Search results for: antimicrobial proteins

Authors: Alma Ramić, Mirjana Skočibušić, Renata Odžak, Tomica Hrenar, Ines Primožič

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In an attempt to identify a new class of antimicrobial agents, the antimicrobial potential of Cinchona alkaloid derivatives was evaluated. The bark of the Cinchona trees is the source of a variety of alkaloids, among which the best known are quinine, quinidine, cinchonine and cinchonidine. They are very useful as organocatalysts in stereoselective synthesis. On the other hand, quinine is traditionally used in the treatment of malaria. Furthermore, Cinchona alkaloids possess various analgesic, anti-inflammatory and anti–arrhythmic properties as well. In this work we present the synthesis of twenty quaternary derivatives of pseudo−enantiomeric Cinchona alkaloid derivatives to evaluate their antibacterial activity. Quaternization of quinuclidine moiety was carried out with groups diverse in their size. The structures of compounds were systematically modified to obtain drug-like properties with proper physical and chemical properties and avoiding toxophore. All compounds were prepared in good yields and were characterized by standard analytical spectroscopy methods (1D and 2D NMR, IR, MS). The antibacterial activities of all compounds were evaluated against series of recent clinical isolates of antibiotic susceptible Gram-positive and resistant Gram-negative pathogens by determining their zone of inhibition and minimum inhibitory concentrations. All compounds showed good to strong broad-spectrum activity, equivalent or better in comparison with standard antibiotics used. Furthermore, seven compounds exhibited significant antibacterial efficiency against Gram-negative isolates. To visualize the results, principal component analysis was used as an additional classification tool. Cytotoxicity of compounds with different cell lines in human cell culture was determined. Based on these results, substituted quaternary Cinchona scaffold can be considered as promising new class of antimicrobials and further investigations should be performed. Supported by Croatian Science Foundation, Project No 3775 ADESIRE.

Keywords: antibacterial efficiency, cinchona alkaloids, cytotoxicity, pseudo‐enantiomers

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Authors: Dalia G. Aseel

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The potato (Solanum tubersum, L.) has become one of the major vegetable crops in Egypt and all over the world. Potato leafroll virus(PLRV) was observed on potato plants collected from different governorates in Egypt. Three cultivars, Spunta, Diamont, and Cara, infected with PLRV were collected; RNA was extracted and subjected to Real-Time PCR using the coat protein gene primers. The results showed that the expression of the coat protein was 39.6-fold, 12.45-fold, and 47.43-fold, respectively, for Spunta, Diamont, and Cara cultivars. Differential Display Polymerase Chain Reaction (DD-PCR) using pathogenesis-related protein 1 (PR-1), β-1,3-glucanases (PR-2), chitinase (PR-3), peroxidase (POD), and polyphenol oxidase (PPO) forward primers for pathogenesis-related proteins (PR). The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, 58 up-regulated and 19 down-regulated genes were detected. Sequence analysis of the up-and down-regulated genes revealed that infected plants were observed in comparison with the healthy control. Sequence analysis of the up-regulated gene was performed, and the encoding sequence analysis showed that the obtained genes include: induced stolen tip protein. On the other hand, two down-regulated genes were identified: disease resistance RPP-like protein and non-specific lipid-transfer protein. In this study, the expressions of PR-1, PR-2, PR-3, POD, and PPO genes in the infected leaves of three potato cultivars were estimated by quantitative real-time PCR. We can conclude that the PLRV-infection of potato plants inhibited the expression of the five PR genes. On the contrary, infected leaves by PLRV elevated the expression of some defense genes. This interaction may also induce and/or suppress the expression of some genes responsible for the plant's defense mechanisms.

