250 research outputs found
Development of peptide inhibitors of lipases and phospholipases by phage display technology
No full textBiološke knjižnice naključnih peptidov, predstavljenih na površini organizmov, nam omogočajo sistematično proučevanje interakcij ciljnih makromolekul. Peptidni modulatorji terapevtsko zanimivih ciljnih proteinov imajo v sodobnem načrtovanju učinkovin vse pomembnejšo vlogo.
V prvem delu smo vpeljali, razvili in optimirali metodologijo bioloških kombinatoričnih knjižnic z uporabo streptavidina kot modelne ciljne makromolekule. V drugem delu pa smo jo uporabili kot orodje za identifikacijo strukturno in funkcionalno optimalnih peptidnih ligandov izbranih ciljnih makromolekul. Primerjalno smo ovrednotili dva tipa bioloških knjižnic, bakterijsko in bakteriofagno predstavitveno knjižnico. Na podlagi lastnih eksperimentov in podatkov, objavljenih v literaturi, smo zaključili, da so bakteriofagne predstavitvene knjižnice sicer tehnično zahtevnejše, vendar v večini primerov vodijo v uspešnejšo selekcijo. Za rešetanje in ovrednotenje posameznih bakteriofagnih klonov smo primerjali dve metodi, encimskoimunski test na trdnem nosilcu in površinsko plazmonsko resonanco. Obe metodi je mogoče uspešno uporabiti za določanje vezavnih lastnosti bakteriofagnih klonov, izbira pa temelji na naravi in količini ciljne makromolekule ter nenazadnje na opremi, ki nam je na voljo. Predvsem kadar je količina ciljne makromolekule omejujoč dejavnik, je metoda izbora površinska plazmonska resonanca, saj raznoliki senzoski čipi omogočajo pritrditev katerekoli makromolekule, ne glede na njene fizikalno-kemijske lastnosti. V tehnologijo bakterifagnega prikaza smo vpeljali uporabo ultrazvoka, ki pri eluciji pripomore tako k pretrganju interakcij med tarčo in peptidom na bakteriofagu, kot tudi desorpciji tarčnega proteina skupaj z bakteriofagom s trdnega nosilca. Uvedba tega inovativnega postopka v afinitetne selekcije bo brez dvoma prispevala k razvoju tehnologije bakteriofagnih predstavitvenih knjižnic. Tehnologijo bioloških peptidnih knjižnic smo uporabili za razvoj novih ligandov in potencialnih inhibitorjev encimov, ki sodelujejo pri razgradnji in absorpciji maščob v prebavnem traktu (pankreasne lipaze, pankreasne fosfolipaze A2). Uporabili smo knjižnico naključnih, cikličnih heptapeptidov in linearnih dodekapetidov izraženih na bakteriofagih. Naredili smo več neodvisnih selekcij, pri katerih smo spreminjali načine spiranja in elucije. Inhibicijo pankreasne lipaze smo dosegli s peptidom D23 (navidezna konstanta inhibicije 16 μM). Ligandi pankreasne fosfolipaze A2 in sorodne fosfolipaze, amoditoksina C, ki smo jih izolirali z različnimi selekcijskimi protokoli, jasno kažejo afiniteto do obeh encimov, neodvisno od ciljne molekule uporabljene pri selekciji. Navkljub vezavi na tarčna proteina, pa peptidi ne vplivajo na njuno aktivnost. Peptidi, pridobljeni s to metodologijo, so ligandi določenih mest ciljnega proteina in lahko predstavljajo spojine vodnice pri razvoju novih učinkovin. Njihova uporabna vrednost je predvsem v sposobnosti selektivnih interakcij s ciljnimi proteinskimi makromolekulami.Libraries of random peptides displayed on the surface of biological systems are an essential tool that enables systematic study of target molecule interactions. Peptide modulators of therapeutically interesting target proteins are gaining an increasingly important role in drug discovery and development. First part comprises of introduction, development and optimization of biological display libraries with the use of a model target molecule, streptavidin. The second part describes the use of such libraries and optimized procedures in a search of novel peptide ligands of selected targets. Bacterial and phage display libraries were compared for their efficiency in the search of model target protein (streptavidin) binding motif. Under similar conditions, phage display library, although technically more demanding, proved to be convincingly better. Efficiency and convenience of enzyme linked immunosorbent assay and surface plasmon resonance were compared for determining the binding properties of peptide-displaying phage clones using streptavidin as a model protein target. Both methods can be successfully used for affinity ranking. The choice therefore depends on the nature and the amount of target protein and the equipment available. Versatile sensor chips enable immobilization of almost any target molecule, regardless of their physiochemical properties and when there is only a small amount of target molecule available, surface plasmon resonance would be a method of choice. An improved non-specific elution strategy was introduced to the phage display technology. This procedure employs ultrasound to break the interaction of high affinity clones with the target and