Towards optimizing the protocol for untargeted profiling of urine volatiles via gas chromatography-ion mobility spectrometry. A pilot study.

The analysis of volatile organic compounds (VOCs) present in various biological samples holds immense potential for non-invasive disease diagnostics and metabolic profiling. One of the biological fluids that are suitable for use in clinical practice is urine. Given the limited quantity of VOCs in the urine headspace, it's imperative to enhance their extraction into the gaseous phase and prevent any degradation of VOCs during the thawing process. The study aimed to test several key parameters (incubation time, temperature, and thawing) that can influence urine volatilome and monitor selected VOCs for their stability. The analysis in this study was performed using a BreathSpec® (G.A.S., Dortmund, Germany) device consisting of a gas chromatograph (GC) coupled with an ion mobility spectrometer (IMS). Testing three different temperatures and incubation times yielded a low number of VOCs (9 out of 34) that exhibited statistically significant differences. However, examining three thawing conditions revealed no VOCs with statistically significant changes. Thus, we conclude that urine composition remains relatively stable despite exposure to various thermal stresses.

Phagocytosis of latex beads by a human monocytic Mono Mac 6 cell line and effects of low-frequency electromagnetic field interaction.

Some studies have shown that electromagnetic fields (EMFs) may impact immune response cells and their functions. The first stage of the defense from pathogens is innate immunity encompassing phagocytosis and phagocytosis-related intracellular effects. Our work aimed to determine the influence of a low-frequency electromagnetic field (7 Hz, 30 mTrms) on the phagocytosis process of latex beads (LBs), the production of reactive oxygen species (ROS), and viability changes in a human monocytic Mono Mac 6 (MM6) cell line as an experimental model of the phagocytosing cells in in vitro cell culture conditions. For these purposes, cells were firstly activated with infectious agents such as lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB), or the proliferatory agent phytohaemagglutinin (PHA), and then a phagocytosis test was performed. Cell viability and range of phagocytosis of latex beads by MM6 cells were measured by flow cytometry, and the level of ROS was evaluated with the use of a cytochrome C reduction test. The obtained results revealed that applied EMF exposure mainly increased the necrosis parameter of cell death when they were pre-stimulated with SEB as an infectious factor and subsequently phagocytosed LBs (P=0.001). Prestimulation with other agents like LPS or PHA preceding phagocytosis resulted in no statistically significant changes in cell death parameters. The level of ROS depended on the used stimulatory agent, phagocytosis, and/or EMF exposure. The obtained effects for EMF exposure indicated only a slight decrease in the ROS level for cells phagocytosing latex beads and being treated with SEB or PHA, while the opposite effect was observed for LPS pre-stimulated cells (data not statistically significant). The results concerning the viability of phagocytosing cells, the effectiveness of the phagocytosis process, and the level of radical forms might result from applied EMF parameters like signal waveform, frequency, flux density, and especially single EMF exposure.

Per- and polyfluoroalkyl substances (PFAS) contamination in a microtidal urban estuary: Sources and sinks.

This study evaluates PFAS contamination and determines the major drainage sources to a temperate microtidal estuary, the Swan Canning Estuary, in Perth Western Australia. We describe how variability in these sources influences PFAS concentrations within this urban estuary. Surface water samples were collected from 20 estuary sites and 32 catchment sites in June and December from 2016 to 2018. Modelled catchment discharge was used to estimate PFAS load over the study period. Three major catchment sources of elevated PFAS were identified with contamination likely resulting from historical AFFF use on a commercial airport and defence base. Estuary PFAS concentration and composition varied significantly with season and spatially with the two different estuary arms responding differently to winter and summer conditions. This study has found that the influence of multiple PFAS sources on an estuary depend on the historical usage timeframe, groundwater interactions and surface water discharge.

Intrinsic cell rheology drives junction maturation.

