Search results for: cationic peptide

Authors: Ravinder Singh, Ankita Gurao, Saroj Bandhan, Sudhir Kumar Kashyap

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The Bos taurus Beta defensin 3 is a defensin peptide secreted by neutrophils and epithelial that exhibits anti-microbial activity. It is one of the crucial components forming an innate defense against intra mammary infections in livestock. The beta defensin 3 by virtue of its anti-microbial activity inhibits major mastitis pathogens including Staphylococcus aureus and Pseudomonas aeruginosa etc, which are also responsible for biofilm formation leading to antibiotic resistance phenomenon. Therefore, the defensin may prove as a non-conventional option to treat mastitis. In this study, computational analysis has been performed including sequence comparison among species and homology modeling of Bos taurus beta defensin 3 protein. The assessments of protein structure were done using the protein structure and model assessment tools integrated in Swiss Model server, which employs various local and global quality evaluation parameters. Further, molecular docking was also carried out between the defensin peptide and the components of biofilm to gain insight into various interactions and structural differences crucial for functionality of this protein.

Keywords: beta defensin 3, bos taurus, docking, homology modeling

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Authors: Jana Tchekalarova, Stela Georgieva, Petia Peneva, Petar Todorov

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In the present study, a series of bioconjugates of N-modified hemorphine analogs containing second pharmacophore cinnamic acids (CA) or caffeic acid (KA) were synthesized by a traditional solid-phase Fmoc chemistry method for peptide synthesis. Electrochemical and fluorometric analysis and in vivo anticonvulsant activity in mice were conducted on the compounds. The three CA (H4-CA, H5-CA, and H7-CA) and three KA (H4-KA, H5-KA, and H7-KA)-conjugated hemorphine derivatives showed dose-dependent anticonvulsant activity in the maximal electroshock test (MES) in mice. The KA-conjugated H5-KA derivate was the only compound that suppressed clonic seizures at the lowest dose of 0.5 µg/mouse in the scPTZ test. The activity against the psychomotor seizures in the 6-Hz test was detected only for the H4-CA (0.5 µg) and H4-KA (0.5 µg and 1 µg), respectively. The peptide derivates did not exhibit neurotoxicity in the rotarod test. Our findings suggest that conjugated CA and KA hemorphine peptides can be used as a background for developing hemorphin-related analogs with anticonvulsant activity. Acknowledgments: This study is funded by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project № BG-RRP-2.004-0002, "BiOrgaMCT".

Keywords: hemorphins, SPSS, caffeic/cinnamic acid, anticonvulsant activity, electrochemistry, fluorimetry

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Authors: Jana Tchekalarova, Stela Georgieva, Petia Peneva, Petar Todorov

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In the present study, a series of bioconjugates of N-modified hemorphine analogs containing second pharmacophore cinnamic acids (CA) or caffeic (KA) were synthesized by a traditional solid-phase Fmoc chemistry method for peptide synthesis. Electrochemical and fluorimetrical analysis and in vivo anticonvulsant activity in mice were conducted on the compounds. The three CA acids (H4-CA, H5-CA, and H7-CA) and three KA acids (H4-KA, H5-KA, and H7-KA)-conjugated hemorphine derivatives showed dose-dependent anticonvulsant activity in the maximal electroshock test (MES) in mice. The KA-conjugated H5-KA derivate was the only compound that suppressed clonic seizures at the lowest dose of 0.5 µg/mouse in the scPTZ test. The activity against the psychomotor seizures in the 6-Hz test was detected only for the H4-CA (0.5 µg) and H4-KA (0.5 µg and 1 µg), respectively. The peptide derivates did not exhibit neurotoxicity in the rotarod test. Our findings suggest that conjugated CA and KA hemorphine peptides can be used as a background for developing hemorphin-related analogs with anticonvulsant activity. Acknowledgements: This study is funded by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project № BG-RRP-2.004-0002, "BiOrgaMCT".

Keywords: hemorphins, caffeic/cinnamic acid, anticonvulsant activity, electrochemistry, fluorimetry

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Authors: Yung-Chih Kuo, Yung-I Lou

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Cationic solid lipid nanoparticles (CSLNs) with anti-melanotransferrin (AMT) and apolipoprotein E (ApoE) were used to carry antimitotic doxorubicin (Dox) across the blood–brain barrier (BBB) for glioblastoma multiforme (GBM) treatment. Dox-loaded CSLNs were prepared in microemulsion, grafted covalently with AMT and ApoE, and applied to human brain microvascular endothelial cells (HBMECs), human astrocytes, and U87MG cells. Experimental results revealed that an increase in the weight percentage of stearyl amine (SA) from 0% to 20% increased the size of AMT-ApoE-Dox-CSLNs. In addition, an increase in the stirring rate from 150 rpm to 450 rpm decreased the size of AMT-ApoE-Dox-CSLNs. An increase in the weight percentage of SA from 0% to 20% enhanced the zeta potential of AMT-ApoE-Dox-CSLNs. Moreover, an increase in the stirring rate from 150 rpm to 450 rpm reduced the zeta potential of AMT-ApoE-Dox-CSLNs. AMT-ApoE-Dox-CSLNs exhibited a spheroid-like geometry, a minor irregular boundary deviating from spheroid, and a somewhat distorted surface with a few zigzags and sharp angles. The encapsulation efficiency of Dox in CSLNs decreased with increasing weight percentage of Dox and the order in the encapsulation efficiency of Dox was 10% SA > 20% SA > 0% SA. However, the reverse order was true for the release rate of Dox, suggesting that AMT-ApoE-Dox-CSLNs containing 10% SA had better-sustained release characteristics. An increase in the concentration of AMT from 2.5 to 7.5 μg/mL slightly decreased the grafting efficiency of AMT and an increase in that from 7.5 to 10 μg/mL significantly decreased the grafting efficiency. Furthermore, an increase in the concentration of ApoE from 2.5 to 5 μg/mL slightly reduced the grafting efficiency of ApoE and an increase in that from 5 to 10 μg/mL significantly reduced the grafting efficiency. Also, AMT-ApoE-Dox-CSLNs at 10 μg/mL of ApoE could slightly reduce the transendothelial electrical resistance (TEER) and increase the permeability of propidium iodide (PI). An incorporation of 10 μg/mL of ApoE could reduce the TEER and increase the permeability of PI. AMT-ApoE-Dox-CSLNs at 10 μg/mL of AMT and 5-10 μg/mL of ApoE could significantly enhance the permeability of Dox across the BBB. AMT-ApoE-Dox-CSLNs did not induce serious cytotoxicity to HBMECs. The viability of HBMECs was in the following order: AMT-ApoE-Dox-CSLNs = AMT-Dox-CSLNs = Dox-CSLNs > Dox. The order in the efficacy of inhibiting U87MG cells was AMT-ApoE-Dox-CSLNs > AMT-Dox-CSLNs > Dox-CSLNs > Dox. A surface modification of AMT and ApoE could promote the delivery of AMT-ApoE-Dox-CSLNs to cross the BBB via melanotransferrin and low density lipoprotein receptor. Thus, AMT-ApoE-Dox-CSLNs have appropriate physicochemical properties and can be a potential colloidal delivery system for brain tumor chemotherapy.