also to detach the target molecule from the immobilization surface. This new approach is able to surmount the incapability of other commonly used non-specific elution techniques and represents an important improvement in a search for novel specific ligands. Phage display technology was used to develop new ligands and potential inhibitors of lipid digestion and absorption enzymes (pancreatic lipase, pancreatic phospholipase A2) in gastrointestinal tract. A random, cyclic heptapetide and a linear dodecapeptide phage display library were used. Several independent selections were performed, differing in washing and elution steps. Inhibition of pancreatic lipase was achieved by peptide D23 (apparent inhibition constant of 16 μM). Peptide ligands of pancreatic phospholipase A2 and structurally related ammodytoxin C, selected by different selection protocols, clearly indicate affinity towards both enzymes, regardless which of the two proteins was used as a target in the selection procedure. Despite clear affinity, none of the peptides had inhibitory activity in vitro.Peptides selected and discovered by screening biological libraries target only a few sites on a given protein, often associated with biological activity. These peptides are modulators of protein function and are a starting point in drug development, their main advantage being the ability of highly specific interaction with protein macromolecules
Chromate-free Pre-treatment of Aluminium for Adhesive Bonding
No full textThe increasing use of aluminium in automotive and transport applications is primarily driven by its high strength to weight ratio, enabling substantially improved fuel economy and reduced CO2 emissions when substituted for heavier materials. However, the change of material presents new challenges with respect to design and methods of joining. Structural adhesive bonding offers several advantages compared with welding, but a major limitation is concern about the durability of joints in wet and corrosive environments. The pre-treatment of the aluminium surface prior to bonding is the key to long service life. Pre-treatments successfully employed by the aerospace industry cannot be used in automotive production, where cheaper and more environmentally friendly pre-treatments are required. Specifically, the use of chromates is unacceptable. Hence, there is a need to develop chromate-free pre-treatments that will consistently provide the required level of performance, while being acceptable both in terms of general engineering practice and economy. To accomplish this task, basic knowledge of the processes occurring on the aluminium surface during pre-treatment, properties of the modified surface, and mechanisms of joint degradation are necessary.
The purpose of the present work has therefore been to contribute to a better understanding of how the aluminium substrate affects the formation and properties of conversion coatings for adhesive bonding. In particular, a commercial chromate-free fluorotitanate/zirconate based process has been investigated and compared with conventional chromate treatment. The materials chosen for this work were commercially extruded AA6060-T6 aluminium and a structural single-part epoxy adhesive. To complement the studies of the commercial alloy, model analogues of the AA6060 aluminium matrix and α-Al(Fe,Mn)Si phase particles present in the commercial alloy were also investigated.
It was observed that the α-Al(Fe,Mn)Si particles played an essential role in the formation and properties of Ti-Zr oxide conversion coatings on AA6060 aluminium. The particles were significantly nobler than the aluminium matrix in the pre-treatment solution. An alkaline diffusion layer therefore developed around the particles during pre-treatment due to oxygen reduction and hydrogen evolution reactions. As Ti-Zr oxide precipitation was favoured at high pH, the conversion layers normally deposited at and in the vicinity of the cathodic particles. The conversion layers formed consequently exhibited considerable lateral variations in thickness. In addition to substrate microstructure, bulk pH and agitation of the conversion bath were important factors controlling the extent of Ti-Zr oxide deposition and its distribution on the surface. On areas well away from the cathodic particles coverage was generally very poor, although a high density of small (<50 nm) oxide particles was deposited, presumably with a composition similar to the continuous conversion layer close to the α-Al(Fe,Mn)Si particles. The cathodic activity of the particles was only slightly reduced by formation of the Ti-Zr oxide conversion coating. In combination with poor coverage of the aluminium matrix, these conversion coatings are therefore not expected to improve the corrosion resistance of aluminium significantly.