A fundamental property of higher eukaryotes that underpins their evolutionary success is stable cell-cell cohesion. Yet, how intrinsic cell rheology and stiffness contributes to junction stabilization and maturation is poorly understood. We demonstrate that localized modulation of cell rheology governs the transition of a slack, undulated cell-cell contact (weak adhesion) to a mature, straight junction (optimal adhesion). Cell pairs confined on different geometries have heterogeneous elasticity maps and control their own intrinsic rheology co-ordinately. More compliant cell pairs grown on circles have slack contacts, while stiffer triangular cell pairs favour straight junctions with flanking contractile thin bundles. Counter-intuitively, straighter cell-cell contacts have reduced receptor density and less dynamic junctional actin, suggesting an unusual adaptive mechano-response to stabilize cell-cell adhesion. Our modelling informs that slack junctions arise from failure of circular cell pairs to increase their own intrinsic stiffness and resist the pressures from the neighbouring cell. The inability to form a straight junction can be reversed by increasing mechanical stress artificially on stiffer substrates. Our data inform on the minimal intrinsic rheology to generate a mature junction and provide a springboard towards understanding elements governing tissue-level mechanics.

Safety of Intranasal Insulin in Type 2 Diabetes on Systemic Insulin: A Double-Blinded Placebo-Controlled Sub-Study of Memaid Trial.

Aims: To determine safety of intranasal insulin (INI) in MemAID trial participants with diabetes treated with systemic insulins. Materials and Methods: This randomized, double-blinded trial consisted of 24-week INI or placebo treatment once daily and 24-week follow-up. Safety outcomes were: 1) Short-term effects on glycemic variability, hypoglycemic episodes on continuous glucose monitoring (CGM) at baseline and on-treatment. 2) Long-term effects on glucose metabolism and weight on INI/placebo treatment and post-treatment follow-up. Of 86 screened subjects, 14 were randomized, 9 (5 INI, 4 Placebo) completed CGM at baseline and on-treatment, and 5 (2 INI, 3 Placebo) completed treatment and follow-up. Results: INI was safe and was not associated with serious adverse events, hypoglycemic episodes or weight gain. INI administration did not acutely affect capillary glucose. Glycemic variability on CGM decreased with INI, compared to baseline. On INI treatment, there was a long-term trend toward lower HbA1c, plasma glucose and insulin. No interactions with subcutaneous insulins were observed. Conclusions: INI is safe in older people with diabetes treated with systemic insulins, and it is not associated with adverse events, hypoglycemia or weight gain. Future studies are needed to determine whether INI administration can reduce glycemic variability, improve insulin sensitivity and thus potentially lessen diabetes burden in this population.

Exchange interactions in ɛ-Fe2O3: GGA + U calculations.

We have studied the origin of magnetic interaction in ɛ-Fe2O3 by ab-initio electronic structure calculations. The exchange integrals of the Heisenberg Hamiltonian have been calculated using the methods based on the density functional theory (DFT) employing generalized gradient approximation (GGA) with orbital dependent potential extension for 3d electrons of Fe (GGA + U method). The calculations confirm the ground antiferromagnetic (AFM) state with two Fe3+ sublattices oriented up (Fe2 and Fe3) and two Fe3+ sublattices oriented down (Fe1 and Fe4). The calculated exchange integrals, including also the intra-sublattice ones, are all of AFM type. Their strength weighted by the number of neighbors is larger between the Fe sublattices with opposite spins than between the sublattices with equal spin directions. The notable exception is a strong exchange integral between the neighboring tetrahedrally-coordinated sites within the Fe4 sublattice, which effectively decreases the molecular field imposed on Fe4 sites by neighboring sites of other sublattices, namely the antiparallelly oriented Fe2 and Fe3. For this reason, the ordered magnetic moment of Fe4 exhibits the fastest decrease with increasing temperature among the sublattices, leading to an uncompensated AFM arrangement in ɛ-Fe2O3. Considering the competition of the inter- and intra-sublattice exchange integrals and applying symmetry arguments, we infer that the collinear AFM ground state of ɛ-Fe2O3 is prone to an intrinsic canting within the sublattices, retaining at the same time the magnetic group symmetry Pna'21'.