Keywords: PLRV, pathogenesis-related proteins (PRs), DD-PCR, sequence, real-time PCR

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Authors: Shivam Yadav, Neelam Atri

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Cyanobacteria are photoautotrophic prokaryotes able to grow in diverse ecological habitats, originated 2.5 - 3.5 billion years ago and brought oxygenic photosynthesis. Since then superoxide dismutases (SODs) acquired great significance due to their ability to catalyze detoxification of byproducts of oxygenic photosynthesis, i.e. superoxide radicals. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of the superoxide dismutases family. In the present study, we extracted information regarding SODs from species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. 144 putative SOD homologues were identified. SODs are present in all cyanobacterial species reflecting their significant role in survival. However, their distribution varies, fewer in unicellular marine strains whereas abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic SODs were conserved well in these proteins. These SODs were classified into three major families according to their domain structures. Interestingly, they lack additional domains as found in proteins of other family. Phylogenetic relationships correspond well with phylogenies based on 16S rRNA and clustering occurs on the basis of structural characteristics such as domain organization. Similar conserved motifs and amino acids indicate that cyanobacterial SODs make use of a similar catalytic mechanism as eukaryotic SODs. Gene gain-and-loss is insignificant during SOD evolution as evidenced by absence of additional domain. This study has not only examined an overall background of sequence-structure-function interactions for the SOD gene family but also revealed variation among SOD distribution based on ecophysiological and morphological characters.

Keywords: comparative genomics, cyanobacteria, phylogeny, superoxide dismutases

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Authors: Recep Keşli, Gülşah Aşık, Cengiz Demir, Onur Türkyılmaz

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Objective: Proteus mirabilis (P. mirabilis) is one of Gram-negative pathogens in human and it causes urinary tract and nosocomial infections. P. mirabilis is susceptible to β-lactams, aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. It was aimed to investigate the resistance status to antimicrobial agents of Proteus mirabilis strains produced from samples sent to Afyon Kocatepe University, ANS Research and Practice Hospital, Microbiology Laboratory from different clinics and polyclinics during the period of 24 months. Methods: Between October 2013 and September 2015, a total of 30 Proteus were isolated from clinical samples of patients were hospitalized in intensive care units and in various departments of Afyon Kocatepe University, ANS Research and Practice Hospital. Identification of the bacteria was determined by conventional methods and VITEK 2 system (bioMérieux, France) was used additionally. Antibacterial susceptibility tests were performed by Kirby Bauer disc (Oxoid, Hempshire, England) diffusion method following the recommendations of CLSI. Results: Of the total 30 Proteus strains isolated from clinical samples, 19 from urine, 7 from wound, 4 from tracheal aspiration materials were isolated. Antimicrobial resistant for these strains were determined to 24,3% for meropenem, 26.2% for imipenem, 20.2% for amikacin 10.5% for cefepim, 33.3% for ciprofloxacin and levofloxacine, 31.6% for ceftazidime, 20% for ceftriaxone, 15.2% for gentamicin and 26.6% for amoxicillin-clavulanate, 26.2% trimethoprim-sulfamethoxale. Conclusion: In the present study, the highest number of clinical isolates of P. mirabilis were isolated from urine (63,3%), followed by the others (36,6%). The distribution of samples P. mirabilis strains to the clinics were as fallows; 16,8% intensive care unit (ICU), 29,9% polyclinics, 53,3% hospital service units The most effective antibiotic on the total of strains were found to be cefepim, the least effective antibiotics on the total of strains were found to be trimethoprim-sulfamethoxale.

Keywords: proteus mirabilis, antibiotic resistance, intensive care unit, Proteus spp.

