Search results for: recombinant E. coli

Authors: Afaf Bouchoucha, Karima Si Larbi, Mohamed Amine Bourouaia, Salah.Boulanouar, Safia.Djabbar

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The synthesis, characterization and comparative biological study of manganese (II) and copper (II) complexes with an heterocyclic ligand used in pharmaceutical field (Scheme 1), were reported. Two kinds of complexes were obtained with derivative sulfonamide, [M (L)₂ (H₂O)₂].H₂O and [M (L)₂ (Cl)₂]3H₂O. These complexes have been prepared and characterized by elemental analysis, FAB mass, ESR magnetic measurements, FTIR, UV-Visible spectra and conductivity. Their stability constants have been determined by potentiometric methods in a water-ethanol (90:10 v/v) mixture at a 0.2 mol l-1 ionic strength (NaCl) and at 25.0 ± 0.1 ºC using Sirko program. DFT calculations were done using B3LYP/6-31G(d) and B3LYP/LanL2DZ. The antimicrobial activity of ligand and complexes against the species Escherichia coli, P. aeruginosa, Klebsiella pneumoniae, S. aureus, Bacillus subtilisan, Candida albicans, Candida tropicalis, Saccharomyces, Aspergillus fumigatus and Aspergillus terreus has been carried out and compared using agar-diffusion method. Also, the toxicity study was evaluated on synchesis complexes using Mice of NMRI strain.

Keywords: hetterocyclic ligand, complex, stability constant, antimicrobial activity, DFT, acute and genotoxicity study

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Authors: Shagufta Jabeen, Faez J. Firdaus Abdullah, Zunita Zakaria, Nurulfiza M. Isa, Yung C. Tan, Wai Y. Yee, Abdul R. Omar

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Pasteurella multocida (PM) is an important veterinary opportunistic pathogen particularly associated with septicemic pasteurellosis, pneumonic pasteurellosis and hemorrhagic septicemia in cattle and buffaloes. P. multocida serotype A has been reported to cause fatal pneumonia and septicemia. Pasteurella multocida subspecies multocida of serotype A Malaysian isolate PMTB2.1 was first isolated from buffaloes died of septicemia. In this study, the genome of P. multocida strain PMTB2.1 was sequenced using third-generation sequencing technology, PacBio RS2 system and analyzed bioinformatically via de novo analysis followed by in-depth analysis based on comparative genomics. Bioinformatics analysis based on de novo assembly of PacBio raw reads generated 3 contigs followed by gap filling of aligned contigs with PCR sequencing, generated a single contiguous circular chromosome with a genomic size of 2,315,138 bp and a GC content of approximately 40.32% (Accession number CP007205). The PMTB2.1 genome comprised of 2,176 protein-coding sequences, 6 rRNA operons and 56 tRNA and 4 ncRNAs sequences. The comparative genome sequence analysis of PMTB2.1 with nine complete genomes which include Actinobacillus pleuropneumoniae, Haemophilus parasuis, Escherichia coli and five P. multocida complete genome sequences including, PM70, PM36950, PMHN06, PM3480, PMHB01 and PMTB2.1 was carried out based on OrthoMCL analysis and Venn diagram. The analysis showed that 282 CDs (13%) are unique to PMTB2.1and 1,125 CDs with orthologs in all. This reflects overall close relationship of these bacteria and supports the classification in the Gamma subdivision of the Proteobacteria. In addition, genomic distance analysis among all nine genomes indicated that PMTB2.1 is closely related with other five Pasteurella species with genomic distance less than 0.13. Synteny analysis shows subtle differences in genetic structures among different P.multocida indicating the dynamics of frequent gene transfer events among different P. multocida strains. However, PM3480 and PM70 exhibited exceptionally large structural variation since they were swine and chicken isolates. Furthermore, genomic structure of PMTB2.1 is more resembling that of PM36950 with a genomic size difference of approximately 34,380 kb (smaller than PM36950) and strain-specific Integrative and Conjugative Elements (ICE) which was found only in PM36950 is absent in PMTB2.1. Meanwhile, two intact prophages sequences of approximately 62 kb were found to be present only in PMTB2.1. One of phage is similar to transposable phage SfMu. The phylogenomic tree was constructed and rooted with E. coli, A. pleuropneumoniae and H. parasuis based on OrthoMCL analysis. The genomes of P. multocida strain PMTB2.1 were clustered with bovine isolates of P. multocida strain PM36950 and PMHB01 and were separated from avian isolate PM70 and swine isolates PM3480 and PMHN06 and are distant from Actinobacillus and Haemophilus. Previous studies based on Single Nucleotide Polymorphism (SNPs) and Multilocus Sequence Typing (MLST) unable to show a clear phylogenetic relatedness between Pasteurella multocida and the different host. In conclusion, this study has provided insight on the genomic structure of PMTB2.1 in terms of potential genes that can function as virulence factors for future study in elucidating the mechanisms behind the ability of the bacteria in causing diseases in susceptible animals.

Keywords: comparative genomics, DNA sequencing, phage, phylogenomics

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Authors: Salem El Ashoor, Fathia M. El-Meheishi, Ibtisam M. Diab

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Zirconium diammin and triammin complexes can be possess biological activities, these complexes were synthesized via the reaction equimolar quantity of (1:10-phenanthroline){NC3H3(C6H2)NC3H3} (L1) or 4-4-amino phenazone {ONC6H5(NH)CH(NH2} (L2) or diphenyl carbizon {HNNCO(NH)2(C6H5)} (L3) with Zirconium Salt {ZrOCl2} in ratio (1:1) to form complexes [{NC3H3(C6H2)NC3H3}ZrOCl2}] [ZrOCl2L1], [{(O2NC6H4(NH)(NH2)}ZrOCl2] [ZrOCl2L2] and [{HNNCO(NH)2(C6H5)ZrOCl2}] [ZrOCl2L3] respectively. The characterization of these complexes were follow by using Fourier Transform Infrared (FT-IR) and UV-Visible spectroscopy. Also a variable temperature study of these complexes has been followed by using UV-Visible spectroscopy to follow electronic transform behaviors under temperature control also DFT study calculation was follow these complexes via the information from FT-IR and UV-Visible spectroscopy. A coordination number of these complexes of types five and six of the geometry can be suggested. These complexes were found to shown deferent inhibition to the growth of bacterial strains of Bacillus spp & Klebsiella spp & E.coli & proteus spp & pseudomona spp) while all complexes were in deferent's concentration (0.001, 0.2 and 1M) and the result as evidenced from the presence. For better understanding these complexes were examined by using Density functional theory (DFT) calculation.

