method, whereas others have been synthesized after additional method development.

The application of the developed method was found to be limited, because poor yield was obtained when a compound with an electron withdrawing substituent on the aniline was synthesized. As a result of this, modification of this method may be required or new methods may have to be developed to synthesize these types of analogues.

Inhibition capability of 5 new spirooxindole dihydroquinazolinones was tested through a biochemical assay. Compound 6e emerged as the most potent inhibitor in the series, with an IC₅₀ value of 0.2 µM. This compound will now serve as a lead compound and should be used as a starting point for future optimization in order to generate more potent IRAP inhibitors.

Abstract Ubiquinone-10 is mostly known for its role as an electron and proton carrier in aerobic cellular respiration and its function as a powerful antioxidant. Accumulating evidence suggest, however, that this well studied membrane component could have several other important functions in living cells. The current study reports on a previously undocumented ability of ubiquinone-10 to modulate the mechanical strength and permeability of lipid membranes. Investigations of DPH fluorescence anisotropy, spontaneous and surfactant induced leakage of carboxyfluorescein, and interactions with hydrophobic and hydrophilic surfaces were used to probe the effects caused by inclusion of ubiquinone-10 in the membrane of phospholipid liposomes. The results show that ubiquinone in concentrations as low as 2&#xa0;mol.% increases the lipid packing order and condenses the membrane. The altered physicochemical properties result in a slower rate of release of hydrophilic components, and render the membrane more resistant towards rupture. As judged from comparative experiments using the polyisoprenoid alcohol solanesol, the quinone moiety is essential for the membrane stabilizing effects to occur. Our findings imply that the influence of ubiquinone-10 on the permeability and mechanical properties of phospholipid membranes is similar to that of cholesterol. The reported data indicate, however, that the molecular mechanisms are different in the two cases.

Biological membranes have developed to separate different compartments of organisms and cells. There is a large number of rather different functions which membranes have to fulfil: (1) they control the material and energy fluxes of metabolic processes, (2) they provide a wrapping protecting the compartments from chemical and physical attacks of the environment, (3) they provide interfaces at which specific biochemical machineries can operate (e.g., membrane bound enzymes), (4) they are equipped for signal transduction, (5) they possess the necessary stability and flexibility to allow cell division, and endo- and exocytosis as well as migration, (6) they present anchoring structures that enable cell-to-cell and cell-to-matrix physical interactions and intercellular communication. These are certainly not all functions of membranes as new functionalities are continuously reported. Since the biological membranes separate essentially aqueous solutions, such separating borders—if they should possess a reasonable stability and also flexibility combined with selective permeability—have to be built up of hydrophobic molecules exposing to both sides a similar interface. It was one of the most crucial and most lucky circumstances for the development and existence of life that certain amphiphilic molecules are able to assemble in bilayer structures (membranes), which—on one side—possess a rather high physical and chemical stability, and—on the other side—are able to incorporate foreign molecules for modifying both the physical properties as well as the permeability of the membranes for defined chemical species. The importance of the chemical function of membranes and all its constituents, e.g., ion channels, pore peptides, transport peptides, etc., is generally accepted. The fluid-mosaic model proposed by Singer and Nicolson [1] is still the basis to understand the biological, chemical, and physical properties of biological membranes. The importance of the purely mechanical properties of membranes came much later into the focus of research. The reasons are probably the dominance of biochemical thinking and biochemical models among biologists and medical researchers, as well as a certain lack of appropriate methods to probe mechanical properties of membranes. The last decades have changed that situation due to the development of techniques like the Atomic Force Microscopy, Fluorescence Microscopy, Micropipette Aspiration, Raman Microspectroscopy, advanced Calorimetry, etc. This chapter is aimed at elucidating how the properties of membranes can be investigated by studying the interaction of vesicles with a very hydrophobic surface, i.e., with the surface of a mercury electrode. This interaction is unique as it results in a complete disintegration of the bilayer membrane of the vesicles and the formation of an island of adsorbed lipid molecules, i.e., a monolayer island. This process can be followed by current-time measurements (chronoamperometry), which allow studying the complete disintegration process in all its details: the different steps of that disintegration can be resolved on the time scale and the activation parameters can be determined. Most interestingly, the kinetics of vesicle disintegration on mercury share important features with the process of vesicle fusion and, thus, sheds light also on mechanisms of endocytosis and exocytosis. Most importantly, not only artificial vesicles (liposomes) can be studied with this approach, but also reconstituted plasma membrane vesicles and even intact mitochondria. Hence, one can expect that the method may provide in future studies also information on the membrane properties of various other vesicles, including exosomes, and may allow investigating various aspects of drug action in relation to membrane properties (transmembrane transport, tissue targeting, bioavailability, etc.), and also the impact of pathophysiological conditions (e.g., oxidative modification) on membrane properties, on a hitherto not or only hardly accessible level.