Low-frequency electromagnetic field influences human oral mucosa keratinocyte viability in response to lipopolysaccharide or minocycline treatment in cell culture conditions.

The aim of the current study was to investigate the influence of low-frequency electromagnetic field (LF-EMF) exposure on viability parameters of oral mucosa keratinocytes cultured in in vitro conditions. The effect of LF-EMF stimulation on cell viability was also specified in the simultaneous presence of lipopolysaccharide (LPS) infectious agent or minocycline (Mino) anti-inflammatory agent. Viability parameters such as early-, late apoptosis and necrosis of keratinocytes were analysed by the flow cytometry method (FCM). The exposure of human oral keratinocyte cell cultures to LF-EMF acting alone or combined with LPS/minocycline agents caused changes in the percentage of cells that undergo programmed or incidental cell death. The overall obtained results are compiled in a graphical form presented in Fig. 1.

Release of insulin granules by simultaneous, high-speed correlative SICM-FCM.

Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic ß-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.

Memory advancement by intranasal insulin in type 2 diabetes (MemAID) randomized controlled clinical trial: Design, methods and rationale.

BACKGROUND: Type 2 diabetes mellitus (T2DM) accelerates brain aging and increases the risk for dementia. Insulin is a key neurotrophic factor in the brain, where it modulates energy metabolism, neurovascular coupling, and regeneration. Impaired insulin-mediated brain signaling and central insulin resistance may contribute to cognitive and functional decline in T2DM. Intranasal insulin (INI) has emerged as a potential therapy for treating T2DM-related cognitive impairment. METHODS/DESIGN: Ongoing from 2015, a prospective, two-center, randomized, double-blind, placebo-controlled trial of 210 subjects (120 T2DM and 90 non-diabetic older adults) randomized into four treatment arms (60 T2DM-INI, 60 T2DM-Placebo, 45 Control-INI, and 45 Control-Placebo) evaluating the long-term effects of daily intranasal administration of 40 International Units (IU) of human insulin, as compared to placebo (sterile saline) over 24 weeks and 24 weeks of post-treatment follow-up. Study outcomes are: 1) long-term INI effects on cognition, daily functionality, and gait speed; 2) identifying a clinically relevant phenotype that predicts response to INI therapy; 3) long-term safety. CONCLUSION: This study addresses an important knowledge gap about the long-term effects of intranasal insulin on memory and cognition in older people with T2DM and non-diabetic controls, and may provide a novel therapeutic target for prevention and treatment of cognitive and functional decline and dementia. Trial Registration NCT02415556.

Proteomic Interactome of C. elegans Mediator Complex Subunit 28 (MDT-28) Reveals Predominant Association with a Restricted Set of Core Mediator Subunits and an Affinity to Additional Structural and Enzymatic Proteins.

Transcription factors exert their regulatory potential on RNA polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation by transcription factors. The Mediator complex consists of 25 subunits in Saccharomyces cerevisiae and 30 or more subunits in multicellular eukaryotes. Mediator subunit 28 (MED28), along with MED30, MED23, MED25 and MED26, belong to presumably evolutionarily new subunits that seem to be absent in unicellular eukaryotes and are likely to have evolved together with multicellularity and cell differentiation. Previously, we have shown that an originally uncharacterized predicted gene, F28F8.5, is the true MED28 orthologue in Caenorhabditis elegans (mdt-28) and showed that it is involved in a spectrum of developmental processes. Here, we studied the proteomic interactome of MDT-28 edited as GFP::MDT-28 using Crispr/Cas9 technology or MDT-28::GFP expressed from extrachromosomal arrays in transgenic C. elegans exploiting the GFPTRAP system and mass spectrometry. The results show that MDT-28 associates with the Head module subunits MDT-6, MDT-8, MDT-11, MDT-17, MDT- 20, MDT-22, and MDT-30 and the Middle module subunit MDT-14. The analyses also identified additional proteins as preferential MDT-28 interactants, including chromatin-organizing proteins, structural proteins and enzymes. The results provide evidence for MDT-28 engagement in the Mediator Head module and support the possibility of physical (direct or indirect) interaction of MDT-28 with additional proteins, reflecting the transcription-regulating potential of primarily structural and enzymatic proteins at the level of the Mediator complex.