Keywords: (1:10-phenanthroline) (L1), 4-4-amino phenazone (L2), diphenyl carbizon (L3), DFT study, antibacterial

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Authors: Jayson Wau, David Timi, Anthony Harakuwe, Bruce Bowden, Cherie Motti, Harry Sakulas, Rag Gubag-Sipou

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The latex of F. botryocarpa fruit is applied on sores, wounds and other skin infections in Papua New Guinea ethnotherapeutic practices. Systematic bioassay guided separation and isolation of subsequent fractions of latex extracts resulted in three bioactive fractions active against Staphylococcus aureus and Escherichia coli. This study reports structural elucidation of the three isolates. Structures were determined by physical (M.pt and Rf values) and spectroscopic (1D-1H NMR, 2D-HSQC NMR, 2D-HMBC NMR) and MS ESI-POS. The two methylene protons (2H-1) and (2H-3) resonate as triplets at δ 3.59 and δ 4.99 respectively. Electron dense δ 4.99 (2H-3) on (C-3) depicts the strong electron-withdrawing component, quaternary nitrogen (=N= +). Protons resonating at δ 3.88 and 3.89 are singlets depicting two methoxy groups. Both δ 3.88 and δ 3.89 are para-aryls substituents. The methines δ 9.13 and 8.60 are singlets depicting two lone protons on the indolizidinium aryl component. All isolates, (1), (2) and (3) were identified to be ficuseptine by comparing 1D-NMR assignments. 2D-NMR and MS of (2) found it to be ficuseptine chloride '2, 3-dihydro-6, 8-bis (4-methoxyphenyl)-, 1H-indolizinium chloride'. Their counter ions of the ficuseptines were not established and provide promising lead for the further investigation.

Keywords: Ficus botryocarpa, antimicrobial activity, ficuseptine, sores

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Authors: Fatbardhë Kurti, Giangiacomo Beretta, Behxhet Mustafa, Fabrizio Gelmini, Avni Hajdari

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Chemical profile, antioxidant and antimicrobial activity of total and fractionated essential oils (EOs) (F1 – hexane, F2 – hexane/diethyl ether, F3 – diethyl ether) derived from five Pinus species (Pinus heldreichii, P. peuce, P. mugo, Pinus nigra, P. sylvestris), were investigated. The hydrodistilled EOs and their chromatographic fractions (direct solid phase extraction, SPE) were analysed by GC-MS and 112 compounds separated and identified. The main constituents were α-pinene, β-pinene, D-limonene, β-caryophyllene, germacrene D, bornyl acetate and 3-carene. The antioxidant activities of total EOs were lower than those of the corresponding fractions, with F2 the strongest in all cases. EOs and fractions showed different degrees of antibacterial efficacy against different microbial pathogens (moderately strong antimicrobial activity against C. albicans and C. krusei ,while low or no activity against E. faecalis and E. coli strains). The detected inhibition zones and MICs for the EOs and fractions were in the range of 14 -35 mm and 0.125 - 1% (v/v), respectively. The components responsible for the antioxidant and antimicrobial activity were oxygenated monoterpenes and sesquiterpenes recovered in the polar EO fractions. These activities seem to be regulated by reciprocal interactions among the different subclasses of phytochemical species present in the EOs.

Keywords: antagonism, antioxidant activity, antibacterial activity, essential oil, fractions, GC-MS, pinus

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Authors: Maryam Farzaneh, Seyyedeh Nafiseh Hassani, Hossein Baharvand

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Objective: Chicken gonadal tissue has a two population such primordial germ cells (PGCs) and stromal cells (somatic cells). PGCs and embryonic germ cells (EGCs) that is a pluripotent type of PGCs in long-term culture are suitable sources for the production of chicken pluripotent stem cell lines, transgenic birds, vaccine and recombinant protein production. In general, the effect of growth factors such bFGF and mouse LIF on derivation of PGCs in vitro are important and in this study we could see the unique effect of small molecules such PD032 and SB43 as a chemical, in comparison to growth factors. Materials and Methods: After incubation of fertilized chicken egg up to 6 days and isolation of primary gonadal tissues and culture of mixed cells like PGCs and stromal cells. PGCs proliferate in the present of fetal calf serum (FCS) and small molecules and in another group bFGF, that these factors are important for PGCs culture and derivation. Somatic cells produce a multilayer feeder under the PGCs in primary culture and PGCs make a small cluster under these cells. Results: In present of small molecules and high volume of FCS (15%), the present of EGCs as a pluripotent stem cells were clear four weeks, that they had a positive immune-staining and periodic acid-Schiff staining (PAS), but in present of growth factors like bFGF without any chemicals, the present of PGCs were clear but after 7 until 10 days, there were disappear. Conclusion: Until now we have seen many researches about derivation and maintenance of chicken PGCs, in the hope of understanding the mechanisms that occur during germline development and production of a therapeutic product by transgenic birds. There are still many unknowns in this area and this project will try to have efficient conditions for identification of suitable culture medium for long-term culture of PGCs in vitro without serum and feeder cells.