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Authors: Debabrata Biswas

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The current trend in the development of antibiotic resistance by multiple bacterial pathogens has resulted in a troubling loss of effective antibiotic options for human. The emergence of multi-drug-resistant pathogens has necessitated higher dosages and combinations of multiple antibiotics, further exacerbating the problem of antibiotic resistance. Zoonotic bacterial pathogens, such as Salmonella, Campylobacter, Shiga toxin-producing Escherichia coli (such as enterohaemorrhagic E. coli or EHEC), and Listeria are the most common and predominant foodborne enteric infectious agents. It was observed that these pathogens gained/developed their ability to survive in the presence of antibiotics either in farm animal gut or farm environment and researchers believe that therapeutic and sub-therapeutic antibiotic use in farm animal production might play an important role in it. The mechanism of action of antimicrobial components used in farm animal production in genomic interplay in the gut and farm environment, has not been fully characterized. Even the risk of promoting the exchange of mobile genetic elements between microbes specifically pathogens needs to be evaluated in depth, to ensure sustainable farm animal production, safety of our food and to mitigate/limit the enteric infection with multiple antibiotic resistant bacterial pathogens. Due to the consumer’s demand and considering the current emerging situation, many countries are in process to withdraw antibiotic use in farm animal production. Before withdrawing use of the sub-therapeutic antibiotic or restricting the use of therapeutic antibiotics in farm animal production, it is essential to find alternative natural antimicrobials for promoting the growth of farm animal and/or treating animal diseases. Further, it is also necessary to consider whether that compound(s) has the potential to trigger the acquisition or loss of genetic materials in zoonotic and any other bacterial pathogens. Development of alternative therapeutic and sub-therapeutic antimicrobials for farm animal production and food processing and preservation and their effective implementation for sustainable strategies for farm animal production as well as the possible risk for horizontal gene transfer in major enteric pathogens will be focus in the study.

Keywords: food safety, natural antimicrobial, sustainable farming, antibiotic resistance

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Authors: K. J. Sannapapamma, H. Malligawad Lokanath, Sakeena Naikwadi

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Eco-friendly textiles are gaining importance among the consumers and textile manufacturers in the healthcare sector due to increased environmental pollution which leads to several health and environmental hazards. Hence, the research was designed to cultivate and develop the organic cotton knit, to prepare and characterize the Vetiver oil microcapsules for textile finishing and to access the wash durability of finished knits. The cotton SAHANA variety grown under organic production systems was processed and spun into 30 single yarn dyed with four natural colorants (Arecanut slurry, Eucalyptus leaves, Pomegranate rind and Indigo) and eco dyed yarn was further used for development of single jersy knitted fabric. Vetiveria zizanioides is an aromatic grass which is being traditionally used in medicine and perfumery. Vetiver essential oil was used for preparation of microcapsules by interfacial polymerization technique subjected to Gas Chromatography Mass Spectrometry (GCMS), Fourier Transform Infrared Spectroscopy (FTIR), Thermo Gravimetric Analyzer (TGA) and Scanning Electron Microscope (SEM) for characterization of microcapsules. The knitted fabric was finished with vetiver oil microcapsules by exhaust and pad dry cure methods. The finished organic knit was assessed for laundering on antimicrobial efficiency and aroma intensity. GCMS spectral analysis showed that, diethyl phthalate (28%) was the major compound found in vetiver oil followed by isoaromadendrene epoxide (7.72%), beta-vetivenene (6.92%), solavetivone (5.58%), aromadenderene, azulene and khusimol. Bioassay explained that, the vetiver oil and diluted vetiver oil possessed greater zone of inhibition against S. aureus and E. coli than the coconut oil. FTRI spectra of vetiver oil and microcapsules possessed similar peaks viz., C-H, C=C & C꞊O stretching and additionally oil microcapsules possessed the peak of 3331.24 cm^-1 at 91.14 transmittance was attributed to N-H stretches. TGA of oil microcapsules revealed that, there was a minimum weight loss (5.835%) recorded at 467.09°C compared to vetiver oil i.e., -3.026% at the temperature of 396.24°C. The shape of the microcapsules was regular and round, some were spherical in shape and few were rounded by small aggregates. Irrespective of methods of application, organic cotton knits finished with microcapsules by pad dry cure method showed maximum zone of inhibition compared to knits finished by exhaust method against S. aureus and E. coli. The antimicrobial activity of the finished samples was subjected to multiple washing which indicated that knits finished with pad dry cure method showed a zone of inhibition even after 20^th wash and better aroma retention compared to knits finished with the exhaust method of application. Further, the group of respondents rated that the 5^th washed samples had the greater aroma intensity in both the methods than the other samples. Thus, the vetiver microencapsulated organic cotton knits are free from hazardous chemicals and have multi-functional properties that can be suitable for medical and healthcare textiles.