Keywords: anti-melanotransferrin, apolipoprotein E, cationic catanionic solid lipid nanoparticle, doxorubicin, U87MG cells

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Authors: Abdul Mutalib Md Jani, Abdul Hadi Mahmud, Mohd Tajuddin Mohd Ali

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The rapid growth of interest in surface modification of nanostructures materials that exhibit improved structural and functional properties is attracting more researchers. The unique properties of highly ordered nanoporous anodic aluminium oxide (NAAO) membrane have been proposed as a platform for biosensing applications. They exhibit excellent physical and chemical properties with high porosity, high surface area, tunable pore sizes and excellent chemical resistance. In this study, NAAO was functionalized with 3-aminopropyltriethoxysilane (APTES) to prepared silane-modified NAAO. Amine functional groups are formed on the surface of NAAO during silanization and were characterized using Fourier Transform Infrared spectroscopy (FTIR). The synthesis of multi segment of peptide on NAAO surfaces can be realized by changing the surface chemistry of the NAAO membrane via click chemistry. By click reactions, utilizing alkyne terminated with amino group, various peptides tagged on NAAO can be envisioned from chiral natural or unnatural amino acids using standard coupling methods (HOBt, EDCI and HBTU). This strategy seemly versatile since coupling strategy of dipeptide with another amino acids, leading to tripeptide, tetrapeptide or pentapeptide, can be synthesized without purification. When an appropriate terminus is selected, multiple segments of amino acids can be successfully synthesized on the surfaces. The immobilized NAAO should be easily separated from the reaction medium by conventional filtration, thus avoiding complicated purification methods. Herein, we proposed to synthesize multi fragment peptide as a model for capturing and attaching various small biomolecules on NAAO surfaces and can be also applied as biosensing device, drug delivery systems and biocatalyst.

Keywords: nanoporous anodic aluminium oxide, silanization, peptide synthesise, click chemistry

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Authors: Zuoyong Zhang, Min Yu, Jinlong Zhao, Shudong He

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The Maillard reaction can produce the flavor enhancing substance through the chemical crosslinking between free amino group of the protein or polypeptide with the carbonyl of the reducing sugar. In this research, solutions of rapeseed meal peptide and D-xylose with or without L-cysteine (RXC or RX) were heated over a range of temperatures (80-140 °C) for 2 h. It was observed that RXs had a severe browning,while RXCs accompanied by more pH decrement with the temperature increasing. Then the correlation among data of quantitative sensory descriptive analysis, free amino acid (FAA) and GC–MS of RXCs and RXs were analyzed using the partial least square regression method. Results suggested that the Maillard reaction product (MRPs) with cysteine formed at 120 °C (RXC-120) had greater sensory properties especially meat-like flavor compared to other MRPs. Meanwhile, it revealed that glutamic and glycine not only had a positive contribution to meaty aroma but also showed a significant and positive influence on umami taste of RXs based on the FAA data. Moreover, the sulfur-containing compounds showed a significant positive correlation with the meat-like flavor of RXCs, while RXs depended on furans and nitrogenous-containing compounds with more caramel-like flavor. Therefore, a MRP with strong meaty flavor could be obtained at 120 °C by addition of cysteine.

Keywords: rapeseed meal, Maillard reaction, sensory characteristics, FAA, GC–MS, partial least square regression

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Authors: Manuela Amorim, Cláudia S. Marques, Maria Conceição Calhau, Hélder J. Pinheiro, Maria Manuela Pintado

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Recently it has been recognized peptides from different food sources with biological activities. However, no relevant study has proven the potential of brewer yeast peptides in the modulation of gut microbiota. The importance of human intestinal microbiota in maintaining host health is well known. Probiotics, prebiotics and the combination of these two components, can contribute to support an adequate balance of the bacterial population in the human large intestine. The survival of many bacterial species inhabiting the large bowel depends essentially on the substrates made available to them, most of which come directly from the diet. Some of these substrates can be selectively considered as prebiotics, which are food ingredients that can stimulate beneficial bacteria such as Lactobacilli or Bifidobacteria growth in the colon. Moreover, conventional food can be used as vehicle to intake bioactive compounds that provide those health benefits and increase people well-being. In this way, the main objective of this work was to study the potential prebiotic activity of brewer yeast peptide extract (BYP) obtained via hydrolysis of yeast proteins by cardosins present in Cynara cardunculus extract for possible use as a functional ingredient. To evaluate the effect of BYP on the modulation of gut microbiota in diet-induced obesity model, Wistar rats were fed either with a standard or a high-fat diet. Quantified via 16S ribosomal RNA (rRNA) expression by quantitative PCR (qPCR), genera of beneficial bacteria (Lactobacillus spp. and Bifidobacterium spp.) and three main phyla (Firmicutes, Bacteroidetes and Actinobacteria) were assessed. Results showed relative abundance of Lactobacillus spp., Bifidobacterium spp. and Bacteroidetes was significantly increased (P < 0.05) by BYP. Consequently, the potential health-promoting effects of WPE through modulation of gut microbiota were demonstrated in vivo. Altogether, these findings highlight the possible intervention of BYP as gut microbiota enhancer, promoting healthy life style, and the incorporation in new food products, leads them bringing associated benefits endorsing a new trend in the improvement of new value-added food products.

Keywords: functional ingredients, gut microbiota, prebiotics, brewer yeast peptide extract

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Authors: Muhammad Mazhar, Yong Zhu, Likang Qin

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Foods contain endogenous components known as dietary fibers, which are classified into soluble and insoluble forms. Dietary fibers are resistant to gut digestive enzymes, modulating anaerobic intestinal microbiota (AIM) and fabricating short-chain fatty acids (SCFAs). Acetate, butyrate, and propionate dominate in the gut, and different pathways, including Wood-Ljungdahl and acrylate pathways, generate these SCFAs. In pancreatic dysfunction, the release of insulin/glucagon is impaired, which leads to hyperglycemia. SCFAs enhance insulin sensitivity or secretion, beta-cell functions, leptin release, mitochondrial functions, and intestinal gluconeogenesis in human organs, which positively affect type 2 diabetes (T2D). Research models presented that SCFAs either enhance the release of peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) from L-cells (entero-endocrine) or promote the release of leptin hormone satiation in adipose tissues through G-protein receptors, i.e., GPR-41/GPR-43. Dietary fibers are the components of foods that influence AIM and produce SCFAs, which may be offering beneficial effects on T2D. This review addresses the effectiveness of SCFAs in modulating gut AIM in the fermentation of dietary fiber and their worth against T2D.

Keywords: dietary fibers, intestinal microbiota, short-chain fatty acids, fermentation, type 2 diabetes

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Authors: Madhu Gupta, Vikas Sharma, Suresh P. Vyas

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CD13 receptors are abundantly overexpressed in tumor cells as well as in neovasculature. The CD13 receptors were selected as a targeted site and polymeric nanoparticles (NPs) as a targeted delivery system. By combining these, a cyclic NGR (cNGR) peptide ligand was coupled on the terminal end of polyethylene glycol-b-poly(lactic-co-glycolic acid) (PEG-b-PLGA) and prepared the dual targeted-NPs (cNGR-PEG-PTX-NPs) to enhance the intracellular delivery of anticancer drug to tumor cells and tumor endothelial cells via ligand-receptor interaction. In-vitro cytotoxicity studies confirmed that the presence of cNGR enhanced the cytotoxic efficiency by 2.8 folds in Human Umbilical Vein Endothelial (HUVEC) cells, while cytotoxicity was improved by 2.6 folds in human fibrosarcoma (HT-1080) cells as compared to non-specific stealth NPs. Compared with other tested NPs, cNGR-PEG-PTX-NPs revealed more cytotoxicity by inducing more apoptosis and higher intracellular uptake. The tumor volume inhibition rate was 59.7% in case of cNGR-PEG-PTX-NPs that was comparatively more with other formulations, indicating that cNGR-PEG-PTX-NPs could more effectively inhibit tumor growth. As a consequence, the cNGR-PEG-PTX-NPs play a key role in enhancing tumor therapeutic efficiency for treatment of CD13 receptor specific solid tumor.