Keywords: chicken gonadal primordial germ cells, pluripotent stem cells, growth factors, small molecules, transgenic birds

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Authors: Rebecca Thombre, Aishwarya Borate

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Chemical synthesis of nanoparticles produces toxic by-products, as a result of which eco-friendly methods of synthesis are gaining importance. The synthesis of nanoparticles using plant derived extracts is economical, safe and eco-friendly. Biostabilized gold nanoparticles were synthesized using extracts of Garcinia indica. The gold nanoparticles were characterized using UV-Vis spectrophotometry and demonstrated a peak at 527 nm. The presence of plant derived peptides and phytoconstituents was confirmed using the FTIR spectra. TEM analysis revealed formation of gold nanopyramids and nanorods. The SAED analysis confirmed the crystalline nature of nanoparticles. The gold nanoparticles demonstrated antibacterial and antifungal activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Aspergillus niger and Pichia pastoris. The cytotoxic activity of gold nanoparticles was studied using HEK, Hela and L929 cancerous cell lines and the apoptosis of cancerous cells were observed using propidium iodide staining. Thus, a simple and eco-friendly method for synthesis of biostabilized gold nanoparticles using fruit extracts of Garcinia indica was developed and the nanoparticles had potent antibacterial, antifungal and anticancer properties.

Keywords: cytotoxic, gold nanoparticles, green synthesis, Garcinia indica, anticancer

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Authors: Ibeku Bernadine Ezenwanyi

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Hospital-Acquired Infections (HAI), also called ‘Nosocomial Infection’ is an infection you get while in the hospital for another reason. They are an important cause of morbidity and mortality. This study examined the epidemiology, clinical outcomes of some instruments used on patients especially catheter that was passed on them during their stay in the hospital. It was discovered that they had catheter–associated urinary tract infection (CAUTI). An observational study was carried out from January to March, 2022, on 180 patients (80 males and 100 females) admitted in the surgical wards, medical wards, dialysis unit and intensive care unit (ICU) of the hospital. The patient’s urine samples were collected for urine culture and the isolation was carried out using plate count agar medium and macconkey agar. Among the 80 males, 45 had Urinary Tract Infections (UTI) mostly proteus infection and among the 100 females, 70 also had (UTI) and the most common was caused by Escherichia coli. Other strain of microorganisms such as Klebsiella, Staphylococcus aureus and Pseudomonas aeruginosa. It is important to follow up patients that these devices were used on with antibiotics to make sure that these infections are not developed because the rate of these infections (UTI) are high especially in females.

Keywords: catheter, urinary tract infection, nosocomial infection, microorganisms

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Authors: Shiraz A. Markarian

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Our systematic studies of diethylsulfoxide (DESO), the nearest homologue of dimethylsulfoxide (DMSO), reveal new physicochemical features. DESO has already received worthy biomedical applications: in some cases even are more pronounced compare with DMSO. The several important physicochemical characteristics of DESO including aqueous solutions have been verified and first reported: melting point of pure substance, density, dielectric relaxation data, vapor pressure and volumetric properties. Analysis of the complete vibrational spectra also leads to the conclusion that very strong interactions take place between DESO and water, even stronger than those between DMSO and water. The simultaneous existence of strong DESO-H₂O and DESO-DESO interactions suggest the coexistence of many types of structural molecular aggregates, the presence of which plays a significant role also in diluted water solutions. Our recent investigations have shown that aqueous solution of DESO could provide amorphous, glassy systems, thus avoiding ice crystallization, in a wide range of concentrations and even at very low cooling rates. The ability of DESO to act as an effective cryoprotectant on E. coli survival was also studied and compared with other commonly used cryoprotective agents. The results also confirm that DESO, more than DMSO, is able to penetrate living tissues without causing significant damage.

Keywords: diethylsulfoxide, dimethylsulfoxide, cryoprotectant, properties

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Authors: Waleed S. Alwaneen

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In many countries, cow dung is used as farm manure and for biogas production. Several bacterial strains associated with cow dung such as Campylobacter, Salmonella sp. and Escherichia coli cause serious human diseases. The objective of the present study was to investigate the use of insect larvae including fruit beetle, waxworms and tiger worms to improve the breakdown of agricultural wastes and reduce their pathogen loads. Fresh cow faeces were collected from a cattle farm and distributed into plastic boxes (100 g/box). Each box was provided with 10 larvae of fruit beetle, Waxworms and Tiger worms, respectively. There were 3 replicates in each treatment including the control. Bacteria were isolated weekly from both control and cow faeces to which larvae were added to determine the bacterial populations. Results revealed that the bacterial load was higher in the cow faeces treated with fruit beetles than in the control, while the bacterial load was lower in the cow faeces treated with waxworms and tiger worms than in the control. The activities of the fruit beetle larvae led to the cow faeces being liquefied which provided a more conducive growing media for bacteria. Therefore, higher bacterial load in the cow faeces treated with fruit beetle might be attributed to the liquefaction of cow faeces.

Keywords: fruit beetle, waxworms, tiger worms, waste conditioning, composting

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Authors: Kinza Khan, Dad Muhmmad, Asif Saleem, Nadia Mukhtar, Tahir Yaqub

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Ethomedicines are more commonly used in underdeveloped and developing countries. These medicines are sometimes more potent in controlling microbial infections than conventional medicines. Medicinal plants have been common practice to cure many diseases for centuries. Murraya koenigii is one of these plants and is commonly used in South Asian countries as a flavoring agent in food. To evaluate its anti-microbial activity, six different bacterial strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Bacillus cereus and Klebsiella pneumonia were used. N-hexane extract of Murraya koenigii leaves shows maximum activity against Bacillus cereus. Acetone extract of Murraya koenigii shoots showed more efficient activity against Pseudomonas aeruginosa Dichloromethane extracts showed maximum activity against Bacillus cereus. Ethanol extract exhibited maximum activity against Pseudomonas aeruginosa and Klebsiella pneumoniae. The methanol extract of Murraya koenigii shoots displayed maximum antibacterial activity against Bacillus cereus. Antifungal activity Ethanol extract was more effective against Candida albicans.