AADvac1, an Active Immunotherapy for Alzheimer's Disease and Non Alzheimer Tauopathies: An Overview of Preclinical and Clinical Development.

Neurofibrillary tau protein pathology is closely associated with the progression and phenotype of cognitive decline in Alzheimer's disease and other tauopathies, and a high-priority target for disease-modifying therapies. Herein, we provide an overview of the development of AADvac1, an active immunotherapy against tau pathology, and tau epitopes that are potential targets for immunotherapy. The vaccine leads to the production of antibodies that target conformational epitopes in the microtubule-binding region of tau, with the aim to prevent tau aggregation and spreading of pathology, and promote tau clearance. The therapeutic potential of the vaccine was evaluated in transgenic rats and mice expressing truncated, non mutant tau protein, which faithfully replicate of human tau pathology. Treatment with AADvac1 resulted in reduction of neurofibrillary pathology and insoluble tau in their brains, and amelioration of their deleterious phenotype. The vaccine was highly immunogenic in humans, inducing production of IgG antibodies against the tau peptide in 29/30 treated elderly patients with mild-to-moderate Alzheimer's. These antibodies were able to recognise insoluble tau proteins in Alzheimer patients' brains. Treatment with AADvac1 proved to be remarkably safe, with injection site reactions being the only adverse event tied to treatment. AADvac1 is currently being investigated in a phase 2 study in Alzheimer's disease, and a phase 1 study in non-fluent primary progressive aphasia, a neurodegenerative disorder with a high tau pathology component.

Temperature modulates estrone degradation and biological effects of exposure in fathead minnows.

Environmental pollutants, including estrogens, are widespread in aquatic environments frequently as a result of treated wastewater effluent discharged. Exposure to estrogens has been correlated with disruption of the normal physiological and reproductive function in aquatic organisms, which could impair the sustainability of exposed populations. However, assessing the effects of estrogen exposure on individuals is complicated by the fact that rates of chemical uptake and environmental degradation are temperature dependent. Because annual temperature regimes often coincide with critical periods of biological activity, temperature-dependent changes in estrogen degradation efficacy during wastewater treatment could modulate biological effects. We examined the interactions between ambient water temperature and degradation of estrone (E1) during wastewater treatment. In addition, we exposed mature fathead minnows (Pimephales promelas) to three environmentally relevant concentrations of E1 at four different water temperatures (15°C, 18°C, 21°C, and 24°C) to reflect natural seasonal variation. E1 degradation occurred with and without the support of robust nitrification at all temperatures; however, the onset of E1 degradation was delayed at cooler water temperatures. In addition, we observed significant interactive effects between temperature and E1 exposure. Female morphometric endpoints were more susceptible to temperature-modulating effects while physiological endpoints were more strongly affected in males. Collectively, the data demonstrate that natural seasonal fluctuations in temperature are sufficient to affect E1 degradation during wastewater treatment and induce sex-dependent physiological and anatomical changes in exposed fish.

Chondrocyte expansion is associated with loss of primary cilia and disrupted hedgehog signalling.