Keywords: cyclic NGR, CD13 receptor, targeted polymeric NPs, solid tumor, intracellular delivery

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Authors: Isaac Quiros-Fernandez, Angel Cid-Arregui

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Tumor-reactive T cell receptors (TCRs) are being subject of intense investigation since they offer great potential in adoptive cell therapies against cancer. However, the identification of tumor-specific TCRs has proven challenging, for instance, due to the limited expansion capacity of tumor-infiltrating T cells (TILs) and the extremely low frequencies of tumor-reactive T cells in the repertoire of patients and healthy donors. We have developed an approach for rapid identification and characterization of neoepitope-reactive TCRs from the T cell repertoire of healthy donors. CD8 T cells isolated from multiple donors are subjected to a first sorting step after staining with HLA multimers carrying the peptide of interest. The isolated cells are expanded for two weeks, after which a second sorting is performed using the same peptide-HLA multimers. The cells isolated in this way are then processed for single-cell sequencing of their TCR alpha and beta chains. Newly identified TCRs are cloned in appropriate expression vectors for functional analysis on Jurkat, NK92, and primary CD8 T cells and tumor cells expressing the appropriate antigen. We have identified TCRs specifically binding HLA-A2 presenting epitopes of tumor antigens, which are capable of inducing TCR-mediated cell activation and cytotoxicity in target cancer cell lines. This method allows the identification of tumor-reactive TCRs in about two to three weeks, starting from peripheral blood samples of readily available healthy donors.

Keywords: cancer, TCR, tumor antigens, immunotherapy

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Authors: Preeti Pal, Anjali Pal

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Chitosan is an effective sorbent for removal of contaminants from wastewater. However, the ability of pure chitosan is specific because of its cationic charge. It causes repulsion in the removal process of various cationic charged molecules. The present study has been carried out for the successful removal of Pb²⁺ ions from aqueous solution by modified chitosan beads. Surface modification of chitosan (CS) beads was performed by using the anionic surfactant (AS), sodium dodecyl sulfate (SDS). Micelle aggregation property of SDS has been utilized for the formation of bilayer over the CS beads to produce surfactant modified chitosan (SMCS) beads. Prepared adsorbents were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) in order to find out their composition and surface morphology. SMCS beads, when compared to the pure CS beads, showed three times higher adsorption. This higher adsorption is believed to be due to the adsolubilization of Pb²⁺ ions on SDS bilayer. This bilayer provides more adsorption sites for quick and effective removal of Pb²⁺ ions from the aqueous phase. Moreover, the kinetic and adsorption isotherm models were employed to the obtained data for the description of the lead adsorption processes. It was found that the removal kinetics follows pseudo-second order model. Adsorption isotherm data fits well to the Langmuir model. The maximum adsorption capacity obtained is 100 mg/g at the dosage of 0.675 g/L for 50 mg/L of Pb²⁺. The adsorption capacity is subject to increase with increasing the Pb²⁺ ions concentration in the solution. The results indicated that the prepared hydrogel beads are efficient adsorbent for removal of Pb²⁺ ions from the aqueous medium.

Keywords: adsolubilisation, anionic surfactant, bilayer, chitosan, Pb²⁺

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Authors: Namsoo Kim, So-Hee Son, Jin-Soo Maeng, Yong-Jin Cho, Chong-Tai Kim

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It has been reported that some enzymes such as trypsin and Alcalase 2.4L are tolerant to a medium high pressure of 300 MPa and preparation of protein hydrolyzates under 300 MPa was advantageous with regard to hydrolysis rate and thus production yield compared with the counterpart under ambient pressure.1,2) In this study, nine fish comprising halibut, soft shell clam and carp were hydrolyzed using Flavourzyme 500MG only, and the combination of Flavourzyme 500 mg, Alcalase 2.4 L, Marugoto E, and Protamex under 300 MPa. Then, the effects of single and multiple protease treatments were determined with respect to contents of soluble solid (SS) and soluble nitrogen, sensory attributes, electrophoretic profiles, and HPLC peak patterns of the fish hydrolyzates (FHs) from various species. The contents of SS of the FHs were quite species-specific and the hydrolyzates of halibut showed the highest SS contents. At this point, multiple protease treatment increased SS content conspicuously in all fish tested. The contents of total soluble nitrogen and TCA-soluble nitrogen were well correlated with those of SS irrespective of fish species and methods of enzyme treatment. Also, it was noticed that multiple protease treatment improved sensory attributes of the FHs considerably. Electropherograms of the FHs showed fast migrating peptide bands that had the molecular masses mostly lower than 1 kDa and this was confirmed by peptide patterns from HPLC analysis for some FHs that had good sensory quality.

Keywords: production, fish hydrolyzates, protease treatments, high pressure

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Authors: Itzel Amaro-Estrada, Eduardo Vergara-Rivera, Virginia Juarez-Flores, Mayra Cobaxin-Cardenas, Rosa Estela Quiroz, Jesus F. Preciado, Sergio Rodriguez-Camarillo

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Bovine anaplasmosis is an infectious, tick-borne disease caused mainly by Anaplasma marginale; typical signs include anemia, fever, abortion, weight loss, decreased milk production, jaundice, and potentially death. Sick bovine can recover when antibiotics are administered; however, it usually remains as carrier for life, being a risk of infection for susceptible cattle. Anaplasma marginale is an obligate intracellular Gram-negative bacterium with genetic composition highly diverse among geographical isolates. There are currently no vaccines fully effective against bovine anaplasmosis; therefore, the economic losses due to disease are present. Vaccine formulation became a hard task for several pathogens as Anaplasma marginale, but peptide-based vaccines are an interesting proposal way to induce specific responses. Phage-displayed peptide libraries have been proved one of the most powerful technologies for identifying specific ligands. Screening of these peptides libraries is also a tool for studying interactions between proteins or peptides. Thus, it has allowed the identification of ligands recognized by polyclonal antiserums, and it has been successful for the identification of relevant epitopes in chronic diseases and toxicological conditions. Protective immune response to bovine anaplasmosis includes high levels of immunoglobulins subclass G2 (IgG2) but not subclass IgG1. Therefore, IgG2 from the serum of protected bovine can be useful to identify ligands, which can be part of an immunogen for cattle. In this work, phage display random peptide library Ph.D. ™ -12 was incubating with IgG2 or blood sera of immunized bovines against A. marginale as targets. After three rounds of biopanning, several candidates were selected for additional analysis. Subsequently, their reactivity with sera immunized against A. marginale, as well as with positive and negative sera to A. marginale was evaluated by immunoassays. A collection of recognized peptides tested by ELISA was generated. More than three hundred phage-peptides were separately evaluated against molecules which were used during panning. At least ten different peptides sequences were determined from their nucleotide composition. In this approach, three phage-peptides were selected by their binding and affinity properties. In the case of the development of vaccines or diagnostic reagents, it is important to evaluate the immunogenic and antigenic properties of the peptides. Immunogenic in vitro and in vivo behavior of peptides will be assayed as synthetic and as phage-peptide for to determinate their vaccine potential. Acknowledgment: This work was supported by grant SEP-CONACYT 252577 given to I. Amaro-Estrada.