Keywords: ethnomedicines, bacteria, fungi, murraya koenigii, antimicrobial activity

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Authors: Abdelkader Basli, Jean-Claude Delaunay, Eric Pedrot, Jean-Michel Mérillon, Jean-Pierre Monti, Khodir Madani, Mohamed Chibane, Tristan Richard

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The antioxidant and antimicrobial activities of Origanum glandulosum collected in Algeria have been studied. Extract was prepared from aerial part of endemic Algerian oregano. The produced extract has been characterized in terms of total phenols (using Folin method), total flavonoid, antioxidant activities (using the DPPH radical scavenging method and ORAC assay) and microbial activity against four bacteria: Streptococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae one yeast: Candida albicans and one fungi: Aspergillus niger. The results pointed the antioxidant activities of the extract of O. glandulosum and antimicrobial activities against all bacteria and C. Candida, but no effect on A. niger. High performance liquid chromatography combined with mass spectrometry (LC-MS) and nuclear magnetic resonance (LC-NMR) were used to separate and identify the major compounds present in the oregano extract. Rosmarinic acid, globoidnan A and B, lithospermic acid B and three flavonoids were identified.

Keywords: origanum glandulosum, antioxidant, microbial activity, polyphenol, LC-MS, LC-NMR

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Authors: Kristina Karapetyan, Flora Tkhruni, Tsovinar Balabekyan, Arevik Israyelyan, Tatyana Khachatryan

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The ability of the lactic acid bacteria (LAB) to prevent and cure a variety of diseases, their protective role against infections and colonization of pathogenic microorganisms in the digestive tract, has lead to the coining of the term probiotics or pro-life. LAB inhibiting the growth of pathogenic and food spoilage microorganisms, maintaining the nutritive quality and improving the shelf life of foods. They have also been used as flavor and texture producers. Enterococcus strains have been used for treatment of diseases such as diarrhea or antibiotic associated diarrhea, inflammatory pathologies that affect colon such as irritable bowel syndrome, or immune regulation, diarrhea caused by antibiotic treatments. The obtaining and investigation of biological properties of proteinoceous antibiotics, on the basis of probiotic LAB shown, that bacteriocins, metabiotics, and peptides of LAB represent bactericides have a broad range of activity and are excellent candidates for development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics. The genotyping by 16S rRNA sequencing for LAB were used. Cell free culture broth (CFC) broth was purified by the Gel filtration method on the Sephadex Superfine G 25 resin. Antimicrobial activity was determined by spot-on-lawn method and expressed in arbitrary units (AU/ml). The diversity of multidrug-resistance (MDR) of pathogenic strains to antibiotics, most widely used for treatment of human diseases in the Republics of Armenia and Nagorno Karabakh were examined. It was shown, that difference of resistance of pathogens to antibiotics depends on their isolation sources. The influences of partially purified antimicrobial preparations (AMP), obtained from the different strains of Enterococcus genus on the growth of MDR pathogenic bacteria were investigated. It was shown, that bacteriocin containing partially purified preparations, obtained from different strains of Enterococcus faecium and durans species, possess bactericidal or bacteriostatic activity against antibiotic resistant intestinal, spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, E. coli, and Salmonella. Endemic strains of LAB, isolated from Matsoni made from donkey, buffalo and goat milk, shown broad spectrum of activity against food spoiling microorganisms, moulds and fungi, such as Salmonella sp., Esherichia coli, Aspergillus and Penicillium species. Highest activity against MDR pathogens shown bacteria, isolated from goat milk products. High stability of the investigated strains of the genus Enerococcus, isolated from samples of matsun from different regions of Nagorno-Karabakh (NKR) to the antibiotics was shown. The obtained data show high stability of the investigated different strains of the genus Enerococcus. The high genetic diversity in Enterococcus group suggests adaptations for specific mutations in different environments. Thus, endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms isolated from different sources and belong to different taxonomic group. Prospect of the use of certain antimicrobial preparations against pathogenic strains is obvious. These AMP can be applied for long term use against different etiology antibiotic resistant pathogens for prevention or treatment of infectional diseases as an alternative to antibiotics.

Keywords: antimicrobial biopreparation, endemic lactic acid bacteria, intra-species diversity, multidrug resistance of pathogens

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Authors: Mahnoor Khan, Bashir Ahmad, Khizar Hayat, Saad Ahmad Khan, Laiba Ahmad, Shumaila Bashir, Abid Ali Khan

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The objective of this study was to synthesize the smallest possible size of ZnO NPs using a modified wet chemical synthesis method and to prepare core shell using polyethylene glycol (PEG) as shell material. Advanced and sophisticated techniques were used to confirm the synthesis, size, and shape of these NPs. Rounded, clustered NPs of size 5.343 nm were formed. Both the plain and core shell NPs were tested against MDR bacteria (E. cloacae, E. amnigenus, Shigella, S. odorifacae, Citrobacter, and E. coli). Both of the NPs showed excellent antibacterial properties, whereas E. cloacae showed maximum zone of inhibition of 16 mm, 27 mm, and 32 mm for 500 μg/ml, 1000 μg/ml, and 1500 μg/ml, respectively for plain ZnO NPs and 18 mm, 28 mm and 35 mm for 500 μg/ml, 1000 μg/ml and 1500 μg/ml for core shell NPs. These NPs were also biocompatible on human red blood cells showing little hemolysis of only 4% for 70 μg/ml for plain NPs and 1.5% for 70 μg/ml for core shell NPs. Core shell NPs were highly biocompatible because of the PEG. Their therapeutic effect as photosensitizers in photodynamic therapy (PDT) for cancer treatment was also monitored. The cytotoxicity of ZnO and PEG-ZnO was evaluated using MTT assay. Our results demonstrated that these NPs could generate ROS inside tumor cells after irradiation which in turn initiates an apoptotic pathway leading to cell death hence proving to be an effective candidate for PDT.