In contrast to the above mechanism, the chromate conversion coating (CCC) formed by a redox reaction between chromate ions and aluminium. A relatively thick, porous chromium oxide layer developed over the aluminium matrix of AA6060, while a significantly thinner film was formed on the α-Al(Fe,Mn)Si particles. The morphology of the CCC covering the matrix was influenced by the hardening Mg2Si phase, primarily by promoting nucleation of the CCC. Despite the thin film (<50 nm) formed on the α-Al(Fe,Mn)Si particles by chromating, the cathodic activity was significantly reduced. Inhibition of the cathodic reactivity at these particles is suggested as an important factor contributing to the high performance of chromate pretreatments on aluminium.
Testing of epoxy-bonded AA6060 aluminium joints in humid environment showed that Ti-Zr based pre-treatment provided improved adhesion relative to alkaline etching and deoxidation only. However, Ti-Zr based pre-treatment was inferior to chromating. Rapid, interfacial crack growth during wedge testing was particularly observed for adherends with a relatively thick Ti-Zr oxide deposit, suggesting that excessive Ti-Zr oxide deposition should be avoided. Furthermore, as the substrate microstructure (i.e. type, area fraction and distribution of cathodic sites) strongly affected the Ti-Zr oxide deposition, the pre-treatment conditions should be adapted to the specific alloy in order to achieve optimum performance.
In the presence of chlorides, degradation of adhesive-bonded joints may be accelerated by a filiform corrosion (FFC) type of mechanism. The α-Al(Fe,Mn)Si particles in AA6060 played a crucial role in promoting FFC, as demonstrated by complete FFC immunity of the iron-free AA6060 model analogue alloy. Ti-Zr based pre-treatment provided less protection against FFC relative to chromate pre-treatment. The good FFC resistance of CCCs was partly attributed to a better inhibition of the cathodic activity at the α-Al(Fe,Mn)Si particles.
The cathodic α-Al(Fe,Mn)Si particles present on the surface of AA6060 aluminium could be effectively removed by different etch treatments. However, selective removal of surface intermetallics did not prevent FFC because filament growth was supported by cathodic activity on particles that become exposed in the filament tail as a result of the corrosion process.
Based on lap shear testing, hot AC anodising in sulphuric acid to a film thickness of about 0.2 µm showed promise as another chromate-free pretreatment for durable adhesive bonding. The performance was better than a conventional chromic-sulphuric acid based etch treatment. While hot AC anodising did not significantly inhibit the cathodic activity on the α-Al(Fe,Mn)Si particles, good resistance against FFC was still obtained due to the oxide film covering the whole aluminium matrix. Based also on separate durability data recently available, hot AC anodising is considered as a robust alternative to chromating for adhesive bonding of aluminium in certain industrial applications.dr.ing.dr.ing
Gas fluxes and biological and physiochemical measurements made across an Arctic forefield.