Tissue engineering-based therapies targeting cartilage diseases, such as osteoarthritis, require in vitro expansion of articular chondrocytes. A major obstacle for these therapies is the dedifferentiation and loss of phenotype accompanying chondrocyte expansion. Recent studies suggest that manipulation of hedgehog signalling may be used to promote chondrocyte re-differentiation. Hedgehog signalling requires the primary cilium, a microtubule-based signalling compartment, the integrity of which is linked to the cytoskeleton. We tested the hypothesis that alterations in cilia expression occurred as consequence of chondrocyte dedifferentiation and influenced hedgehog responsiveness. In vitro chondrocyte expansion to passage 5 (P5) was associated with increased actin stress fibre formation, dedifferentiation and progressive loss of primary cilia, compared to primary (P0) cells. P5 chondrocytes exhibited ~50 % fewer cilia with a reduced mean length. Cilia loss was associated with disruption of ligand-induced hedgehog signalling, such that P5 chondrocytes did not significantly regulate the expression of hedgehog target genes (GLI1 and PTCH1). This phenomenon could be recapitulated by applying 24 h cyclic tensile strain, which reduced cilia prevalence and length in P0 cells. LiCl treatment rescued cilia loss in P5 cells, partially restoring hedgehog signalling, so that GLI1 expression was significantly increased by Indian hedgehog. This study demonstrated that monolayer expansion disrupted primary cilia structure and hedgehog signalling associated with chondrocyte dedifferentiation. This excluded the possibility to use hedgehog ligands to stimulate re-differentiation without first restoring cilia expression. Furthermore, primary cilia loss during chondrocyte expansion would likely impact other cilia pathways important for cartilage health and tissue engineering, including transforming growth factor (TGF), Wnt and mechanosignalling.

Ligand influence in Li-ion battery hybrid active materials: Ni methylenediphosphonate vs. Ni dimethylamino methylenediphosphonate.

The influence of a ligand on the structural, morphological and electrochemical properties of organic-inorganic hybrid nickel diphosphonates was assessed using Ni methylenediphosphonate (NiMeDP) and Ni dimethylamino methylenediphosphonate (NiDMAMDP) as model electrode materials.

Pre-emptive analgesic effect of lornoxicam in mandibular third molar surgery: a prospective, randomized, double-blind clinical trial.

The aim of this study was to establish whether the pre-emptive use of lornoxicam (16mg) in third molar surgery ensures successful postoperative analgesia and reduces rescue analgesic intake when compared to postoperative application, and in comparison with placebo. Ninety patients were split randomly into three groups: group A received lornoxicam 60min before surgery and placebo 60min after surgery; group B received placebo 60min before surgery and lornoxicam 60min after surgery; group C received placebo 60min before surgery and placebo 60min after surgery. Postoperative pain was recorded on a visual analogue scale and on a numerical rating scale at 1, 2, 4, 6, 8, 12, and 24h after surgery. The patients recorded total dose of paracetamol intake during the 24h after the procedure. The efficacy of postoperative analgesia was greater in lornoxicam groups when compared to the placebo group; there was no difference between the two lornoxicam groups (A and B). Patients in group C took their first rescue analgesic dose earlier after surgery than patients in the two lornoxicam groups. The average dose of paracetamol taken in group C was 1000mg, while it was500 mg in the lornoxicam groups.

Molecular profile of 5-fluorouracil pathway genes in colorectal carcinoma.

BACKGROUND: This study addresses involvement of major 5-fluorouracil (5-FU) pathway genes in the prognosis of colorectal carcinoma patients. METHODS: Testing set and two validation sets comprising paired tumor and adjacent mucosa tissue samples from 151 patients were used for transcript profiling of 15 5-FU pathway genes by quantitative real-time PCR and DNA methylation profiling by high resolution melting analysis. Intratumoral molecular profiles were correlated with clinical data of patients. Protein levels of two most relevant candidate markers were assessed by immunoblotting. RESULTS: Downregulation of DPYD and upregulation of PPAT, UMPS, RRM2, and SLC29A1 transcripts were found in tumors compared to adjacent mucosa in testing and validation sets of patients. Low RRM2 transcript level significantly associated with poor response to the first-line palliative 5-FU-based chemotherapy in the testing set and with poor disease-free interval of patients in the validation set irrespective of 5-FU treatment. UPP2 was strongly methylated while its transcript absent in both tumors and adjacent mucosa. DPYS methylation level was significantly higher in tumor tissues compared to adjacent mucosa samples. Low intratumoral level of UPB1 methylation was prognostic for poor disease-free interval of the patients (P = 0.0002). The rest of the studied 5-FU genes were not methylated in tumors or adjacent mucosa. CONCLUSIONS: The observed overexpression of several 5-FU activating genes and DPYD downregulation deduce that chemotherapy naïve colorectal tumors share favorable gene expression profile for 5-FU therapy. Low RRM2 transcript and UPB1 methylation levels present separate poor prognosis factors for colorectal carcinoma patients and should be further investigated.