Keywords: ZnO, hemolysis, cytotoxiciy assay, photodynamic therapy, antibacterial

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Authors: Ali Amiri, Arash Esfandiari, Elham Zarenezhad

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We report here an efficient and rapid method for the preparation of 3,4-dihydro-1H-quinoxalin-2-ones and 1H‑quinolin-2-ones that involves grinding of o-, m-, or p‑phenylenediamine and three dialkyl acetylenedicarboxylates using a pestle and mortar. This solvent-free approach requires only a few minutes of reaction time. This type of reaction is expected to be the most economical method since neither catalyst nor solvent is used. Finally, all synthesised compounds were screened for antimicrobial activity against two Gram-positive bacteria (Pseudomonas aeruginosa PTCC 1077, Escherichia coli PTCC1330) and two Gram-negative bacteria (Staphylococcus aureus PTCC 1133, Bacillus cereus PTCC 1015) and their activity. Compared with gentamycin and ampicillin as reference drugs for Gram-negative and Gram-positive bacteria, respectively. The minimum inhibitory concentration (MIC) of the synthesised compounds and reference drugs were determined by the microdilution method. Good antibacterial activity was observed for 3,4-dihydro-1H-quinoxalin-2-ones against all species of Gram-positive and Gram-negative bacteria, and1H‑quinolin-2-ones showed good antibacterial activity against two Gram-positive bacteria.

Keywords: quinolin, quinoxalin, anti-bacterial activity, minimum inhibitory concentration (MIC)

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Authors: Daniel Aberdam

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Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia-related keratopathy (ARK), caused by a combination of factors including limbal stem-cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub-epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation found in patients into one allele of the PAX6 gene into limbal stem cells. Resulting mutated clones, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency were further characterized. Sequencing analysis showed that no off-target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of soluble recombinant PAX6 protein into the culture medium was sufficient to activate endogenous PAX6 gene and, as a consequence, rescue the phenotype. It strongly suggests that our in vitro model recapitulates well the epithelial defect and becomes a powerful tool to identify drugs that could rescue the corneal defect in patients. Furthermore, we demonstrate that the homeotic transcription factor Pax6 is able to be uptake naturally by recipient cells to function into the nucleus.

Keywords: Pax6, crispr/cas9, limbal stem cells, aniridia, gene therapy

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Authors: Nabaa M. Abdul Rahim, Mohammed A.Kadhim, Fadhil K. Fuliful

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A biocompatible layer is prepared from calcium phosphate, which plays a role in building damaged bones and is used in many applications. In this research, calcium phosphate is coated on stainless steel substrates (SS 316) by using the flame coating. FE-SEM images show that the behavior of the sample surfaces varies with distance, at 3cm, appeared with nanostructures of bumps shaped of diameter about 317 nm. The contents of the elements are analyzed by energy-dispersive X-ray spectroscopy (EDX). The chemical elements C, Ca, Fe, Ni, Cr, Mn and O corresponding to calcium phosphate and the alloy are revealed by EDX analysis of the coating layer. XRD patterns for the calcium phosphate layers indicate the formation of the Hap layer on the deposited layers. The samples are immersed in a solution of simulated body fluids (SBF) for 21 days to examine the biocompatibility, as the tests show that the calcium phosphate ratio of 1.65 is the appropriate and biocompatible ratio in the human body. The assays show antibacterial activity using the diffusion disk procedure. On the surface of the agar, observed infested E.coli bacteria and incubated for 24 hours at 37°C. Bacteria grow on the entire agar rather than in some areas around some samples at a distance of 3 cm from the flame hole.

Keywords: biomaterial, flame coating, antibacterial activity, stainless steel

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Authors: F. Madani, M. Doudi, L. Rahimzadeh Torabi

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The irregular consumption of current antibiotics contributes to an escalation in antibiotic resistance among urinary pathogens on a global scale. The objective of this research was to investigate the process of biologically synthesized silver nanoparticles through the utilization of Zataria multiflora extract. Additionally, the study aimed to evaluate the efficacy of these synthesized nanoparticles in inhibiting the growth of multi-drug resistant negative bacillus bacteria, which commonly contribute to urinary tract infections. The botanical specimen utilized in the current research investigation was Z. multiflora, and its extract was produced employing the Soxhlet extraction technique. The study examined the green synthesis conditions of silver nanoparticles by considering three key parameters: the quantity of extract used, the concentration of silver nitrate salt, and the temperature. The particle dimensions were ascertained using the Zetasizer technique. In order to identify synthesized Silver nanoparticles TEM, XRD, and FTIR methods were used. For evaluating the antibacterial effects of nanoparticles synthesized through a biological method, different concentrations of silver nanoparticles were studied on 140 cases of Multiple drug resistance (MDR) bacteria strains Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris,Citrobacter freundii, Acinetobacter bumanii and Pseudomonas aeruginosa, (each genus of bacteria, 20 samples), which all were MDR and cause urinary tract infections, for identification of bacteria were used of PCR test and laboratory methods (Agar well diffusion and Microdilution methods) to assess their sensitivity to Nanoparticles. The data were subjected to analysis using the statistical software SPSS, specifically employing nonparametric Kruskal-Wallis and Mann-Whitney tests. This study yielded noteworthy findings regarding the impacts of varying concentrations of silver nitrate, different quantities of Z. multiflora extract, and levels of temperature on nanoparticles. Specifically, it was observed that an increase in the concentration of silver nitrate, extract amount, and temperature resulted in a reduction in the size of the nanoparticles synthesized. However, the impact of the aforementioned factors on the index of particle diffusion was found to be statistically non-significant. According to the transmission electron microscopy (TEM) findings, the particles exhibited predominantly spherical morphology, with a diameter spanning from 25 to 50 nanometers. Nanoparticles in the examined sample. Nanocrystals of silver. FTIR method illustrated that the spectrums of Z. multiflora and synthesized nanoparticles had clear peaks in the ranges of 1500-2000, and 3500 - 4000. The obtained results of antibacterial effects of different concentrations of silver nanoparticles on according to agar well diffusion and microdilution method, biologically synthesized nanoparticles showed 1000 mg /ml highest and lowest mean inhibition zone diameter in E. coli, A. bumanii 23 and 15mm, respectively. MIC was observed for all of bacteria 125 mg/ml and for A. bumanii 250 mg/ml. Comparing the growth inhibitory effect of chemically synthesized the results obtained from the experiment indicated that both nanoparticles and biologically synthesized nanoparticles exhibit a notable growth inhibition effect. Specifically, the chemical method of synthesizing nanoparticles demonstrated the highest level of growth inhibition at a concentration of 62.5 mg/mL The present study demonstrated an inhibitory effect on bacterial growth, facilitating the causative factors of urine infection and multidrug resistance (MDR).