Keywords: exhaust and pad dry cure finishing, interfacial polymerization, organic cotton knits, vetiver oil microcapsules

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Authors: Sarmiza Stanca, Wolfgang Fritzsche, Christoph Krafft, Jürgen Popp

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Independent of the cell type a thin layer of a few nanometers thickness surrounds the cell interior as the cellular membrane. The transport of ions and molecules through the membrane is achieved in a very precise way by pores. Understanding the process of opening and closing the pores due to an electrochemical gradient across the membrane requires knowledge of the pore constitutive proteins. Recent reports prove the access to the molecular level of the cellular membrane by atomic force microscopy (AFM). This technique also permits an electrochemical study in the immediate vicinity of the tip. Specific molecules can be electrochemically localized in the natural cellular membrane. Our work aims to recognize the protein domains of the pores using an AFM probe as a miniaturized amperometric sensor, and to follow the protein behavior while changing the applied potential. The intensity of the current produced between the surface and the AFM probe is amplified and detected simultaneously with the surface imaging. The AFM probe plays the role of the working electrode and the substrate, a conductive glass on which the cells are grown, represent the counter electrode. For a better control of the electric potential on the probe, a third electrode Ag/AgCl wire is mounted in the circuit as a reference electrode. The working potential is applied between the electrodes with a programmable source and the current intensity in the circuit is recorded with a multimeter. The applied potential considers the overpotential at the electrode surface and the potential drop due to the current flow through the system. The reported method permits a high resolved electrochemical study of the protein domains on the living cell membrane. The amperometric map identifies areas of different current intensities on the pore depending on the applied potential. The reproducibility of this method is limited by the tip shape, the uncontrollable capacitance, which occurs at the apex and a potential local charge separation.

Keywords: AFM, sensor, membrane, pores, proteins

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Authors: Zehour Rahmani, Messouda Dekmouche, Mohamed Hadjadj, Mokhtar Saidi

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In the last decades of the nineteenth century, the study of disease – causing microorganisms became concentrated on bacteria and largely institutionalized. In earlier years, the scientists interested in bacteria had originally been chemists like Pasteur, physicists like Tyndall, or botanists like Cohn and ward. For this reason, the objective of this research was to evaluate the potential of some dithiolethiones on standard microorganism strains as well as multi-drug resistant bacteria, which were isolated from hospitals. Recent studies have demonstrated, that several dithiolethione compounds, particularly (3H-1,2-dithiole-3-thione), exhibit the biological activities against several bacteria.

Keywords: bacteria, dithiolethiones, microorganism, potential

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Authors: Erfan Rahimi, Hadi Bahari Far, Mojgan Shikhpour

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Background: Urinary tract infection is one of the most common nosocomial infections, especially among women. E. coli is one of the main causes of urinary tract infections and one of the most common antibiotics to fight this bacterium is ampicillin. As conventional antibiotics led to bacterial antibiotic resistance, modification of the pure drugs can address this issue. The aim of this study was to prepare nanofluids containing carbon nanotubes conjugated with ampicillin to improve drug performance and reduce antibiotic resistance. Methods: Multi-walled carbon nanotubes (MWCNTs) were activated with thionyl chloride by reflux system and nanofluids containing antibiotics were prepared by ultrasonic method. The properties of the prepared nano-drug were investigated by general element analysis, infrared spectroscopy, Raman spectroscopy, scanning electron microscopy and transmission electron microscopy. After the treatment of the desired strain with nanofluid, microbial studies were performed to evaluate the antibacterial effects and molecular studies were carried out to measure the expression of the resistance gene AcrAB. Result: We have shown that the antimicrobial effect of ampicillin-functionalized MWCNTs at low concentrations performed better than that of the conventional drug in both resistant and ATCC strains. Also, a decrease in antibiotic resistance of bacteria treated with ampicillin-functionalized MWCNTs compared to the pure drug was observed. Also, ampicillin-functionalized MWCNTs downregulated the expression of AcrAB in treated bacteria. Conclusion: Because carbon nanotubes are capable of destroying the bacterial wall, which provides antibiotic resistance features in bacteria, their usage in the form of nanofluids can make lower dosages (about three times less) than that of the pure drug more effective. Additionally, the expression of the bacterial resistance gene AcrAB decreased, thereby reducing antibiotic resistance and improving drug performance against bacteria.