Lipodisks, planar lipid bilayer structures stabilized by PEG-ylated lipids, were in the present study covalently bound and immobilized onto sensors for quartz crystal microbalance with dissipation monitoring (QCM-D) studies. It is shown that the modified sensors can be used to characterize the interaction of lipodisks with α-helical amphiphilic peptides with an accuracy similar to that obtained with well established fluorimetric approximations. The method presented has the great advantage that it can be used with peptides in their native form even if no fluorescent residues are present. The potential of the method is illustrated by determining the parameters describing the association of melittin, mastoparan X, and mastoparan with immobilized lipodisks. Both thermodynamic and kinetic analyses are possible. The presented method constitutes a useful tool for fundamental studies of peptide–membrane interactions and can also be applied to optimize the design of lipodisks, for example, for sustained release of antimicrobial peptides in therapeutic applications.

In this study we demonstrate the importance of proper data processing in adsorption isotherm estimations. This was done by investigating and reprocessing data from five cases on two closely related platforms: liquid chromatography (LC) and biosensors. The previously acquired adsorption data were reevaluated and reprocessed using a three-step numerical procedure: (i) preprocessing of adsorption data, (ii) adsorption data analysis and (iii) final rival model fit. For each case, we will discuss what we really measure and what additional information can be obtained by numerical processing of the data. These cases clearly demonstrate that numerical processing of LC and biosensor data can be used to gain deeper understanding of molecular interactions with adsorption media. This is important because adsorption data, especially from biosensors, is often processed using old and simplified methods.

This short review describes how the theory of electrochemical metal nucleation considering non-stationary effects due to the activation of latent nucleation sites has been successfully translated and applied to describe phenomena observed on lipid membranes. This rather unexpected connection is merely formal, but has resulted in a completely new approach in liposome research. It has been proposed that hydrophobic active sites spontaneously and constantly appear and disappear on lipid membranes. These sites control the affinity of liposomes for hydrophobic surfaces and determine the permeability of the lipid membrane to small hydrophilic molecules. Thus, the kinetic models for liposome adhesion on hydrophobic substrates and for the spontaneous leakage of liposomal content are identical to that of non-stationary nucleation mentioned above. Therefore, the broad scope of the available work on metal nucleation has facilitated the interpretation of the data obtained in liposome research. Future applications of the nucleation model in the realm of liposomes are also discussed.

Concerns regarding poor aqueous solubility, high toxicity and lack of specificity impede the translation of many hydrophobic anticancer agents into safe and effective anticancer drugs. The application of colloidal drug delivery systems, and in particular the use of lipid-based nanocarriers, has been identified as a promising means to overcome these issues. PEG-stabilized lipid nanodisks (lipodisks) have lately emerged as a novel type of biocompatible, nontoxic and adaptable drug nanocarrier. In this study we have explored the potential of lipodisks as a platform for formulation and tumour targeted delivery of hydrophobic anticancer agents. Using curcumin as a model compound, we show that lipodisks can be loaded with substantial amounts of hydrophobic drugs (curcumin/lipid molar ratio 0.15). We demonstrate moreover that by deliberate choice of preparation protocols the lipodisks can be provided with relevant amounts of targeting proteins, such as epidermal growth factor (EGF). Data from in vitro cell studies verify that such EGF-decorated curcumin-loaded lipodisks are capable of EGF-receptor specific targeting of human A-431 tumour cells, and strongly suggest that the interaction between the lipodisks and the tumour cells results in receptor-mediated internalization of the disks and their cargo.

Issues concerning non-specificity, degradation and hemolysis severely hamper the development of membranolytic amphiphilic peptides into safe and efficient anticancer agents. To increase the therapeutic potential, we have previously developed a strategy based on formulation of the peptides in biocompatible nanosized lipodisks. Studies using melittin as model peptide show that the proteolytic degradation and hemolytic effect of the peptide are substantially reduced upon loading in lipodisks. Here, we explored the possibilities to increase the specificity and boost the cytotoxicity of melittin to tumor cells by use of targeting lipodisk. We demonstrate that small (~20 nm) EGF-targeted lipodisks can be produced and loaded with substantial amounts of peptide (lipid/peptide molar ratio >7) by means of a simple and straightforward preparation protocol. In vitro cell studies confirm specific binding of the peptide-loaded disks to tumor cells and suggest that cellular internalization of the disks results in a significantly improved cell-killing effect.

Problem: Preterm birth (PTB) is a leading cause of infant mortality and morbidity. The pathogenesis of PTB is complex and involves many factors, including socioeconomy, inflammation and infection. Asymmetric dimethylarginine, ADMA and symmetric dimethylarginine, SDMA are involved in labor as inhibitors of nitric oxide, a known relaxant of the uterine smooth muscles. Arginines are scarcely studied in relation to PTB and we aimed to investigate arginines (ADMA, SDMA and L-arginine) in women with spontaneous PTB and term birth.

Methods of the Study: The study was based on data from the population-based, prospective cohort BASIC study conducted in Uppsala County, Sweden, between September 2009 and November 2018. Arginines were analyzed by Ultra-High Performance Liquid Chromatography using plasma samples taken at the onset of labor from women with spontaneous PTB (n = 34) and term birth (n = 45). We also analyzed the inflammation markers CRP, TNF-R1 and TNF-R2 and GDF-15.