Keywords: multiple drug resistance, negative bacillus bacteria, urine infection, Zataria multiflora

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Authors: Patsarawadee Paojinda, Waranya Imprasittichai, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

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Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, can be stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to 3 orders of magnitude, while the Km was not much affected and remained at low µM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivaties and metabolic functions on molecular evolution.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

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Authors: Ali Mohamadi Sani

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The aim of this study was to determine the antimicrobial effect of Helichrysum arenarium L. essential oil in "in-vitro" condition on the growth of seven microbial species including Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Saccharomyces cereviciae, Candida albicans, Aspergillus flavus and Aspergillus parasiticus using microdilution method. The minimum inhibitory concentration (MIC) and minimum bactericidal or fungicidal concentration (MBC, MFC) were determined for the essential oil at ten concentrations. Finally, the sensitivity of tested microbes to the essential oil of H. arenarium was investigated. Results showed that Bacillus subtilis (MIC=781.25 and MBC=6250 µg/ml) was more resistance than two other bacterial species. Among the tested yeasts, Saccharomyces cereviciae (MIC=97.65 and MFC=781.25 µg/ml) was more sensitive than Candida albicans, while among the fungal species, growth of Aspergillus parasiticus inhibited at lower concentration of oil than the Aspergillus flavus. The extracted essential oil exhibited the same MIC value in the liquid medium against all fungal strains (48.82 µg/ml), while different activity against A. flavus and A. parasiticus was observed in this medium with MFC values of 6250 and 390.625µg/ml, respectively. The results of the present study indicated that Helichrysum arenarium L essential oil had significant (P<0.05) antimicrobial activity; therefore, it can be used as a natural preservation to increase the shelf life of food products.

Keywords: Helichrysum arenarium, antimicrobial, essential oil, MIC

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Authors: Maria Giovanna Lupo, Marina Camera, Marcello Rattazzi, Nicola Ferri

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A complex interplay among chronic kidney disease, lipid metabolism and aortic calcification has been recognized starting from results of many clinical and experimental studies. Here we investigated the influence of kidney function on PCSK9 levels, both in uremic rats and in clinical observation study, and its potential direct action on cultured smooth muscle cells (SMCs) calcification. In a cohort of 594 subjects enrolled in a single centre, observational, cross-sectional and longitudinal study, a negative association between GFR and plasma PCSK9 was found. Atherosclerotic cardiovascular disease (ASCVD), as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats, induced aortic calcification and increased total cholesterol and PCSK9 levels in plasma, livers and kidneys. Immunohistochemical analysis confirmed PCSK9 expression in aortic SMCs. SMCs overexpressing PCSK9 (SMCsPCSK9), cultured for 7-days in a pro-calcification environment (2.0mM or 2.4mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to mocked SMCs. Under the same experimental conditions, the addition of exogenous recombinant PCSK9 did not increase the extracellular calcification of SMCs. By flow cytometry analysis we showed that SMCsPCSK9, in response to 2.4mM Pi, released higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALPL), compared to EVs from mocks SMCs. In conclusion, our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated to CKD condition. This effect appears to be mediated by a positive effect of endogenous PCSK9 on the release of EVs containing Ca2+ and ALP, which facilitate the deposition inorganic calcium phosphate crystals.

Keywords: PCSK9, calcification, extracellular vesicles, chronic kidney disease

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Authors: Mondal Indranil, Bhakat Debjyoti, Mukhopadayay Asish K., Chatterjee Nabendu S.

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CS6 is the prevalent CF in our region and deciphering its molecular regulators would play a pivotal role in reducing the burden of ETEC pathogenesis. In prokaryotes, most of the genes are under the control of one operon and the promoter present upstream of the gene regulates the transcription of that gene. Here the promoter of CS6 was characterized by computational method and further analyzed by β-galactosidase assay and sequencing. Promoter constructs and deletions were prepared as required to analyze promoter activity. The effect of different additives on the CS6 promoter was analysed by the β-galactosidase assay. Bioinformatics analysis done by Softberry/BPROM predicted fur, lrp, and crp boxes, -10 and -35 region upstream of the CS6 gene. The promoter construction in no promoter plasmid pTL61T showed that region -573 to +1 is actually the promoter region as predicted. Sequential deletion of the region upstream of CS6 revealed that promoter activity remains the same when -573bp to -350bp is deleted. But after the deletion of the upstream region -350 bp to -255bp, promoter expression decreases drastically to 26%. Further deletion also decreases promoter activity up to a little range. So the region -355bp to -255bp holds the promoter sequence for the CS6 gene. Additives like iron, NaCl, etc., modulate promoter activity in a dose-dependent manner. From the promoter analysis, it can be said that the minimum region lies between -254 and +1. Important region(s) lies between -350 bp to -255 bp upstream in the promoter, which might have important elements needed to control CS6 gene expression.