No full textHalocarbon fluxes measured using dynamic flux chambers across a retreating glacier's forefield, at Midtre Lovenbreen, Svalbard. Also included in this dataset, are physiochemical and biological parameters of the soil where each chamber measurement was conducted. If using this data, please cite: Macdonald, M.L., Wadham, J.L., Young, D., O'Dohery, S., Lunder, C., Hermansen, O., Lamarche-Gagnon, G., (2019). Consumption of CH3Cl, CH3Br and CH3I and emission of CHCl3, CHBr3 and CH2Br2 from a retreating Arctic glacier’s forefield. JOURNAL, VOL, ISSUE. doi: XXX
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Application Process for Seeking 501(c)(3) Tax-Exempt Status
No full textCharities and other entities seeking tax-exempt status as 501(c)(3) organizations generally must apply to the Internal Revenue Service. This report provides an overview of the application process
Presence of bilitranslocase in the vascular system and its role in the vasodilatation activity of anthocyanins
No full textBackground: Flavonoids are well known for their vasodilata- tion activity. Their mechanism of action is still to be elucidated. Bilitranslocase is a bilirubin-specific membrane carrier that is also responsible for the ATP-independent transport of flavonoids across the cell membrane [1]. Recently, the expression of bilitranslocase in the endothelium has been characterized [2]. The aim of the study was to examine the possible role of bilitranslocase in the vasodilatation activity of flavonoids. As a source of flavonoids we used bilberries, which are abundant in anthocyanins that show strong affinity for bilitranslocase. Methods: A bilberry extract, of which the anthocyanin content was quantified by HPLC-DAD analysis, was used. Anthocyanins were expressed as a standard of cyanidin 3-glucoside (mg/L). Thoracic aortic rings obtained from male Wistar rats were mounted in standard organ baths filled with Krebs-Henseleit solution, maintained at 37°C with a 95% O2 / 5% CO2 mixture. Rings were divided into four groups: control group (intact aortic rings), endothelium denuded rings, rings with nitric oxide synthase (NOS) activity inhibited by application of L-NNA (0.1 mmol/L) and rings with inhibited bilitranslocase-mediated membrane transport. In the last group, aortic rings were pre-incubated for 30 min with mono-specific polyclonal bilitranslocase antibodies (0.24 mg/mL). After inducing submaximal contraction (60 mmol/L KCl) in all studied groups, a chemically characterized bilberry extract was applied in increasing concentrations (0.5-20 mg/L). Vascular tone was measured isometrically by a mechano-electrical transducer. Further tests were done to check the expression of bilitranslocase in the vascular system (endothelial cell line EA.hy 926 and vascular smooth muscle cell line A7r5) by Western blot analysis using bilitranslocase antibodies.
Results: Western blot analysis showed the presence of bilitranslocase on both endothelial and smooth muscle cells. Bilberry extract relaxed aortic rings in a concentration- dependent manner in the control group, but neither in endothelium-denuded aortic rings nor in rings with inhibited NOS. The maximum relaxation (19.00 ± 2.01%, n = 5) observed at 20 mg/L in the group with inhibited bilitranslocase activity was significantly lower (p < 0.001) compared to the control group (34.95 ± 3.11%, n = 10).
Conclusion: Our results show that the vasodilatation activity of anthocyanins in bilberry extract is partly dependent on the bilitranslocase-mediated transport of flavonoids into the endothe- lium, followed by the activation of NOS. However, even though the bilitranslocase is also expressed on smooth muscle cells, its role in the vasodilatation activity on these cells remains negligible. References
1. Passamonti S, Terdoslavich M, Franca R, Vanzo A, Tramer F, Braidot E, Petrussa E and Vianello A: Bioavailability of flavonoids: a review of their membrane transport and the function of bilitranslocase in animal and plant organisms. Curr Drug Metab 2009, 10:369–394.
