Evaluation and improvement of the structural mimicry of gp120 mimics

Despite 30 years of research dedicated to the development of an HIV 1 vaccine, a successful candidate capable of elicitation of neutralizing antibodies against the virus has not yet emerged. The Env gp120 mutation rate is very rapid, however in order to maintain its binding site towards CD4 receptor, certain crucial residues must be conserved within the viral strains, making it an interesting therapeutic target. This thesis describes the synthesis of discontinuous gp120 epitope mimics with a novel cyclisation linker, which was applied to improve the solubility and purification of the constructs. For the mimicry of gp120, cyclic peptides mimicking the discontinuous epitopes were assembled on a molecular scaffold. The synthesized linear and cyclic peptides, and final constructs with cyclic peptides assembled on a molecular scaffold were analysed by 1H NMR and CD spectroscopy. These spectroscopic methods allowed to gain a deeper understanding of the secondary structure of the synthesized compounds. It was found that cyclisation constrained the peptides, when compared to their linear counterparts. The NMR spectra of the final gp120 mimics were superimposed on each other and it was found, that two out of three compounds were almost identical. CD spectrometry confirmed the results obtained by NMR. Since binding studies of the synthesised gp120 mimics required access to CD4 and gp120 proteins, therefore approaches towards expression, purification and characterisation of these proteins were made. However, the challenging refolding of CD4D12 resulted in obtaining a heterogenous mixture of properly folded and misfolded protein. The expression of gp120 in mammalian cells was low-yielding and this protein could not be produced in an amount needed for binding studies. A reliable and reproducible technique was needed to evaluate binding of gp120 mimics to CD4 receptor. SPR was chosen to study the kinetics of the constructs. Careful optimisation of the experimental conditions yielded in a highly reproducible method for the evaluation of gp120 discontinuous mimics

Oxidized LDLs inhibit TLR-induced IL-10 production by monocytes : a new aspect of pathogen-accelerated atherosclerosis

It is widely accepted that oxidized low-density lipoproteins and local infections or endotoxins in circulation contribute to chronic inflammatory process at all stages of atherosclerosis. The hallmark cells of atherosclerotic lesions—monocytes and macrophages—are able to detect and integrate complex signals derived from lipoproteins and pathogens, and respond with a spectrum of immunoregulatory cytokines. In this study, we show strong inhibitory effect of oxLDLs on anti-inflammatory interleukin-10 production by monocytes responding to TLR2 and TLR4 ligands. In contrast, pro-inflammatory tumor necrosis factor secretion was even slightly increased, when stimulated with lipopolysaccharide from Porphyromonas gingivalis—an oral pathogen associated with atherosclerosis. The oxLDLs modulatory activity may be explained by altered recognition of pathogen-associated molecular patterns, which involves serum proteins, particularly vitronectin. We also suggest an interaction between vitronectin receptor, CD11b, and TLR2. The presented data support a novel pathway for pathogen-accelerated atherosclerosis, which relies on oxidized low-density lipoprotein-mediated modulation of anti-inflammatory response to TLR ligands. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10753-012-9472-3) contains supplementary material, which is available to authorized users

The synthesis of potential selective ligands of ionotropic receptors of glutamic acid in the group of phenylalanine derivatives

Niniejsza praca jest kontynuacją badań mających na celu poszukiwanie potencjalnych selektywnych ligandów jonotropowych receptorów kwasu glutaminowego w grupie pochodnych fenyloalaniny. W części teoretycznej przedstawiono podział, budowę oraz mechanizm działania jonotropowych receptorów glutaminergicznych i ich rolę w procesie ekscytotoksyczności. Szczególną uwagę poświęcono najnowszym publikacjom dotyczącym antagonistów receptorów AMPA i KA. Opisany został tutaj również mechanizm reakcji Suzuki-Miyaura oraz jej przegląd w syntezie różnorodnych związków, gdyż reakcja ta stanowiła kluczowy etap w syntezie zaplanowanych związków. Celem pracy była synteza nowych, potencjalnych, kompetytywnych antagonistów jonotropowych receptorów AMPA i KA oraz optymalizacja warunków reakcji Suzuki-Miyaura w celu ich otrzymania.W części eksperymentalnej opisano syntezę bifenylowych pochodnych fenyloalaniny oraz przebieg przeprowadzonego skriningu. W wyniku przeprowadzonych prac otrzymano trzy z pięciu zaplanowanych związków. Tożsamość i czystość wszystkich otrzymanych połączeń potwierdzono metodą chromatografii cienkowarstwowej (TLC) oraz przy użyciu spektroskopii 1H-NMR i 13C-NMR. Uzyskane związki zostały przekazane do badań farmakologicznych in vitro mających na celu ocenę ich powinowactwa do natywnych receptorów AMPA, KA i NMDA, a także powinowactwo do sklonowanych homomerycznych receptorów (iGluA1-4) w celu wykrycia selektywnego wiązania do danego podtypu receptora.This work is a continuation of the research focused on searching for potential, selective ligands of ionotropic glutamate receptors in the group of phenylalanine derivatives.The theoretical part of the work presents the classification, structure and mechanism of action of the ionotropic glutamate receptors and their role in the process of excitotoxicity. Special attention was paid to the latest publications concerning antagonists of AMPA and KA receptors. The mechanism of Suzuki-Miyaura reaction and the review of its application in the synthesis of variety of compounds was described due to its crucial role in the synthesis of desired compounds. The aim of this study was to synthetize novel, potential, competitive antagonists of ionotropic AMPA and KA receptors and to optimize the conditions of Suzuki-Miyaura reaction in order to obtain them.The experimental part of the work describes the synthesis of biphenyl derivatives of phenylalanine and the process of screening. As a result of conducted research three out of five desired compounds were obtained. Identity and purity of all obtained compounds was confirmed by Thin Layer Chromatography (TLC) method and by using 1H-NMR and 13C-NMR Spectroscopy. Obtained compounds were passed to pharmacological in vitro studies that aim to evaluate their affinity to the native AMPA, KA and NDMA receptors and also their affinity to the cloned, homomeric receptors (iGluA1-4) in order to detect selective bonding to the particular type of receptor