Keywords: microbiology, promoter, colonization factor, ETEC

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Authors: Adjebli Ahmed, Messis Abdelaziz, Ayeche Riad, Tighilet Karim, Talbi Melissa, Smaili Yanis, Lehri Mokrane, Louardiane Mustapha

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Colorectal cancer is one of the most common types of cancer worldwide, and its incidence has been increasing in recent years. Data and fecal samples from colorectal cancer patients were collected at the Amizour Public Hospital's oncology department (Bejaia, Algeria). Microbiological and cohort study were conducted at the Biological Engineering of Cancers laboratory at the Faculty of Medicine of the University of Bejaia. All the data showed that patients aged between 50 and 70 years were the most affected by colorectal cancer, while the age categories of [30-40] and [40-50] were the least affected. Males were more likely to be at risk of contracting colorectal cancer than females. The most common types of colorectal cancer among the studied population were sigmoid cancer, rectal cancer, transverse colon cancer, and ascending colon cancer. The hereditary factor was found to be more dominant than other risk factors. Bacterial identification revealed the presence of certain pathogenic and opportunistic bacterial genera, such as E. coli, K. pneumoniae, Shigella sp, and Streptococcus group D. These results led us to conclude that dysbiosis of the intestinal microbiome is strongly present in colorectal cancer patients at the EPH of Amizour.

Keywords: microbiome, colorectal cancer, risk factors, bacterial identification

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Authors: Anita Vidacs, Robert Rajko, Csaba Vagvolgyi, Judit Krisch

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With the optimization of a new disinfectant the number of tests could be decreased and the cost of processing too. Good sanitizers are eco-friendly and allow no resistance evolvement of bacteria. The essential oils (EOs) are natural antimicrobials, and most of them have the Generally Recognized As Safe (GRAS) status. In our study, the effect of the EOs cinnamon, marjoram, and thyme was investigated against mixed species bacterial biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida, and Staphylococcus aureus. The optimal concentration of EOs, disinfection time and level of pH were evaluated with the aid of Response Surface Box-Behnken Design (RSD) on 1 day and 7 days old biofilms on metal, plastic, and wood surfaces. The variable factors were in the range of 1-3 times of minimum bactericide concentration (MBC); 10-110 minutes acting time and 4.5- 7.5 pH. The optimized EO disinfectant was compared to industrial used chemicals (HC-DPE, Hypo). The natural based disinfectants were applicable; the acting time was below 30 minutes. EOs were able to eliminate the biofilm from the used surfaces except from wood. The disinfection effect of the EO based natural solutions was in most cases equivalent or better compared to chemical sanitizers used in food industry.

Keywords: biofilm, Box-Behnken design, disinfectant, essential oil

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Authors: Khrizelle Angelique Sablan, Rizalinda De Leon, Jaeyoung Lee, Joey Ocon

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The impending water shortage drives us to find alternative sources of water. One of the possible solutions is desalination of seawater. There are numerous processes by which it can be done and one if which is capacitive deionization. Capacitive deionization is a relatively new technique for water desalination. It utilizes the electric double layer for ion adsorption. Carbon-based materials are commonly used as electrodes for capacitive deionization. In this study, carbon nanotubes (CNTs) were treated in a mixture of nitric and sulfuric acid. The silver addition was also facilitated to incorporate antimicrobial action. The acid-treated carbon nanotubes (f-CNTs) and silver-decorated f-CNTs (Ag@f-CNTs) were used as electrode materials for seawater deionization and compared with CNT and acid-treated CNT. The synthesized materials were characterized using TEM, EDS, XRD, XPS and BET. The electrochemical performance was evaluated using cyclic voltammetry, and the deionization performance was tested on a single cell with water containing 64mg/L NaCl. The results showed that the synthesized Ag@f-CNT-10 H could have better performance than CNT and a-CNT with a maximum ion removal efficiency of 50.22% and a corresponding adsorption capacity of 3.21 mg/g. It also showed antimicrobial activity against E. coli. However, the said material lacks stability as the efficiency decreases with repeated usage of the electrode.

Keywords: capacitive deionization, carbon nanotubes, desalination, acid functionalization, silver

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Authors: Salwa A. H. Hamdi, Maha A. M. El-Shazly, Mona Fathi Fol, Hanan S. Mossalem, Mosad A. Ghareeb, Amina M. Ibrahim

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One of the most distinctive and defining features of cephalopods squid, cuttlefish, and Octopus is their inking behavior. Their ink, which is blackened by melanin but also contains other constituents, has been used by humans in various ways for millennia. The present study aims to investigate the chemical profiling of the Octopus vulgaris ink extract and to evaluate its antioxidant, antimicrobial, and anti-schistosomal activities. The present results showed that GC-MS examination of Octopus vulgaris ink comprises 21 compounds. The main detected compounds are (E)-1, 2, 3, 4-Tetra (4-phenylphenyl)-2-butene-1,4-dione, Lipo-3-episapelin A, and 5,10-Dihexyltetrabenzoporphyrin. Results showed that the octopus ink had antioxidant capacity and the capability to mask DPPH free radicals in comparison with ascorbic acid. Octopus Vulgaris ink extract had inhibitory action against three gram-positive bacteria, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis, and three gram-negative bacteria, Neisseria gonorrhoeae, Escherichia coli, and Pseudomonas aeuroginosa. Additionally, the extracted ink revealed antifungal activity against Aspergillus flavus and yeast as Candida albicans. The obtained data indicated the effectiveness of ink extract in pharmaceutical industries as an antioxidant, antimicrobial and antischistosomicidal