Keywords: urinary tract infection, antibiotic resistance, carbon nanotube, nanofluid

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Authors: Clotilde Guyon, Nada Jmari, Yen-Chin Li, Jean Denoyel, Noriyuki Fujikado, Christophe Blanchet, David Root, Matthieu Giraud

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Aire induces ectopic expression of a large repertoire of tissue-specific antigen (TSA) genes in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in maturing T cells. Although important mechanisms of Aire-induced transcription have recently been disclosed through the identification and the study of Aire’s partners, the fine transcriptional functions underlied by a number of them and conferred to Aire are still unknown. Alternative cleavage and polyadenylation (APA) is an essential mRNA processing step regulated by the termination complex consisting of 85 proteins, 10 of them have been related to Aire. We evaluated APA in MECs in vivo by microarray analysis with mRNA-spanning probes and RNA deep sequencing. We uncovered the preference of Aire-dependent transcripts for short-3’UTR isoforms and for proximal poly(A) site selection marked by the increased binding of the cleavage factor Cstf-64. RNA interference of the 10 Aire-related proteins revealed that Clp1, a member of the core termination complex, exerts a profound effect on short 3’UTR isoform preference. Clp1 is also significantly upregulated in the MECs compared to 25 mouse tissues in which we found that TSA expression is associated with longer 3’UTR isoforms. Aire-dependent transcripts escape a global 3’UTR lengthening associated with MEC differentiation, thereby potentiating the repressive effect of microRNAs that are globally upregulated in mature MECs. Consistent with these findings, RNA deep sequencing of actinomycinD-treated MECs revealed the increased stability of short 3’UTR Aire-induced transcripts, resulting in TSA transcripts accumulation and contributing for their enrichment in the MECs.

Keywords: Aire, central tolerance, miRNAs, transcription termination

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Authors: M. C. O. Ezeibe, F. I. O. Ezeibe