Results: Women with spontaneous PTB had higher plasma levels of ADMA (p < 0.001), and L-Arginine (p = 0.03). In addition, inflammation marker, TNF-R1 (p = 0.01) was higher in spontaneous PTB compared to term birth. Further, in spontaneous PTB, no significant correlations could be observed when comparing levels of arginines with inflammation markers, except ADMA versus CRP.

Conclusions: These findings provide novel evidence for the potential involvement of arginines in the pathogenesis of spontaneous PTB and it seems that arginine levels at labor vary independently of several inflammatory markers. Further research is warranted to investigate the potential of arginines as therapeutic targets in the prevention and management of spontaneous PTB.

PROBLEM: The exact biochemical mechanisms that initiate labor are not yet fully understood. Nitric oxide is a potent relaxant of uterine smooth muscles until labor starts, and its precursor is L-arginine. Asymmetric (ADMA) and symmetric (SDMA) dimethylarginines, are potent NO-inhibitors. However, arginines (dimethylarginines and L-arginine) are scarcely studied in relation to labor and childbirth. We aimed to investigate arginines in women with spontaneous (SLVB) and induced (ILVB) term labor with vaginal birth and in women undergoing elective caesarean section (ECS).

METHOD OF STUDY: Women at gestational week 16-18 were recruited to the population-based prospective cohort study BASIC at the Uppsala University Hospital, Sweden. Plasma samples taken at start of labor were analyzed for arginines, from SLVB (n = 45), ILVB (n = 45), and ECS (n = 45), using Ultra-High Performance Liquid Chromatography. Between-group differences were assessed using Kruskal-Wallis and Mann-Whitney U-test.

RESULTS: Women with SLVB and ILVB had higher levels of ADMA (p < .0001), SDMA (p < .05) and lower L-arginines (p < .01), L-arginine/ADMA (p < .0001), and L-arginine/SDMA (p < .01, respectively <.001) compared to ECS. However, ILVB had higher ADMA (p < .0001) and lower L-arginine (p < .01), L-arginine/ADMA (p < .0001), and L-arginine/SDMA (p < .01) compared to SLVB. Results are adjusted for gestational length at birth and cervical dilatation at sampling.

CONCLUSION: Our novel findings of higher levels of dimethylarginines in term vaginal births compared to ECS give insights into the biochemical mechanisms of labor. These findings might also serve as a basis for further studies of arginines in complicated pregnancies and labor.

Single cell metabolomics is an emerging and rapidly developing field that complements developments in single cell analysis by genomics and proteomics. Major goals include mapping and quantifying the metabolome in sufficient detail to provide useful information about cellular function in highly heterogeneous systems such as tissue, ultimately with spatial resolution at the individual cell level. The chemical diversity and dynamic range of metabolites poses particular challenges for detection, identification and quantification. In this review we discuss both significant technical issues of measurement and interpretation, and progress toward addressing them, with recent examples from diverse biological systems. We provide a framework for further directions aimed at improving workflow and robustness so that such analyses may become commonly applied, especially in combination with metabolic imaging and single cell transcriptomics and proteomics.

Different extracts of viable therapeutic Saccharomyces boulardii cells were evaluated for their antimicrobial activities against Escherichia coli and Candida albicans. Water, methanol, isopropanol, n-butanol and ethanol were used as solvents for extraction. Ethanol-extract exhibited the highest antimicrobial activity towards both strains, followed by water-extract. No antimicrobial activity could be detected on testing methanol-extract towards both strains. Ethanol- and water-extracts, cells remaining after water and ethanol extraction and broth were also tested for their antimicrobial activities against Gram-positive, Gram-negative, non-filamentous and filamentous fungi and showed considerable amounts of antimicrobial activities. Ethanol extracts exhibited the highest antimicrobial activity against all the tested strains, was then fractionated on a Sephadex G-100 column and the obtained fractions were examined using the agar-well diffusion method against Staphylococcus aureus, E.coli, C. albicans and Aspergillus niger. Results obtained indicate the presence of different scattered active fractions with different potencies against the four tested microorganisms. A large scale fermentation process was conducted using a BioFlo benchtop-15L Fermentor/ Bioreactor and the products were evaluated for their antimicrobial activities.