Keywords: bovine anaplasmosis, peptides, phage display, veterinary vaccines

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Authors: Ceren Karaman, Onur Karaman

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The adsorption of Remazol Black B (RBB), an anionic dye, onto dried orange pulp (DOP) adsorbent prepared by only drying and by treating with cetyltrimetylammonium bromide (CTAB), a cationic surfactant, surface-modified orange pulp (SMOP) was studied in a stirred batch experiments system at 25°C. The adsorption of RBB on each adsorbent as a function of surfactant dosage, initial pH of the solution and initial dye concentration was investigated. The optimum amount of CTAB was found to be 25g/l. For RBB adsorption studies, while working pH value for the DOP adsorbent system was determined as 2.0, it was observed that this value shifted to 8.0 when the 25 g/l CTAB treated-orange pulp (SMOP) adsorbent was used. It was obtained that the adsorption rate and capacity increased to a certain value, and the adsorption efficiency decreased with increasing initial RBB concentration for both DOP and SMOP adsorbents at pH 2.0 and pH 8.0. While the highest adsorption capacity for DOP was determined as 62.4 mg/g at pH 2.0, and as 325.0 mg/g for SMOP at pH 8.0. As a result, it can be said that permanent cationic coating of the adsorbent surface by CTAB surfactant shifted the working pH from 2.0 to 8.0 and it increased the dye adsorption rate and capacity of orange pulp much more significantly at pH 8.0. The equilibrium RBB adsorption data on each adsorbent were best described by the Langmuir isotherm model. The adsorption kinetics of RBB on each adsorbent followed a pseudo-second-order model. Moreover, the intraparticle diffusion model was used to describe the kinetic data. It was found that diffusion is not the only rate controlling step. The adsorbent was characterized by the Brunauer–Emmett–Teller (BET) analysis, Fourier-transform-infrared (FTIR) spectroscopy, and scanning-electron-microscopy (SEM). The mechanism for the adsorption of RBB on the SMOP may include hydrophobic interaction, van der Waals interaction, stacking and electrostatic interaction.

Keywords: adsorption, Cetyltrimethylammonium Bromide (CTAB), orange pulp, Remazol Black B (RBB), surface modification

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Authors: Doriane Delafosse, Dominique Fontvieille

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Background: Wastewater treatment plants (WWTPs) generally display significant efficacy in virus retention yet, are sometimes highly variable, partly in relation to large fluctuating loads at the head of the plant and partly because of episodic dysfunctions in some treatment processes. The problem is especially sensitive when human enteric viruses, such as human Noroviruses Genogroup I or Adenoviruses, are in concern: their release downstream WWTP, in environments often interconnected to recreational areas, may be very harmful to human communities even at low concentrations. It points out the importance of WWTP permanent monitoring from which their internal treatment processes could be adjusted. One way to adjust primary treatments is to add coagulants and flocculants to sewage ahead settling tanks to improve decantation. In this work, sludge produced by three coagulants (two organics, one mineral), four flocculants (three cationic, one anionic), and their combinations were studied for their efficacy in human enteric virus retention. Sewage samples were coming from a WWTP in the vicinity of the laboratory. All experiments were performed three times and in triplicates in laboratory pilots, using Murine Norovirus (MNV-1), a surrogate of human Norovirus, as an internal control (spiking). Viruses were quantified by (RT-)qPCR after nucleic acid extraction from both treated water and sediment. Results: Low values of sludge virus retention (from 4 to 8% of the initial sewage concentration) were observed with each cationic organic flocculant added to wastewater and no coagulant. The largest part of the virus load was detected in the treated water (48 to 90%). However, it was not counterbalancing the amount of the introduced virus (MNV-1). The results pertained to two types of cationic flocculants, branched and linear, and in the last case, to two percentages of cations. Results were quite similar to the association of a linear cationic organic coagulant and an anionic flocculant, though suggesting that differences between water and sludges would sometimes be related to virus size or virus origins (autochthonous/allochthonous). FeCl₃, as a mineral coagulant associated with an anionic flocculant, significantly increased both auto- and allochthonous virus retention in the sediments (15 to 34%). Accordingly, virus load in treated water was lower (14 to 48%) but with a total that still does not reach the amount of the introduced virus (MNV-1). It also appeared that the virus retrieval in a bare 0.1M NaCl suspension varied rather strongly according to the FeCl₃ concentration, suggesting an inhibiting effect on the molecular analysis used to detect the virus. Finally, no viruses were detected in both phases (sediment and water) with the combination branched cationic coagulant-linear anionic flocculant, which was later demonstrated as an effect, here also, of polymers on the virus detection-molecular analysis. Conclusions: The combination of FeCl₃-anionic flocculant gave its highest performance to the decantation-based virus removal process. However, large unbalanced values in spiking experiments were observed, suggesting that polymers cast additional obstacles to both elution buffer and lysis buffer on their way to reach the virus. The situation was probably even worse with autochthonous viruses already embedded into sewage's particulate matter. Polymers and FeCl₃ also appeared to interfere in some steps of molecular analyses. More attention should be paid to such impediments wherever chemical additives are considered to be used to enhance WWTP processes. Acknowledgments: This research was supported by the ABIOLAB laboratory (Montbonnot Saint-Martin, France) and by the ASPOSAN association. Field experiments were possible thanks to the Grand Chambéry WWTP authorities (Chambéry, France).

Keywords: flocculants-coagulants, polymers, enteric viruses, wastewater sedimentation treatment plant

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Authors: Edite Kulmane, Mara Pilmane, Romans Lacis