Keywords: antimicrobial, antioxidant, ink, octopus vulgaris

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Authors: M. O. Eyankware, D. O. Ufomata

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Groundwater samples were collected from Otovwodo-Ughelli and Environ with the aim of assessing groundwater quality of the area. Twenty (20) water samples from Boreholes (BH) (six) and Hand Dug Wells (HDW) (fourteen) were randomly sampled and were analysed for different physiochemical and bacteriological parameters. The following 16 parameters have been considered viz: pH, electrical conductivity, temperature, total hardness, total dissolved solids, dissolved oxygen, biological oxygen demand, phosphate, sulphate, chloride, nitrate, calcium, sodium, chloride, magnesium, and total suspended solids. On comparing the results against drinking quality standards laid by World Health Organization and Nigeria industrial standard, it was found that the water quality parameters were not above the (WHO, 2011 and NIS, 2007) permissible limit. Microbial analysis reveals the presence of coliform and E.coli in two hand-dug well (HDW7 and 13) and one borehole well (BH20). These contaminations are perhaps traceable to have originated from human activities (septic tanks, latrines, dumpsites) and have affected the quality of groundwater in Otovwodo-Ughelli. From the piper trilinear diagram, the dominant ionic species is alkali bicarbonate water type, with bicarbonate as the predominant ion (Na+ + K+)-HCO3.

Keywords: groundwater, surface water, Ughelli, Nigeria industrial standard, who standard

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Authors: Thangaraj Arasakumar, Athar Ata, Palathurai Subramaniam Mohan

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A series of novel 4-quinolone-3-carboxylic acid grafted spiropyrrolidines as new type of antibacterial agents were synthesized via multicomponent 1,3-dipolar cycloaddition reaction of an azomethine ylides with a newly prepared (E)-4-oxo-6-(3-phenyl-acryloyl)-1,4-dihydro-quinoline-3-carboxylic acids in high regioselectivity with good yields. The structure of cycloadduct characterized by FT IR, mass, 1H, 13C, 2D NMR techniques and elemental analysis. Structure and spectrometry of compound 8a has been investigated theoretically by using HF and DFT approach at B3LYP, M05-2x/6-31G* levels of theories. The optimized geometries and calculated vibrational frequencies are evaluated via comparison with experimental values. A good agreement is found between the measured and calculated values. The DFT studies support the molecular mechanism of this cycloaddition reaction and determine the molecular electrostatic potential and thermodynamic properties. Furthermore, the antibacterial activities of synthesized compounds were evaluated against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria strains (Escherichia coli, Klebsiella pneumoniae). Among 21 compounds screened, 8f and 8p were found to be more active against tested bacteria.

Keywords: antibacterial activity, azomethine ylide, DFT calculation, spirooxindole

Procedia PDF Downloads 178

Authors: Sanam Pudasaini, A. T. K. Perera, Ahmed Syed Shaheer Uddin, Sum Huan Ng, Chun Yang

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Development of high-efficiency and environment friendly bacterial inactivation methods is of great importance for preventing waterborne diseases which are one of the leading causes of death in the world. Traditional bacterial inactivation methods (e.g., ultraviolet radiation and chlorination) have several limitations such as longer treatment time, formation of toxic byproducts, bacterial regrowth, etc. Recently, an electroporation-based inactivation method was introduced as a substitute. Here, an electroporation-based continuous flow microfluidic device equipped with an array of micropillars is developed, and the device achieved high bacterial inactivation performance ( > 99.9%) within a short exposure time ( < 1 s). More than 99.9% reduction of Escherichia coli bacteria was obtained for the flow rate of 1 mL/hr, and no regrowth of bacteria was observed. Images from scanning electron microscope confirmed the formation of electroporation-induced nano-pore within the cell membrane. Through numerical simulation, it has been shown that sufficiently large electric field strength (3 kV/cm), required for bacterial electroporation, were generated using PDMS micropillars for an applied voltage of 300 V. Further, in this method of inactivation, there is no involvement of chemicals and the formation of harmful by-products is also minimum.

Keywords: electroporation, high-efficiency, inactivation, microfluidics, micropillar

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Authors: Salahaedin Waiezi, Nik Ahmad Nizam Nik Malek, Hassan Abdelmagid Elzamzami, Shahrulnizahana Mohammad Din

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A green and safe approach to the synthesis of silver nanoparticles (AgNP) can be performed using plant leaf extract as the reducing agent. Hence, this paper reports the biosynthesis of AgNP using Clinacanthus nutans plant extract. C. nutans is known as belalai gajah in Malaysia and is widely used as a medicinal herb locally. The biosynthesized AgNP, using C. nutans aqueous extract at pH 10, with the reaction temperature of 70°C and 48 h reaction time, was characterized by UV-Vis spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX), and transmission electron microscope (TEM). A peak appeared in the UV-Vis spectra at around 400 nm, while XRD confirmed the crystal structure of AgNP, with the average size between 20 to 30 nm, as shown in FESEM and TEM. The antibacterial activity of the biosynthesized AgNP, which was performed using the disc diffusion technique (DDT) indicated effective inhibition against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. In contrast, minimal antibacterial activity was detected against Enterococcus faecalis and methicillin-resistant Staphylococcus aureus (MRSA). In general, AgNP produced using C. nutans leaf extract possesses potential antibacterial activity.

Keywords: silver nanoparticles, Clinacanthus nutans, antibacterial agent, biosynthesis

Procedia PDF Downloads 168