The kynurenine pathway (KP) is the main path for tryptophan metabolism, and it represents a multitude of potential sites for drug discovery in neuroscience, including pain, stroke, and epilepsy. L-kynurenine (LKYN), the first active metabolite in the pathway, emerges to be a prodrug targeting glutamate receptors. The safety, tolerability, pharmacokinetics, and pharmacodynamics of LKYN in humans have not been previously investigated. In an open-label, single ascending dose study, six participants received an intravenous infusion of 50, 100, and 150 µg/kg LKYN and new six participants received an intravenous infusion of 0.3, 0.5, 1, and 5 mg/kg LKYN. To compare the pharmacological effects between species, we investigated in vivo the vascular effects of LKYN in rats. In humans, LKYN was safe and well-tolerated at all dose levels examined. After infusion, LKYN plasma concentration increased significantly over time 3.23 ± 1.12 µg/mL (after 50 µg/kg), 4.04 ± 1.1 µg/mL (after 100 µg/kg), and 5.25 ± 1.01 µg/mL (after 150 µg/kg) (p ≤ 0.001). We observed no vascular changes after infusion compared with baseline. In rats, LKYN had no effect on HR and MAP and caused no dilation of dural and pial arteries. This first-in-human study of LKYN showed that LKYN was safe and well-tolerated after intravenous infusion up to 5 mg/kg over 20 minutes. The lack of change in LKYN metabolites in plasma suggests a relatively slow metabolism of LKYN and no or little feed-back effect of LKYN on its synthesis. The therapeutic potential of LKYN in stroke and epilepsy should be explored in future studies in humans.

Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.

Vitamin D-3 is a pro-hormone, which is sequentially activated by 25- and 1 alpha-hydroxylation to form 25-hydroxyvitamin D-3 [25(OH)D-3] and 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(OH)2D(3)], respectively. Subsequent inactivation is performed by 24-hydroxylation. These reactions are carried out by a series of CYP450 enzymes. The 25-hydroxylation involves mainly CYP2R1 and CYP27A1, whereas 1 alpha-hydroxylation and 24-hydroxylation are catalyzed by CYP27B1 and CYP24A1, respectively, and are tightly regulated to maintain adequate levels of the active vitamin D hormone, 1 alpha,25(OH)(2)D-3. Altered circulating vitamin D levels, in particular 25(OH)D-3, have been linked to several disorders of the nervous system, e.g., schizophrenia and Parkinson disease. However, little is known about the mechanisms of vitamin D actions in the neurons. In this study, we examined vitamin D metabolism and its regulation in a murine motor neuron-like hybrid cell line, NSC-34. We found that these cells express mRNAs for the four major CYP450 enzymes involved in vitamin D activation and inactivation, and vitamin D receptor (VDR) that mediates vitamin D actions. We also found high levels of CYP24A1-dependent 24,25-dihydroxyvitamin D-3 [24,25(OH)(2)D-3] production, that was inhibited by the well-known CYP enzyme inhibitor ketoconazole and by several inhibitors that are more specific for CYP24A1. Furthermore, CYP24A1 mRNA levels in NSC-34 cells were up-regulated by 1 alpha,25(OH)(2)D-3 and its synthetic analogs, EB1089 and tacalcitol. Our results suggest that NSC-34 cells could be a novel model for the studies of neuronal vitamin D metabolism and its mechanism of actions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.

In this paper, an LC-MS/MS method for the simultaneous identification and quantification of cyanotoxins with hydrophilic and lipophilic properties in edible bivalves is presented. The method includes 17 cyanotoxins comprising 13 microcystins (MCs), nodularin (NOD), anatoxin-a (ATX-a), homoanatoxin (h-ATX) and cylindrospermopsin (CYN). A benefit to the presented method is the possibility for the MS detection of MC-LR-[Dha7] and MC-LR-[Asp3] as separately identified and MS-resolved MRM signals, two congeners which were earlier detected together. The performance of the method was evaluated by in-house validation using spiked mussel samples in the quantification range of 3.12-200 mu g/kg. The method was found to be linear over the full calibration range for all included cyanotoxins except CYN for which a quadratic regression was used. The method showed limitations for MC-LF (R-2 = 0.94), MC-LA (R-2 <= 0.98) and MC-LW (R-2 <= 0.98). The recoveries for ATX-a, h-ATX, CYN, NOD, MC-LF and MC-LW were lower than desired (<70%), but stable. Despite the given limitations, the validation results showed that the method was specific and robust for the investigated parameters. The results demonstrate the suitability of the method to be applied as a reliable monitoring tool for the presented group of cyanotoxins, as well as highlight the compromises that need to be included if multi-toxin methods are to be used for the analysis of cyanotoxins with a broader range of chemical properties. Furthermore, the method was used to analyze 13 samples of mussels (Mytilus edulis) and oysters (Magallana gigas) collected in the 2020-2022 summers along the coast of Bohuslan (Sweden). A complementary qualitative analysis for the presence of cyanotoxins in phytoplankton samples collected from marine waters around southern Sweden was performed with the method. Nodularin was identified in all samples and quantified in bivalve samples in the range of 7-397 <= g/kg. Toxins produced by cyanobacteria are not included in the European Union regulatory monitoring of bivalves; thus, the results presented in this study can be useful in providing the basis for future work including cyanotoxins within the frame of regulatory monitoring to increase seafood safety.