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Introduction: Acquired heart diseases remain one of the leading health care problems in the world. Changes in myocardium of the diseased hearts are complex and pathogenesis is still not fully clear. The aim of this study was to identify appearance and distribution of apoptosis, homeostasis regulating factors, and innervation and ischemia markers in right atrial tissue in different acquired heart diseases. Methods: During elective open heart surgery were taken right atrial tissue fragments from 12 patients. All patients were operated because of acquired heart diseases- aortic valve stenosis (5 patients), coronary heart disease (5 patients), coronary heart disease and secondary mitral insufficiency (1 patient) and mitral disease (1 patient). The mean age was (mean±SD) 70,2±7,0 years (range 58-83 years). The tissues were stained with haematoxylin and eosin methods for routine light-microscopical examination and for immunohistochemical detection of protein gene peptide 9.5 (PGP 9.5), human atrial natriuretic peptide (hANUP), vascular endothelial growth factor (VEGF), chromogranin A and endothelin. Apoptosis was detected by TUNEL method. Results: All specimens showed degeneration of cardiomyocytes with lysis of myofibrils, diffuse vacuolization especially in perinuclear region, different size of cells and their nuclei. The severe invasion of connective tissue was observed in main part of all fragments. The apoptotic index ranged from 24 to 91%. One specimen showed region of newly performed microvessels with cube shaped endotheliocytes that were positive for PGP 9.5, endothelin, chromogranin A and VEGF. From all fragments, taken from patients with coronary heart disease, there were observed numerous PGP 9.5-containing nerve fibres, except in patient with secondary mitral insufficiency, who showed just few PGP 9.5 positive nerves. In majority of specimens there were regions observed with cube shaped mixed -VEGF immunoreactive endocardial and epicardial cells. Only VEGF positive endothelial cells were observed just in few specimens. There was no significant difference of hANUP secreting cells among all specimens. All patients operated due to the coronary heart disease moderate to numerous number of chromogranin A positive cells were seen while in patients with aortic valve stenosis tissue demonstrated just few factor positive cells. Conclusions: Complex detection of different factors may indicate selectively disordered morphopathogenetical event of heart disease: decrease of PGP 9.5 nerves suggests the decreased innervation of organ; increased apoptosis indicates the cell death without ingrowth of connective tissue; persistent presence of hANUP proves the unchanged homeostasis of cardiomyocytes probably supported by expression of chromogranins. Finally, decrease of VEGF detects the regions of affected blood vessels in heart affected by acquired heart disease.

Keywords: heart, apoptosis, protein-gene peptide 9.5, atrial natriuretic peptide, vascular endothelial growth factor, chromogranin A, endothelin

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Authors: Henry Memczak, Marc Hovestaedt, Bernhard Ay, Sandra Saenger, Thorsten Wolff, Frank F. Bier

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The influenza viruses cause flu epidemics every year and serious pandemics in larger time intervals. The only cost-effective protection against influenza is vaccination. Due to rapid mutation continuously new subtypes appear, what requires annual reimmunization. For a correct vaccination recommendation, the circulating influenza strains had to be detected promptly and exactly and characterized due to their antigenic properties. During the flu season 2016/17, a wrong vaccination recommendation has been given because of the great time interval between identification of the relevant influenza vaccine strains and outbreak of the flu epidemic during the following winter. Due to such recurring incidents of vaccine mismatches, there is a great need to speed up the process chain from identifying the right vaccine strains to their administration. The monitoring of subtypes as part of this process chain is carried out by national reference laboratories within the WHO Global Influenza Surveillance and Response System (GISRS). To this end, thousands of viruses from patient samples (e.g., throat smears) are isolated and analyzed each year. Currently, this analysis involves complex and time-intensive (several weeks) animal experiments to produce specific hyperimmune sera in ferrets, which are necessary for the determination of the antigen profiles of circulating virus strains. These tests also bear difficulties in standardization and reproducibility, which restricts the significance of the results. To replace this test a peptide-based assay for influenza virus subtyping from corresponding virus samples was developed. The differentiation of the viruses takes place by a set of specifically designed peptidic recognition molecules which interact differently with the different influenza virus subtypes. The differentiation of influenza subtypes is performed by pattern recognition guided by machine learning algorithms, without any animal experiments. Synthetic peptides are immobilized in multiplex format on various platforms (e.g., 96-well microtiter plate, microarray). Afterwards, the viruses are incubated and analyzed comparing different signaling mechanisms and a variety of assay conditions. Differentiation of a range of influenza subtypes, including H1N1, H3N2, H5N1, as well as fine differentiation of single strains within these subtypes is possible using the peptide-based subtyping platform. Thereby, the platform could be capable of replacing the current antigenic characterization of influenza strains using ferret hyperimmune sera.

Keywords: antigenic characterization, influenza-binding peptides, influenza subtyping, influenza surveillance

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Authors: Ava Faridi, Pouya Faridi, Aleksandr Kakinen, Ibrahim Javed, Thomas P. Davis, Pu Chun Ke

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Human islet amyloid polypeptide (IAPP) is a hormone associated with glycemic control and type 2 diabetes. Biophysically, the chirality of IAPP fibrils has been little explored with respect to the aggregation and toxicity of the peptide. Biochemically, it remains unclear as for how protein expression in pancreatic beta cells may be altered by cell exposure to the peptide, and how such changes may be mitigated by nanoparticle inhibitors for IAPP aggregation. In this study, we first demonstrated the elimination of the IAPP nucleation phase and shortening of its elongation phase by silica nanoribbons. This accelerated IAPP fibrillization translated to reduced toxicity, especially for the right-handed silica nanoribbons, as revealed by cell viability, helium ion microscopy, as well as zebrafish embryo survival, developmental and behavioral assays. We then examined the proteomes of βTC6 pancreatic beta cells exposed to the three main aggregation states of monomeric, oligomeric and amyloid fibrillar IAPP, and compared that with cellular protein expression modulated by graphene quantum dots (GQDs). A total of 29 proteins were significantly regulated by different forms of IAPP, and the majority of these proteins were nucleotide-binding proteins. A regulatory capacity of GQDs against aberrant protein expression was confirmed. These studies have demonstrated the great potential of employing nanomaterials targeting the mesoscopic enantioselectivity and protein expression dysregulation in pancreatic beta cells.

Keywords: graphene quantum dots, IAPP, silica nanoribbons, protein expression, toxicity

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Authors: Choukri Lekbir, Mira Mokhtari

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Hydrogen-Materials interaction and mechanisms can be modeled at nano scale by quantum methods. In this work, the effect of hydrogen on the electronic properties of a cluster material model «nickel» has been studied by using of density functional theoretical (DFT) method. Two types of clusters are optimized: Nickel and hydrogen-nickel system. In the case of nickel clusters (n = 1-6) without presence of hydrogen, three types of electronic structures (neutral, cationic and anionic), have been optimized according to three basis sets calculations (B3LYP/LANL2DZ, PW91PW91/DGDZVP2, PBE/DGDZVP2). The comparison of binding energies and bond lengths of the three structures of nickel clusters (neutral, cationic and anionic) obtained by those basis sets, shows that the results of neutral and anionic nickel clusters are in good agreement with the experimental results. In the case of neutral and anionic nickel clusters, comparing energies and bond lengths obtained by the three bases, shows that the basis set PBE/DGDZVP2 is most suitable to experimental results. In the case of anionic nickel clusters (n = 1-6) with presence of hydrogen, the optimization of the hydrogen-nickel (anionic) structures by using of the basis set PBE/DGDZVP2, shows that the binding energies and bond lengths increase compared to those obtained in the case of anionic nickel clusters without the presence of hydrogen, that reveals the armor effect exerted by hydrogen on the electronic structure of nickel, which due to the storing of hydrogen energy within nickel clusters structures. The comparison between the bond lengths for both clusters shows the expansion effect of clusters geometry which due to hydrogen presence.

Keywords: binding energies, bond lengths, density functional theoretical, geometry optimization, hydrogen energy, nickel cluster

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Authors: Arefeh Jafarian, Mohammad Taghikani, Saied Abroun, Amir Allahverdi, Masoud Soleimani

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Introduction: The miRNAs have key roles in control of pancreatic islet development and insulin secretion. In this regards, current study investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9. Findings: After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose as well as extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. In derived IPCs by miR-375 alone are capable to express insulin and other endocrine specific transcription factors, the cells lack the machinery to respond to glucose. The differentiated hMSCs by miR-375 and anti-miR-9 lentiviruses could secrete insulin and c-peptide in a glucose-regulated manner. Conclusion: It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.