[The Influence of Palliative Chemotherapy on the Quality of Life of Patients with Gastric Cancer]. / Vliv paliativní chemoterapie na kvalitu zivota nemocných s karcinomem zaludku.

BACKGROUND: Gastric cancer is a malignant disease with a poor prognosis. The incidence of gastric cancer in the Czech Republic in 2013 was 14.34 cases per 100,000 citizens. Unfortunately, most patients are dia-gnosed with advanced stage disease and therefore undergo palliative treatment. Some patients undergo surgery and a very small percentage undergo palliative chemotherapy. The five year survival rate for those with advanced gastric cancer ranges from 5-15%. METHODS: This is a prospective study of patients undergoing chemotherapy for advanced gastric cancer. The aim was to assess the quality of life of those undergoing chemotherapy. RESULTS: The results showed that chemotherapy reduced the quality of life for these patients. DISCUSSION: Although palliative chemotherapy prolonged time to progression, it had little impact on overall survival. Conversely, chemotherapy reduced quality of life. Thus, clinicians and patients must decide whether to begin palliative chemotherapy. The final decision should be made by the patient after discussion with the treating clinician. CONCLUSION: Treatment of patients with gastric cancer must be undertaken on an individual basis. Those undergoing palliative treatment must play an active role in the decision process regarding chemotherapy and assess the potential benefits and drawbacks. Because chemotherapy treatment has a detrimental effect on quality of life, the decision should be based on factors that predict the likely therapeutic effect of chemotherapy. A definitive decision can then be made as to whether chemotherapy is indicated. KEY WORDS: gastric cancer - palliative chemotherapy - chemotherapy - quality of life - WHOQOL-BREFThis study was supported by grant of Internal Grant Agency of the Czech Ministry of Health No. NS14227-3.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 10. 1. 2016Accepted: 8. 6. 2016.

Multi-parametric analysis assists in STN localization in Parkinson's patients.

BACKGROUND: Initial subthalamic nucleus (STN) localization is based on MRI and an anatomical atlas and then refined intraoperatively using electrophysiological mapping with microelectrode recordings (IOA - intraoperative multi-unit activity) during deep brain stimulation (DBS) in Parkinson's disease (PD). IOA is time consuming and subjective. The purpose of this study was to assess the value of high frequency multi-unit background activity (MUA, frequency >500Hz), and local field potentials (LFP, frequency 5-500Hz) in detection of the STN borders. METHODS: This was a retrospective, single center study. 18 leads in ten PD patients that underwent STN DBS surgery were evaluated. IOA, MUA and LFP have been compared in detection of the STN. IOA using single train spikes analysis have been used as a gold standard. RESULTS: Both LFP in beta range (20-35Hz) and MUA increased as the microelectrode entered the STN and their increase correlated with dorsal/ventral STN borders. The differences (mean±sd) were: between IOA and MUA of the dorsal/ventral border 0.20±0.76/0.28±0.30mm; between IOA and LFP of the dorsal/ventral border 0.08±0.94/0.05±0.53mm. Using Bland-Altman statistics, only 2/36 (5.6%) differences between IOA and MUA and also 2/36 differences between IOA and LFP (one for the dorsal border and one for the ventral border) were out of ±1.96 SD line of measurement differences. Correlation between dorsal border/ventral border positions obtained by IOA and MUA was 0.86, p<0.000005/0.97, p<10(-11); by IOA and LFP was 0.78, p<0.00015/0.88, p<0.000001. CONCLUSIONS: Both MUA and LFP are characteristically elevated in the STN compared to neighboring structures. They may provide fast, real-time, objective and reliable markers of STN borders.