Introduction: Measuring testosterone concentrations is of interest both in clinical situations and for research, the latter expanding rapidly during recent years. An increased demand for convenient methods has prompted a number of companies to develop enzyme-linked immunosorbent assay (ELISA) kits to measure testosterone concentrations in saliva. However, the inter-comparability of kits from different manufacturers have yet to be determined. Aim of study: The aim of this study was to compare commercially available ELISA kits from four different manufacturers (Salimetrics, IBL, DRG and Demeditec). Methods: Saliva was collected from 50 participants (25 men and 25 women). Each sample was analysed by the four ELISA kits. Results: The correlations between the ELISA kits from Demeditec, DRG and Salimetrics were moderate to high with r-values >.77; however, proportional errors between the methods calls for caution. The ELISA kit from IBL malfunctioned and no results from this kit was obtained. Conclusions: Results from studies using the ELISA kits from Demeditec, DRG and Salimetrics are generally comparable; however, translation using the formulae presented in the current study could increase the accuracy of these comparisons.

Chromatography media (resins) are very important for and widely used by the biopharma industry in large scale production of biopharmaceuticals, e.g. monoclonal antibodies. Today there are several hundred biopharmaceuticals released globally on the healthcare market. This thesis discusses various strategies and methods for the characterisation of chemical and functional stability of chromatography media. In addition, various analytical techniques used in these areas were evaluated and applied. Further, more specific physical and chemical characterisation methods were evaluated and applied to explore different properties of various chromatography media.

In Papers I-III, established methodologies for performing chemical and functional stability studies were used. Mainly agarose-based chromatography media were investigated. For fast screening of the chemical stability, the total organic carbon analysis technique was evaluated and applied. This technique that measures the carbon leakage from the chromatography media at different conditions, proved to be very suitable and robust. For detection and/or identification of leakage compounds responsible for or for part of the measured carbon leakage, different methods such as (high performance) liquid chromatography and gas chromatography mass spectrometry were used.

In Papers IV-VII, different properties (i.e. functional performance, ligand content and surface chemistry) were evaluated for different agarose-based chromatography media. Standard chromatographic methods (ion exchange chromatography) and spectroscopic methods (e.g. Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry) were evaluated and applied. Chemometric methods were used for efficient evaluation of data.

Information of chemical, functional and leakage data of chromatography media are valuable and important for the biopharmaceutical companies to be able to fulfil the regulatory requirements of biopharmaceuticals. In addition, information of various chemical, functional and physical properties of chromatography media is likewise important during development and set up of new biopharmaceutical processes.

Paints mostly consist of three major components which are binder, pigment/filler andsolvent. Many other components are added in smaller amount and these are calledadditives. One of these additives is defoamers which are added to the paint todecrease foam which can cause defects in the dried paint for example as pores. Thisstudy was about investigating if the defoamers can be identified and quantified withhigh performance liquid chromatography coupled to mass spectrometry. This includessample preparation, chromatographic separation and detector settings. Calibrationcurves where constructed for paints containing different concentrations of defoamerand for a paint with 0% defoamer where different concentration of defoamer whereadded. Standard addition was done for a paint. Matrix effects were investigated bycomparing signal from defoamer in MeOH compared in paint. This study showed thatthe sample preparation of paints should involve dilution in MeOH or water followedby adding of formic acid and centrifugation and filtration to avoid problems in theinstrument. It is possible to identify if a defoamer is present in paint. Quantificationhas not been achieved, due to possible matrix effects and different response whendefoamer is added to the paint before analysis compared to when the defoamer isadded in the manufacturing process.

The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellularmembraneswhen overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE)was detected to be themost affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.

Protein modifications such as oxidations have a strong impact on protein function and activity in various organisms. High-resolution mass spectrometric techniques in combination with various sample preparation methodologies allow for the in-detail characterization of protein structures and strongly contribute to a greater understanding of the impact of protein modifications in nature. This paper outlines the general workflows for the characterization of oxidation sites in proteins by mass spectrometry (MS). Different types of oxidations are taken into consideration; both qualitative and quantitative aspects of MS-based approaches are presented with respect to oxidized proteins. Both bottom-up and top-down MS approaches are described and evaluated; a wide range of the particular applications corresponding to these techniques is also presented. Furthermore, the common advantages and downsides of these techniques are assessed. The approaches for enrichment of low-abundance oxidized proteins are extensively presented for different cysteine oxidations and protein carbonylations. A short description about databases and bioinformatic software solutions for oxidative protein prediction, identification, and biological interpretation is also given in this review.