Keywords: diabetes, differentiation, MSCs, insulin producing cells, miR-375, miR-9

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Authors: Vrushali Guhe, Shailza Singh

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Cutaneous leishmaniasis is neglected tropical disease present worldwide caused by the protozoan parasite Leishmania major, the therapeutic armamentarium for leishmaniasis are showing several limitations as drugs are showing toxic effects with increasing resistance by a parasite. Thus identification of novel therapeutic targets is of paramount importance. Previous studies have shown that autophagy, a cellular process, can either facilitate infection or aid in the elimination of the parasite, depending on the specific parasite species and host background in leishmaniasis. In the present study, our objective was to target the essential autophagy protein ATG8, which plays a crucial role in the survival, infection dynamics, and differentiation of the Leishmania parasite. ATG8 in Leishmania major and its homologue, LC3, in Homo sapiens, act as autophagic markers. Present study manifested the crucial role of ATG8 protein as a potential target for combating Leishmania major infection. Through bioinformatics analysis, we identified non-conserved motifs within the ATG8 protein of Leishmania major, which are not present in LC3 of Homo sapiens. Against these two non-conserved motifs, we generated a peptide library of 60 peptides on the basis of physicochemical properties. These peptides underwent a filtering process based on various parameters, including feasibility of synthesis and purification, compatibility with Selective Reaction Monitoring (SRM)/Multiple reaction monitoring (MRM), hydrophobicity, hydropathy index, average molecular weight (Mw average), monoisotopic molecular weight (Mw monoisotopic), theoretical isoelectric point (pI), and half-life. Further filtering criterion shortlisted three peptides by using molecular docking and molecular dynamics simulations. The direct interaction between ATG8 and the shortlisted peptides was confirmed through Surface Plasmon Resonance (SPR) experiments. Notably, these peptides exhibited the remarkable ability to penetrate the parasite membrane and exert profound effects on Leishmania major. The treatment with these peptides significantly impacted parasite survival, leading to alterations in the cell cycle and morphology. Furthermore, the peptides were found to modulate autophagosome formation, particularly under starved conditions, suggesting their involvement in disrupting the regulation of autophagy within Leishmania major. In vitro, studies demonstrated that the selected peptides effectively reduced the parasite load within infected host cells. Encouragingly, these findings were corroborated by in vivo experiments, which showed a reduction in parasite burden upon peptide administration. Additionally, the peptides were observed to affect the levels of LC3II within host cells. In conclusion, our findings highlight the efficacy of these novel peptides in targeting Leishmania major’s ATG8 and disrupting parasite survival. These results provide valuable insights into the development of innovative therapeutic strategies against leishmaniasis via targeting autophagy protein ATG8 of Leishmania major.

Keywords: ATG8, leishmaniasis, surface plasmon resonance, MD simulation, molecular docking, peptide designing, therapeutics

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Authors: Marcin Stasiulewicz, Sebastian Filipkowski, Aneta Panuszko

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Background: The hallmark of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, is an aggregation of the abnormal forms of peptides and proteins. Water is essential to functioning biomolecules, and it is one of the key factors influencing protein folding and misfolding. However, the hydration studies of proteins are complicated due to the complexity of protein systems. The use of model compounds can facilitate the interpretation of results involving larger systems. Objectives: The goal of the research was to characterize the properties of the hydration water surrounding the two three-residue K peptide fragments INS (Isoleucine - Asparagine - Serine) and NSR (Asparagine - Serine - Arginine). Methods: Fourier-transform infrared spectra of aqueous solutions of the tripeptides were recorded on Nicolet 8700 spectrometer (Thermo Electron Co.) Measurements were carried out at 25°C for varying molality of solute. To remove oscillation couplings from water spectra and, consequently, obtain narrow O-D semi-heavy water bands (HDO), the isotopic dilution method of HDO in H₂O was used. The difference spectra method allowed us to isolate the tripeptide-affected HDO spectrum. Results: The structural and energetic properties of water affected by the tripeptides were compared to the properties of pure water. The shift of the values of the gravity center of bands (related to the mean energy of water hydrogen bonds) towards lower values with respect to the ones corresponding to pure water suggests that the energy of hydrogen bonds between water molecules surrounding tripeptides is higher than in pure water. A comparison of the values of the mean oxygen-oxygen distances in water affected by tripeptides and pure water indicates that water-water hydrogen bonds are shorter in the presence of these tripeptides. The analysis of differences in oxygen-oxygen distance distributions between the tripeptide-affected water and pure water indicates that around the tripeptides, the contribution of water molecules with the mean energy of hydrogen bonds decreases, and simultaneously the contribution of strong hydrogen bonds increases. Conclusions: It was found that hydrogen bonds between water molecules in the hydration sphere of tripeptides are shorter and stronger than in pure water. It means that in the presence of the tested tripeptides, the structure of water is strengthened compared to pure water. Moreover, it has been shown that in the vicinity of the Asparagine - Serine - Arginine, water forms stronger and shorter hydrogen bonds. Acknowledgments: This work was funded by the National Science Centre, Poland (grant 2017/26/D/NZ1/00497).

Keywords: amyloids, K-peptide, hydration, FTIR spectroscopy

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Authors: Suwapitch Chalongkulasak, Teerasak E-Kobon, Pramote Chumnanpuen

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Acne vulgaris is a common skin disease mainly caused by the Gram–positive pathogenic bacterium, Propionibacterium acnes. This bacterium stimulates inflammation process in human sebaceous glands. Giant African snail (Achatina fulica) is alien species that rapidly reproduces and seriously damages agricultural products in Thailand. There were several research reports on the medical and pharmaceutical benefits of this snail mucus peptides and proteins. This study aimed to in silico predict multifunctional bioactive peptides from A. fulica mucus peptidome using several bioinformatic tools for determination of antimicrobial (iAMPpred), anti–biofilm (dPABBs), cytotoxic (Toxinpred), cell membrane penetrating (CPPpred) and anti–quorum sensing (QSPpred) peptides. Three candidate peptides with the highest predictive score were selected and re-designed/modified to improve the required activities. Structural and physicochemical properties of six anti–P. acnes (APA) peptide candidates were performed by PEP–FOLD3 program and the five aforementioned tools. All candidates had random coiled structure and were named as APA1–ori, APA2–ori, APA3–ori, APA1–mod, APA2–mod and APA3–mod. To validate the APA activity, these peptide candidates were synthesized and tested against six isolates of P. acnes. The modified APA peptides showed high APA activity on some isolates. Therefore, our biomimetic mucus peptides could be useful for preventing acne vulgaris and further examined on other activities important to medical and pharmaceutical applications.