Effect of the Addition of Edible Insect Flour from Yellow Mealworm (<i>Tenebrio molitor</i>) on the Sensory Acceptance, and the Physicochemical and Textural Properties of Sponge Cake

The aim of this study was to evaluate the possibility of using insect flour for sponge cake supplementation. Consumer acceptance, chemical composition, textural properties, and microbiological characteristics were determined. The addition of mealworm flour significantly increased the content of nutrients, especially protein, ash, lipid, and dietary fiber. Mealworm flour influenced the color parameters as well as textural properties. The fatty acid profile was dominated by monounsaturated fatty acids, ranging from 9.72 g/100 g for wheat flour sponge cake to 41.82 g/100 g for sponge cake with 20% addition of mealworm flour. The amino acid profile of mealworm flour was characterized by a significantly higher content of essential amino acids compared to wheat flour and sponge cakes. Sponge cake supplementation resulted in a good nutritional value of protein except for lysine which was the limiting amino acid in all samples. However, the limiting amino acid index was 63.04-63.10% compared to 30.38% for the mealworm and wheat sponge cake, respectively. The presence of insect flour reduced the organoleptic properties of the obtained sponge cakes, regardless of its quantity. The addition of mealworm flour contributed to a significant reduction in the hardness and fracturability of the sponge cakes on the baking day and during the 30-day storage. Insect flour addition did not reduce the microbiological safety of the final product. The study results indicate the possibility of using mealworm flour in the production of confectionery products

Phenolic Profile, Antioxidant Activity and Amino Acid Composition of Moringa Leaves Fermented with Edible Fungal Strains

Solid-state fermentation (SSF) is widely recognised as a technique to increase the bioactive potential and nutritional value of plant materials. However, the effect of this biotreatment differs for individual substrates. This study aimed to evaluate the impact of SSF with filamentous fungi (Rhizopus, Aspergillus, and Neurospora) on a moringa leaf phenolic profile, antioxidant activity, and amino acid composition. A total of 43 phenolic compounds were determined in the dried leaves analysed by HPLC-ESI-TOF-MS. The leaves contained 11.79 mg/g of free phenolics: flavonols (80.6%, mainly quercetin and kaempferol glycosides), hydroxycinnamic acid derivatives (12.3%), vitexin and vicenin (6.9%), and a small amount of lignan (isolariciresinol isomers). The result of the 1-day fermentation was a slight enhancement in the concentration of individual free phenolics (flavones) and the antioxidant activity of the leaves. However, extending the incubation period caused a significant decrease in those parameters and cannot be recommended for obtaining a food fortificant from moringa leaves. In contrast, the 3-day fermentation with N. intermedia led to a 26% average accumulation of individual amino acids. Therefore, the SSF with Neurospora can be a promising method for improving the nutritional composition of moringa leaves and needs further investigation

Chemical Composition, Nutritional Value, and Acceptance of Nut Bars with the Addition of Edible Insect Powder

Six types of nut-based bars with the addition of edible insect flour were obtained. Flours made from three different insects (Tenebrio molitor L., Acheta domesticus L., Alphitobius diaperinus P.) were used at two different additive levels (15% and 30%) in relation to the weight of the nuts. The addition of insect flour significantly increased protein content and the insoluble fraction of dietary fiber. The largest amount of these compounds was found in bars with 30% cricket flour, 15.51 g/100 g and 6.04 g/100 g, respectively, in comparison to standard bars, 10.78 g/100 g and 3.14 g/100 g, respectively. The greatest consumer acceptance was found in relation to bars with buffalo worm flour. The overall acceptance of these bars was 6.26&ndash;6.28 points compared to 6.48 for standard bars. Bars and raw materials were characterized by the high biological value of the protein. Cis linoleic acid dominated among unsaturated fatty acids. The percentage of this compound was in the range of 69.56%, for bars with a 30% addition of buffalo worm flour, to 73.88%, for bars with 15% cricket flour. Instrumental analysis of taste and smell compounds showed the presence of compounds such as 3-methylbutanoic acid, hexanal, and 2,3-pentanedione

Chemical Composition, Antioxidant Properties and Sensory Aspects of Sponge Cakes Supplemented with Edible Insect Flours

The chemical composition, antioxidant properties, and sensory aspects of sponge cakes with the addition of flours from edible insects (buffalo worm, cricket, and mealworm) were evaluated. The addition of edible-insect flours increased the protein, fat, and dietary fiber content in all cases. The utilization of edible insects demonstrated a notable augmentation in the phenolic compounds (especially protocatechuic acid and protocatechuic aldehyde, and syringic, ferulic, and sinapic acids). This resulted in an increase in the antioxidant activity measured against the ABTS radical cation, the DPPH radical, and ferric ions. The antioxidant potential, assessed by four different methods, unequivocally confirmed that the aforementioned polyphenolic compounds found in edible insects provide significant radical-scavenging and antioxidant activity in sponge cakes containing them. The polyunsaturated fatty acid contents were significantly lower in cakes with insect flour compared to the standard wheat cakes. Products and raw materials exhibited high values of the n − 6/n − 3 ratio, which may be associated with negative health effects, with a high oleic acid content. The amino acid score (AAS) for the essential amino acids exceeded 100% for all obtained products. The sponge cakes were accepted by consumers and the taste was the most important predictor for overall acceptability, whereas the structure and appearance had less impact