UNLABELLED: Sophisticated strategies to analyze cell surface proteins are indispensable to study fundamental biological processes, such as the response of cells to environmental changes or cell-cell communication. Herein, we describe a refined mass spectrometry-based approach for the specific characterization and quantitation of cell surface proteins expressed in the female reproductive tract. The strategy is based on in situ biotinylation of rabbit oviducts, affinity enrichment of surface exposed biotin tagged proteins and dimethyl labeling of the obtained tryptic peptides followed by LC-MS/MS analysis. This approach proved to be sensitive enough to analyze small sample amounts (<1mug) and allowed further to trace the dynamic composition of the surface proteome of the oviductal epithelium in response to male gametes. The relative protein expression ratios of 175 proteins were quantified. Thirty-one of them were found to be altered over time, namely immediately, 1h and 2h after insemination compared to the time-matched control groups. Functional analysis demonstrated that structural reorganization of the oviductal epithelial cell surface was involved in the early response of the female organ to semen. In summary, this study outlines a workflow that is capable to monitor alterations in the female oviduct that are related to key reproductive processes in vivo. BIOLOGICAL SIGNIFICANCE: The proper interaction between the female reproductive tract, in particular, the oviduct and the male gametes, is fundamental to fertilization and embryonic development under physiological conditions. Thereby the oviductal epithelial cell surface proteins play an important role. Besides their direct interaction with male gametes, these molecules participate in signal transduction and, thus, are involved in the mandatory cellular response of the oviductal epithelium. In this study we present a refined LC-MS/MS based workflow that is capable to quantitatively analyze the expression of oviductal epithelial cell surface proteins in response to insemination in vivo. A special focus was on the very early interaction between the female organ and the male gametes. At first, this study clearly revealed an immediate response of the surface proteome to semen, which was modulated over time. The described methodology can be applied for studies of further distinct biological events in the oviduct and therefore contribute to a deeper insight into the formation of new life.

Intravenous use of a psychostimulant drug containing methcathinone (ephedrone) and manganese causes an irreversible extrapyramidal syndrome in drug abusers. We aimed to reproduce the syndrome in mice to evaluate dopaminergic damage. C57/B6 mice were intraperitoneally injected once a day with the study drug or saline for a period of 27 weeks. Motor activity was recorded in an automated motility-box. After 13 and 27 weeks of treatment, ex vivo digital autoradiography was performed using [C-11]dihydrotetrabenazine ([C-11]DTBZ). After 27 weeks of treatment [C-11]DTBZ autoradiography demonstrated a significant increase in the striatum to -cerebellum binding ratio compared with saline treated controls. At the same time point, there was no evident change in motor activity. Increased [C-11]DTBZ binding may indicate vesicular monoamine transporter type 2 (VMAT2) function is altered. The lack of extrapyramidal symptoms in animals could be attributed to low dosing regimen or high metabolic rate.

An intravenously injectable illicit drug made by mixing pseudoephedrine, potassium permanganate, vinegar and water, yielding methcathinone (Mcat) and manganese (Mn), induces an extrapyramidal syndrome with parkinsonism, dystonia, gait and balance disorders similar to manganism. Although the cause of the syndrome is largely attributed to Mn, the interaction of the drug's individual components is not known and the role of Mcat is possibly underestimated. Aim of the present study was to analyze dose-dependent behavioral effects of the mixture and its two main active components Mcat and Mn in an acute setting and determine the lethal doses of each substance. Three groups of C57BL/6 mice were injected intraperitoneally with (1) the drug mixture containing 10, 25, 50, 100 or 150 mg of Mcat and respectively 1.6, 3.8, 6.9, 17.1 and 22.6 mg of Mn per kilogram of body weight; (2) 10, 25, 50, 100, 150, 200 or 300 mg of racemic Mcat/kg of body weight; (3) MnCl2 10, 25 or 50 mg/kg of body weight. Locomotor activity of the animals, various signs and time of death were recorded. Lower doses (10 and 25 mg/kg) of Mcat had a clear motor activity stimulating effect and this was clearly dose-dependent. High doses of Mcat produced epileptic seizures in 74% of the animals and became lethal with the highest doses. Similarly, the mixture had a clear dose-dependent stimulating effect and the higher doses became lethal. The LD50 of the pseudoephedrine mixture was 110.2 mg of Mcat/kg and for pure Mcat 201.7 mg/kg. Mn did not prove to be lethal in doses up to 50 mg/kg, but had a strong dose dependent inhibitory effect on the animals' behavior. Our data reveal that both Mn and Mcat have a significant role in the toxicity of the mixture.

The degradation and transformation of organic carbon (C) in inland waters result in significant CO2 emissions from inland waters. Even though most of the C in inland waters occurs as dissolved organic carbon (DOC), studies on particulate organic carbon (POC) and how it influences the overall reactivity of organic C in transport are still scarce. We sampled 30 aquatic ecosystems following an aquatic continuum including peat surface waters, streams, rivers, and lakes. We report DOC and POC degradation rates, relate degradation patterns to environmental data across these systems, and present qualitative changes in dissolved organic matter and particulate organic matter during degradation. Microbial degradation rates of POC were approximately 15 times higher compared to degradation of DOC, with POC half-lives of only 17 +/- 3 (mean +/- SE) days across all sampled aquatic ecosystems. Rapid POC decay was accompanied by a shift in particulate C: N ratios, whereas dissolved organic matter composition did not change at the time scale of incubations. The faster degradation of the POC implies a constant replenishment to sustain natural POC concentrations. We suggest that degradation of organic matter transported through the inland water continuum might occur to a large extent via transition of DOC into more rapidly cycling POC in nature, for example, triggered by light. In this way, particles would be a dominant pool of organic C processing across the boreal aquatic continuum, partially sustained by replenishment via flocculation of DOC.