Keywords: Propionibacterium acnes, Achatina fulica, peptidomes, antibacterial peptides, snail mucus

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Authors: Weiwei Cao, Xiaodong Xing

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In order to effectively resolve the microbial pollution and contamination, synthetic nano-antibacterial materials are widely used in daily life. Among them, nanodiamonds (NDs) have recently been demonstrated to hold promise as useful materials in biomedical applications due to their high specific surface area and biocompatibility. In this work, the copolymer, poly(4-vinylpyridine-co-2-hydroxyethyl methacrylate) was applied for the surface functionalization of NDs to produce the quaternized poly(4-vinylpyridine-co-2-hydroxyethyl methacrylate)-functionalized NDs (QNDs). Then, QNDs were used as a substrate for silver nanoparticles (AgNPs) to produce a QND@Ag hybrid. The composition and morphology of the resultant nanostructures were confirmed by Fourier transform infrared spectra (FT-IR), transmission electron microscope (TEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The mass fraction of AgNPs in the nanocomposites was about 35.7%. The antibacterial performances of the prepared nanocomposites were evaluated with Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus by minimum inhibitory concentration (MIC), inhibition zone testing and time-kill study. As a result, due to the synergistic antibacterial activity of QND and AgNPs, this hybrid showed substantially higher antibacterial activity than QND and polyvinyl pyrrolidone (PVP)-stabilized AgNPs, and the AgNPs on QND@Ag were more stable than the Ag NPs on PVP, resulting in long-term antibacterial effects. More importantly, this hybrid showed excellent water solubility and low cytotoxicity, suggesting the great potential application in biomedical applications. The present work provided a simple strategy that successfully turned NDs into nanosized antibiotics with simultaneous superior stability and biocompatibility, which would broaden the applications of NDs and advance the development of novel antibacterial agents.

Keywords: cationic copolymer, nanodiamonds, silver nanoparticles, dual antibacterial activity, lower cytotoxicity

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Authors: Sophio Kobauri, Temur Kantaria, Nina Kulikova, David Tugushi, Ramaz Katsarava

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Polymeric nanoparticles-based drug delivery systems and therapeutics have a great potential in the treatment of a numerous diseases, due to they are characterizing the flexible properties which is giving possibility to modify their structures with a complex definition over their structures, compositions and properties. Important characteristics of the polymeric nanoparticles (PNPs) used as drug carriers are high particle’s stability, high carrier capacity, feasibility of encapsulation of both hydrophilic and hydrophobic drugs, and feasibility of variable routes of administration, including oral application and inhalation; NPs are especially effective for intracellular drug delivery since they penetrate into the cells’ interior though endocytosis. A variety of PNPs based drug delivery systems including charged and neutral, degradable and non-degradable polymers of both natural and synthetic origin have been developed. Among these huge varieties the biodegradable PNPs which can be cleared from the body after the fulfillment of their function could be considered as one of the most promising. For intracellular uptake it is highly desirable to have positively charged PNPs since they can penetrate deep into cell membranes. For long-lasting circulation of PNPs in the body it is important they have so called “stealth coatings” to protect them from the attack of immune system of the organism. One of the effective ways to render the PNPs “invisible” for immune system is their PEGylation which represent the process of pretreatment of polyethylene glycol (PEG) on the surface of PNPs. The present work deals with constructing PNPs from amino acid based biodegradable polymers – regular poly(ester amide) (PEA) composed of sebacic acid, leucine and 1,6-hexandiol (labeled as 8L6), cationic PEA composed of sebacic acid, arginine and 1,6-hexandiol (labeled as 8R6), and comb-like co-PEA composed of sebacic acid, malic acid, leucine and 1,6-hexandiol (labeled as PEG-PEA). The PNPs were fabricated using the polymer deposition/solvent displacement (nanoprecipitation) method. The regular PEA 8L6 form stable negatively charged (zeta-potential within 2-12 mV) PNPs of desired size (within 150-200 nm) in the presence of various surfactants (Tween 20, Tween 80, Brij 010, etc.). Blending the PEAs 8L6 and 8R6 gave the 130-140 nm sized positively charged PNPs having zeta-potential within +20 ÷ +28 mV depending 8L6/8R6 ratio. The PEGylating PEA PEG-PEA was synthesized by interaction of epoxy-co-PEA [8L6]0,5-[tES-L6]0,5 with mPEG-amine-2000 The stable and positively charged PNPs were fabricated using pure PEG-PEA as a surfactant. A firm anchoring of the PEG-PEA with 8L6/8R6 based PNPs (owing to a high afinity of the backbones of all three PEAs) provided good stabilization of the NPs. In vitro biocompatibility study of the new PNPs with four different stable cell lines: A549 (human), U-937 (human), RAW264.7 (murine), Hepa 1-6 (murine) showed they are biocompatible. Considering high stability and cell compatibility of the elaborated PNPs one can conclude that they are promising for subsequent therapeutic applications. This work was supported by the joint grant from the Science and Technology Center in Ukraine and Shota Rustaveli National Science Foundation of Georgia #6298 “New biodegradable cationic polymers composed of arginine and spermine-versatile biomaterials for various biomedical applications”.

Keywords: biodegradable poly(ester amide)s, cationic poly(ester amide), pegylating poly(ester amide), nanoparticles

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Authors: Anita K. Kovacs, Peter Hegyes, Zsolt Bozso, Gabor Toth

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Fluorination has been used extensively by the pharmaceutical industry as a strategy to improve the pharmacokinetics of drugs due to its effectiveness in increasing the potency of antimicrobial peptides (AMPs). Multiple-fluorinated indole-ring-containing tryptophan derivatives have the potential of having better antimicrobial activity than the widely used mono-fluorinated indole-ring containing tryptophan derivatives, but they are not available commercially. Therefore, our goal is to synthesize multiple-fluorinated indole-ring containing tryptophan derivatives to incorporate them into AMPs to enhance their antimicrobial activity. During our work, we are trying several methods (classical organic synthesis, enzymic synthesis, and solid phase peptide synthesis) for the synthesis of the said compounds, with mixed results. With classical organic synthesis (four different routes), we did not get the desired results. The reaction of serin with substituted indole in the presence of acetic anhydride led to racemic tryptophane; with the reaction of protected serin with indole in the presence of nickel complex was unsuccessful; the reaction of serin containing protected dipeptide with disuccinimidyl carbonate we achieved a tryptophane containing dipeptide, its chiral purity is being examined; the reaction of alcohol with substituted indole in the presence of copper complex was successful, but it was only a test reaction, we could not reproduce the same result with serine. The undergoing tryptophan-synthase method has shown some potential, but our work has not been finished yet. The successful synthesis of the desired multiple-fluorinated indole-ring-containing tryptophan will be followed by solid phase peptide synthesis in order to incorporate it into AMPs to enhance their antimicrobial activity. The successful completion of these phases will mean the possibility of manufacturing new, effective AMPs.

Keywords: halogenation, fluorination, tryptophan, enhancement of antimicrobial activity

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Authors: Charmi Chande, Ravindra Phadke

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Microfluidics devices are fabricated by using multiple fabrication methods. Photolithography is one of the common methods wherein SU8 is widely used for making master which in turn is used for making working chip by the process of soft lithography. The high-aspect ratio features of SU-8 makes it suitable to be used as micro moulds for injection moulding, hot embossing, and moulds to form polydimethylsiloxane (PDMS) structures for bioMEMS (Microelectromechanical systems) applications. But due to high cost, difficulty in procuring and need for clean room, restricts the use of this polymer especially in developing countries and small research labs. ‘Bisphenol –A’ based polymers in mixture with curing agent are used in various industries like Paints and coatings, Adhesives, Electrical systems and electronics, Industrial tooling and composites. We present the novel use of ‘Bisphenol – A’ based polymer in fabricating micro channels for Lab On Chip(LOC) devices. The present paper describes the prototype for production of microfluidics chips using range of ‘Bisphenol-A’ based polymers viz. GY 250, ATUL B11, DER 331, DER 330 in mixture with cationic photo initiators. All the steps of chip production were carried out using an inexpensive approach that uses low cost chemicals and equipment. This even excludes the need of clean room. The produced chips using all above mentioned polymers were validated with respect to height and the chip giving least height was selected for further experimentation. The lowest height achieved was 7 micrometers by GY250. The cost of the master fabricated was $ 0.20 and working chip was $. 0.22. The best working chip was used for morphological identification and profiling of microorganisms from environmental samples like soil, marine water and salt water pan sites. The current chip can be adapted for various microbiological screening experiments like biochemical based microbial identification, studying uncultivable microorganisms at single cell/community level.