2. Maestro A, Terdoslavich M, Vanzo A, Kuku A, Tramer F, Nicolin V, Micali F, Decorti G and Passamonti S: Expression of bilitranslocase in the vascular endothelium and its function as a flavonoid transporter. Cardiovasc Res 2009 in press
sj-pdf-3-pmj-10.1177_02692163221142950 – Supplemental material for Healthcare use and healthcare costs for patients with advanced cancer; the international ACTION cluster-randomised trial on advance care planning
No full textSupplemental material, sj-pdf-3-pmj-10.1177_02692163221142950 for Healthcare use and healthcare costs for patients with advanced cancer; the international ACTION cluster-randomised trial on advance care planning by Ida J Korfage, Suzanne Polinder, Nancy Preston, Johannes JM van Delden, A)JLM Geraerds, Lesley Dunleavy, Kristof Faes, Guido Miccinesi, Giulia Carreras, Caroline Moeller Arnfeldt, Marijke C Kars, Giuseppe Lippi, Urska Lunder, Ceu Mateus, Kristian Pollock, Luc Deliens, Mogens Groenvold, Agnes van der Heide and Judith AC Rietjens in Palliative Medicine</p
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501(c)(4)s and Campaign Activity: Analysis Under Tax and Campaign Finance Laws
No full textThis report discusses 501(c)(4) social welfare organizations, including the requirements necessary to maintain 501(c)(4) status and the requirements for annual reporting to the Internal Revenue Service and Federal Election Commission
Effect of Oral Semaglutide on Cardiovascular Parameters and Their Mechanisms in Patients with Type 2 Diabetes: Rationale and Design of the Semaglutide Anti-Atherosclerotic Mechanisms of Action Study (SAMAS)
No full textIntroduction Type 2 diabetes (T2D) management has reached a point where not only optimal glycaemic control is necessary, but also additional interventions with proven cardiovascular risk reduction benefit. Subcutaneous semaglutide has been shown to provide cardiovascular protection, but its use may be limited by its injection formulation. To overcome this limitation, an oral semaglutide tablet has been developed, which could potentially be of the same value as its injection counterpart, but in a much wider group of patients with T2D, thereby allowing for broader cardiovascular risk reduction in this vulnerable patient population. Methods A total of 100 consecutive patients with T2D and a disease duration of up to 10 years, without manifest cardiovascular disease, who are treated with metformin (+/- sulphonylurea) and optimal cardioprotective therapy, will be recruited in a single-blinded, randomized trial named "Semaglutide Anti-atherosclerotic Mechanisms of Action Study (SAMAS)." After 1:1 randomization, patients will receive either oral semaglutide 14 mg daily or placebo for 1 year. The primary outcome comprises changes in atherosclerosis-related structural and functional characteristics of the arterial wall, namely: reduction of the carotid intima-media thickness, improvement of endothelial function and decrease in arterial stiffness. Secondary outcomes are changes in atherogenic small dense low-density lipoproteins, glucose control (HbA1c) and inflammatory markers (hsCRP). Possible correlations between primary endpoints and changes in lipids, HbA1c and high-sensitivity C-reactive protein will be sought. Discussion This is the first study to investigate the direct and indirect anti-atherosclerotic mechanisms of oral semaglutide. The results are expected to confirm the position of oral semaglutide in the multifactorial management of T2D with an emphasis on cardiovascular disease prevention
sj-pdf-6-pmj-10.1177_02692163221142950 – Supplemental material for Healthcare use and healthcare costs for patients with advanced cancer; the international ACTION cluster-randomised trial on advance care planning
No full textSupplemental material, sj-pdf-6-pmj-10.1177_02692163221142950 for Healthcare use and healthcare costs for patients with advanced cancer; the international ACTION cluster-randomised trial on advance care planning by Ida J Korfage, Suzanne Polinder, Nancy Preston, Johannes JM van Delden, A)JLM Geraerds, Lesley Dunleavy, Kristof Faes, Guido Miccinesi, Giulia Carreras, Caroline Moeller Arnfeldt, Marijke C Kars, Giuseppe Lippi, Urska Lunder, Ceu Mateus, Kristian Pollock, Luc Deliens, Mogens Groenvold, Agnes van der Heide and Judith AC Rietjens in Palliative Medicine</p
sj-pdf-5-pmj-10.1177_02692163221142950 – Supplemental material for Healthcare use and healthcare costs for patients with advanced cancer; the international ACTION cluster-randomised trial on advance care planning
No full textSupplemental material, sj-pdf-5-pmj-10.1177_02692163221142950 for Healthcare use and healthcare costs for patients with advanced cancer; the international ACTION cluster-randomised trial on advance care planning by Ida J Korfage, Suzanne Polinder, Nancy Preston, Johannes JM van Delden, A)JLM Geraerds, Lesley Dunleavy, Kristof Faes, Guido Miccinesi, Giulia Carreras, Caroline Moeller Arnfeldt, Marijke C Kars, Giuseppe Lippi, Urska Lunder, Ceu Mateus, Kristian Pollock, Luc Deliens, Mogens Groenvold, Agnes van der Heide and Judith AC Rietjens in Palliative Medicine</p
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