Halogenated dodecaborates, and especially dodecaiodododecaborate(2−), are found to trigger effectively the release of the contents of phospholipid liposomes, including liposomes containing distearoylphosphatidylcholine and cholesterol, which are used clinically in cancer therapy. The basis of the release is studied through differential scanning calorimetry, cryo-transmission electron microscopy, and atomic force microscopy. Upon administration at high concentrations, drastic morphological changes are induced by the dodecaborates. Their possible use in triggered release is suggested.

Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of reliability and delay, a small anti-EPO monolith membrane contained in a disposable column (anti-EPO monolith column) has been successfully used and validated (n = 10). This new sample preparation, combined with LC-FAIMS-MS/MS, has been performed on plasma and urine samples collected from one horse which received an Eprex[registered sign] treatment during six consecutive days and a second one with a single injection of Aranesp[registered sign]. This inventive technology allowed the possibility to confirm the presence of rHuEPO within one day with a limit of detection validated for both urine and plasma at 250 pg mL-1 by means of a disposable, ready to use immunoaffinity column. The lower limit of detection (LLOD) obtained for each matrix was 100 pg mL-1. These results provide an important improvement for rHuEPO doping control in horseracing especially the possibility to confirm these banned molecules in both matrices, urine and plasma, with a confidence of two specific target peptides.

Hybrid liposomes resulting from co-assembly of dipalmitoylphosphatidylcholine and polyglycidol-derivatized lipids were prepared. The latter were composed of a lipid-mimetic residue to which a linear polyglycidol chain (degree of polymerization, DP, in the 23–110 range) was conjugated. Formulations with varying copolymer type and content were prepared by film hydration technique followed by extrusion. The hybrid structures were studied by means of dynamic and electrophoretic light scattering, cryogenic transmission electron microscopy, and fluorescence spectroscopy. Cytotoxicity towards OPM-2 (multiple myeloma) and EJ (human urinary bladder carcinoma) cell lines was assessed as well. Predominantly unilamellar liposomes with mean hydrodynamic diameters in the 113–134 nm range and neutral to slightly negative surface potential were prepared. The integrity of liposomes containing copolymers with DP of the polyglycidol chain 23 and 30 was preserved at copolymer contents up to 10 mol%. Bilayer disks were observed at somewhat lower contents of the copolymers of the highest DP of the polyglycidol chain. The hybrid structures were less leaky than the plain liposomes, which was attributed to formation of a strongly hydrogen-bonded polyglycidol layer around the bilayer membrane. They exhibited low toxicological potential, favorable physicochemical characteristics, and ability to act as containers for sustained release.

Objective

To investigate plasma steroid hormone levels in postmenopausal breast cancer patients with and without adjuvant endocrine therapy and in healthy postmenopausal women.

Methods

Steroid hormone levels in postmenopausal breast cancer patients treated with aromatase inhibitors (n = 32) were compared with breast cancer patients treated with tamoxifen (n = 34), breast cancer patients without adjuvant endocrine therapy (n = 15), and healthy postmenopausal women (n = 56). Pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone (DHEA), androstenedione, total testosterone, dihydrotestosterone, estrone and estradiol were measured using liquid chromatography-tandem mass spectrometry. Sex hormone binding globulin was measured by solid-phase chemiluminescent immunometric assays, and the free androgen index was calculated.

Results

Aromatase inhibitor users did not differ in dihydrotestosterone, total testosterone, androstenedione, DHEA, or free androgen index levels from healthy controls or untreated breast cancer patients. The highest total testosterone levels were found in tamoxifen-treated women, who had significantly higher plasma concentrations than both women treated with aromatase inhibitors and breast cancer patients without adjuvant treatment. Concentrations of cortisol and cortisone were significantly greater in aromatase inhibitor users as well as tamoxifen users, in comparison with healthy controls and untreated breast cancer patients. Aromatase inhibitor users had lower estrone and estradiol plasma concentrations than all other groups.

Conclusion

Adjuvant treatment with aromatase inhibitors or tamoxifen was associated with increased cortisol and cortisone plasma concentrations as well as decreased estradiol concentrations. Androgen levels were elevated in tamoxifen-treated women but not in aromatase inhibitor users.

The biophysical analysis of the aggregates formed by different chemotypes of bacterial lipopolysaccharides (LPS) before and after challenge by two different anti-endotoxic antimicrobial peptides (LL37 and bovine lactoferricin), was performed in order to determine their effect on the morphology of LPS aggregates. Small-angle neutron scattering (SANS) and cryogenic transmission electron microscopy (cryoTEM) were used to examine the structures formed by both smooth and rough LPS chemotypes and the effect of the peptides, by visualization of the aggregates and analysis of the scattering data by means of both mathematical approximations and defined models. The data showed that the structure of LPS determines the morphology of the aggregates and inuences the binding activity of both peptides. The morphologies of the worm-like micellar aggregates formed by the smooth LPS were relatively unaltered by the presence of the peptides due to their pre-existing high degree of positive curvature being little affected by their association with either peptide. On the other hand the aggregates formed by the rough LPS chemotypes, showed marked morphological changes from lamellar structures to ordered micellar networks, induced by the increase in positive curvature engendered upon association with the peptides. The combined use of cryoTEM and SANS proved to be a very useful tool for studying the aggregation properties of LPS in solution at biologically relevant concentrations.