Keywords: bisphenol–A based epoxy, cationic photoinitiators, microfabrication, photolithography

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Authors: B. J. Mahen, N. E. Lloyd-Gale, S. Johnson, W. P. Rakowicz, M. J. Harris, A. D. Miller

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Background: Migraine is a leading cause of disability in the world. Calcitonin gene-related peptide (CGRP) receptor antagonists offer an approach to migraine prophylaxis by inhibiting the inflammatory and vasodilatory effects of CGRP. In recent years, NICE licensed the use of three CGRP-receptor antagonists: Fremanezumab, Galcanezumab, and Erenumab. Here, we present the outcomes of CGRP-antagonist treatment in a cohort of patients who suffer from episodic or chronic migraine and have failed at least three oral prophylactic therapies. Methods: We offered CGRP antagonists to 86 patients who met the NICE criteria to start therapy. We recorded the number of headache days per month (HDPM) at 0 weeks, 3 months, and 12 months. Of those, 26 patients were switched to an alternative treatment due to poor response or side effects. Of the 112 total cases, 9 cases did not sufficiently maintain their headache diary, and 5 cases were not followed up at 3 months. We have therefore included 98 sets of data in our analysis. Results: Fremanezumab achieved a reduction in HDPM by 51.7% at 3 months (p<0.0001), with 63.7% of patients meeting NICE criteria to continue therapy. Patients trialed on Galcanezumab attained a reduction in HDPM by 47.0% (p=0.0019), with 51.6% of patients meeting NICE criteria to continue therapy. Erenumab, however, only achieved a reduction in HDPM by 17.0% (p=0.29), and this was not statistically significant. Furthermore, 34.4%, 9.7%, and 4.9% of patients taking Fremanezumab, Galcanezumab, and Erenumab, respectively, continued therapy beyond 12 months. Of those who attempted drug holidays following 12 months of treatment, migraine symptoms relapsed in 100% of cases. Conclusion: We observed a significant improvement in HDPM amongst episodic and chronic migraine patients following treatment with Fremanezumab or Galcanezumab.

Keywords: migraine, CGRP, fremanezumab, galcanezumab, erenumab

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Authors: Dafna Knani, Sarit S. Sivan

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Aggrecan, one of the main components of the intervertebral disc (IVD), belongs to the family of proteoglycans (PGs) that are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein. Its primary function is to maintain tissue hydration and hence disc height under the high loads imposed by muscle activity and body weight. Significant PG loss is one of the first indications of disc degeneration. A possible solution to recover disc functions is by injecting a synthetic hydrogel into the joint cavity, hence mimicking the role of PGs. One of the hydrogels proposed is GAG-analogues, based on sulfate-containing polymers, which are responsible for hydration in disc tissue. In the present work, we used molecular dynamics (MD) to study the effect of the hydrogel crosslinking (type and degree) on the swelling behavior of the suggested GAG-analogue biomimetics by calculation of cohesive energy density (CED), solubility parameter, enthalpy of mixing (ΔEmix) and the interactions between the molecules at the pure form and as a mixture with water. The simulation results showed that hydrophobicity plays an important role in the swelling of the hydrogel, as indicated by the linear correlation observed between solubility parameter values of the copolymers and crosslinker weight ratio (w/w); this correlation was found useful in predicting the amount of PEGDA needed for the desirable hydration behavior of (CS)₄-peptide. Enthalpy of mixing calculations showed that all the GAG analogs, (CS)₄ and (CS)₄-peptide are water-soluble; radial distribution function analysis revealed that they form interactions with water molecules, which is important for the hydration process. To conclude, our simulation results, beyond supporting the experimental data, can be used as a useful predictive tool in the future development of biomaterials, such as disc replacement.

Keywords: molecular dynamics, proteoglycans, enthalpy of mixing, swelling

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Authors: Mahmut Sozmen, Alparslan Kadir Devrim, Yonca Betil Kabak, Tuba Devrim

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Acute myocardial infarction is the leading cause of deaths in the worldwide. Mature cardiomyocytes do not have the ability to regenerate instead fibrous tissue proliferate and granulation tissue to fill out. Periostin is an extracellular matrix protein from fasciclin family and it plays an important role in the cell adhesion, migration, and growth of the organism. Periostin prevents apoptosis while stimulating cardiomyocytes. The main objective of this project is to investigate the effects of the recombinant murine periostin peptide administration for the cardiomyocyte regeneration in a rat model of acute myocardial infarction. The experiment was performed on 84 male rats (6 months old) in 4 group each contains 21 rats. Saline applied subcutaneously (1 ml/kg) two times with 24 hours intervals to the rats in control group (Group 1). Recombinant periostin peptide (1 μg/kg) dissolved in saline applied intraperitoneally in group 2 on 1, 3, 7, 14 and 21. days on same dates in group 4. Isoproterenol dissolved in saline applied intraperitoneally (85mg/kg/day) two times with 24 hours intervals to the groups 3 and 4. Rats in group 4 further received recombinant periostin peptide (1 μg/kg) dissolved in saline intraperitoneally starting one day after the final isoproterenol administration on days 1, 3, 7, 14 and 21. Following the final application of periostin rats continued to feed routinely with pelleted chow and water ad libitum for further seven days. At the end of 7th day rats sacrificed, blood and heart tissue samples collected for the immunohistochemical and biochemical analysis. Angiogenesis in response to tissue damage, is a highly dynamic process regulated by signals from the surrounding extracellular matrix and blood serum. In this project, VEGF, ANGPT, bFGF, TGFβ are the key factors that contribute to cardiomyocyte regeneration were investigated. Additionally, the relationship between mitosis and apoptosis (Bcl-2, Bax, PCNA, Ki-67, Phopho-Histone H3), cell cycle activators and inhibitors (Cyclin D1, D2, A2, Cdc2), the origin of regenerating cells (cKit and CD45) were examined. Present results revealed that periostin stimulated cardiomyocye cell-cycle re-entry in both normal and MCA damaged cardiomyocytes and increased angiogenesis. Thus, periostin contributes to cardiomyocyte regeneration during the healing period following myocardial infarction which provides a better understanding of its role of this mechanism, improving recovery rates and it is expected to contribute the lack of literature on this subject. Acknowledgement: This project was financially supported by Turkish Scientific Research Council- Agriculture, Forestry and Veterinary Research Support Group (TUBİTAK-TOVAG; Project No: 114O734), Ankara, TURKEY.

Keywords: cardiotoxicity, immunohistochemistry, isoproterenol, periostin

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