Mass Spectrometry Imaging (MSI) characterizes changes in chemical composition between regions of biological samples such as tissues. One goal of statistical analysis of MSI experiments is class comparison, i.e. determining analytes that change in abundance between conditions more systematically than as expected by random variation. To reach accurate and reproducible conclusions, statistical analysis must appropriately reflect the initial research question, the design of the MSI experiment, and all the associated sources of variation. This manuscript highlights the importance of following these general statistical principles. Using the example of two case studies with complex experimental designs, and with different strategies of data acquisition, we demonstrate the extent to which choices made at key points of this workflow impact the results, and provide suggestions for appropriate design and analysis of MSI experiments that aim at detecting differentially abundant analytes. (C) 2018 Elsevier B.V. All rights reserved.

Efficiency, emission, and uptime of vehicles in production and use are three factors that Scania CV AB focuses on to minimise the environmental impact brought about by the automotive industry. Optimising the use of engine oil is valued as a step in obtaining such improvements. This thesis focuses on learning more about how the sensor signals of a Fluid Property Sensor (FPS) detect soot and oxidation in engine oil samples, with comparison to a traditional laboratory analysis with Fourier Transform Infrared Spectroscopy (FTIR). A laboratory test rig was used to conduct measurements of engine oil samples of the grade 5W-20, using the FPS. FTIR was used to determine the oil sample’s soot concentration and oxidation level. Plotting the sensor signals against soot concentration and oxidation levels respectively, revealed that the soot concentration had a strong correlation with the sensor signals and that the oxidation level had a weaker correlation with the sensor signals. An analysis of variance (ANOVA) confirmed that the experiments were different from one another. Principal component analysis (PCA) was performed to study trends and relations between variables in the data and found strong relations between soot, oxidation, dielectric constant, and density. Linear Multivariate Regressions at different temperature intervals demonstrated that density is the sensor parameter with the strongest influence on both soot and oxidation. From Partial Least Squares Regression modelling of the data, it was concluded that the sensor signals had a greater influence on modelling oxidation; however, the soot regression had a better fit to the data points and would perform well on unseen data.

Objective: The progesterone receptor modulator (PRM) mifepristone holds the potential to be developed for regular contraception. However, long-term treatment can cause thickening of the endometrium and PRM-associated endometrial changes (PAEC). The objective of this study was to explore the molecular expression of endometrium displaying PAEC after mifepristone treatment in order to understand the future implications of PAEC and safety of long-term use. Study design: Endometrial biopsies were obtained from premenopausal women following 3 months of continuous mifepristone treatment. The biopsies were evaluated regarding occurrence of PAEC and followed up by a comparative analysis of gene expression in PAEC endometrium (n=7) with endometrium not displaying PAEC (n=4). Methods used included microarray analysis, Ingenuity Pathway Analysis (IPA) and real-time polymerase chain reaction. Results: Three genes relevant within endometrial function were up-regulated with PAEC: THY1 (p=.02), ADAM12 (p=.04) and TN-C (p=.04). The proliferation marker MKi67 was not altered (p=.31). None of the differentially regulated genes were involved in the endometrial cancer-signaling pathway (based on IPA knowledge database). Conclusion: The genes altered in endometrium displaying PAEC after 3 months of mifepristone exposure are mainly involved in the structural architecture of tissue. Implications: PAEC features may be explained by the altered genes and their networks affecting tissue architecture although not involved in endometrial cancer signaling pathways, and thus, treatment with mifepristone at this dosage does not show any adverse effect at endometrial level.

Despite our growing understanding of the global carbon cycle, scientific consensus on the drivers and mechanisms that control dissolved organic carbon (DOC) turnover in aquatic systems is lacking, hampered by the mismatch between research that approaches DOC reactivity from either intrinsic (inherent chemical properties) or extrinsic (environmental context) perspectives. Here we propose a conceptual view of DOC reactivity in which the combination of intrinsic and extrinsic factors controls turnover rates and determines which reactions will occur. We review three major types of reactions (biological, photochemical, and flocculation) from an intrinsic chemical perspective and further define the environmental features that modulate the expression of chemically inherent reactivity potential. Finally, we propose hypotheses of how extrinsic and intrinsic factors together shape patterns in DOC turnover across the land-to-ocean continuum, underscoring that there is no intrinsic DOC reactivity without environmental context. By acknowledging the intrinsic–extrinsic control duality, our framework intends to foster improved modeling of DOC reactivity and its impact on ecosystem services.