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Reasons viral diseases (including AI, HIV/AIDS, and COVID-19), tumors (including Cancers and Prostrate enlargement), and antimicrobial-resistant infections (AMR) are difficult to cure are features of the pathogens which normal cells do not have or need (biomedical markers) have not been identified; medicines that can counter the markers have not been invented; strategies and mechanisms for their treatments have not been developed. When cells become abnormal, they acquire negative electrical charges, and viruses are either positively charged or negatively charged, while normal cells remain neutral (without electrical charges). So, opposite charges' electrostatic attraction is a treatment mechanism for viral diseases and tumors. Medicines that have positive electrical charges would mop abnormal (infected and tumor) cells and DNA viruses (negatively charged), while negatively charged medicines would mop RNA viruses (positively charged). Molecules of Aluminum-magnesium silicate [AMS: Al₂Mg₃ (SiO₄)₃], an approved medicine and pharmaceutical stabilizing agent, consist of nanoparticles which have both positive electrically charged ends and negative electrically charged ends. The very small size (0.96 nm) of the nanoparticles allows them to reach all cells in every organ. By stabilizing antimicrobials, AMS reduces the rate at which the body metabolizes them so that they remain at high concentrations for extended periods. When drugs remain at high concentrations for longer periods, their efficacies improve. Again, nanoparticles enhance the delivery of medicines to effect targets. Both remaining at high concentrations for longer periods and better delivery to effect targets improve efficacy and make lower doses achieve desired effects so that side effects of medicines are reduced to allow the immunity of patients to be enhanced. Silicates also enhance the immune responses of treated patients. Improving antimicrobial efficacies and enhancing patients` immunity terminate infections so that none remains that could develop resistance. Some countries do not have natural deposits of AMS, but they may have Aluminum silicate (AS: Al₄ (SiO₄)₃) and Magnesium silicate (MS: Mg₂SiO₄), which are also approved medicines. So, AS and MS were used to formulate an AMS-brand, named Medicinal synthetic AMS {Al₄ (SiO₄)₃ + 3Mg₂SiO₄ → 2Al₂Mg₃ (SiO₄)₃}. To overcome the challenge of AMS, AS, and MS being un-absorbable, Dextrose monohydrate is incorporated in MSAMS-formulations for the simple sugar to convey the electrically charged nanoparticles into blood circulation by the principle of active transport so that MSAMS-antimicrobial formulations function systemically. In vitro, MSAMS reduced (P≤0.05) titers of viruses, including Avian influenza virus and HIV. When used to treat virus-infected animals, it cured Newcastle disease and Infectious bursa disease of chickens, Parvovirus disease of dogs, and Peste des petits ruminants disease of sheep and goats. A number of HIV/AIDS patients treated with it have been reported to become HIV-negative (antibody and antigen). COVID-19 patients are also reported to recover and test virus negative when treated with MSAMS. PSA titers of prostate cancer/enlargement patients normalize (≤4) following treatment with MSAMS. MSAMS has also potentiated ampicillin trihydrate, sulfadimidin, cotrimoxazole, piparazine citrate and chloroquine phosphate to achieve ≥ 95 % infection-load reductions (AMR-prevention). At 75 % of doses of ampicillin, cotrimoxazole, and streptomycin, supporting MSAMS-formulations' treatments with antioxidants led to the termination of even already resistant infections.

Keywords: electrical charges, viruses, abnormal cells, aluminum-magnesium silicate

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Authors: M. I. Tyletc, O. I. Podgornya, T. G. Shaposhnikova, S. V. Shabelnikov, A. G. Mittenberg, M. A. Daugavet

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Ascidiacea is the most numerous class of the Tunicata subtype. These chordates' distinctive feature of the anatomical structure is a tunic consisting of cellulose fibrils, protein molecules, and single cells. The mechanisms of the tunic formation are not known in detail; tunic formation could be used as the model system for studying the interaction of cells with the extracellular matrix. Our model species is the ascidian Styela rustica, which is prevalent in benthic communities of the White Sea. As previously shown, the tunic formation involves morula blood cells, which contain the major 48 kDa protein p48. P48 participation in the tunic formation was proved using antibodies against the protein. The nature of the protein and its function remains unknown. The current research aims to determine the amino acid sequence of p48, as well as to clarify its role in the tunic formation. The peptides that make up the p48 amino acid sequence were determined by mass spectrometry. A search for peptides in protein sequence databases identified sequences homologous to p48 in Styela clava, Styela plicata, and Styela canopus. Based on sequence alignment, their level of similarity was determined as 81-87%. The correspondent sequence of ascidian Styela canopus was used for further analysis. The Styela rustica p48 sequence begins with a signal peptide, which could indicate that the protein is secretory. This is consistent with experimentally obtained data: the contents of morula cells secreted in the tunic matrix. The isoelectric point of p48 is 9.77, which is consistent with the experimental results of acid electrophoresis of morula cell proteins. However, the molecular weight of the amino acid sequence of ascidian Styela canopus is 103 kDa, so p48 of Styela rustica is a shorter homolog. The search for conservative functional domains revealed the presence of two Ca-binding EGF-like domains, thrombospondin (TSP1) and tyrosinase domains. The p48 peptides determined by mass spectrometry fall into the region of the sequence corresponding to the last two domains and have amino acid substitutions as compared to Styela canopus homolog. The tyrosinase domain (pfam00264) is known to be part of the phenoloxidase enzyme, which participates in melanization processes and the immune response. The thrombospondin domain (smart00209) interacts with a wide range of proteins, and is involved in several biological processes, including coagulation, cell adhesion, modulation of intercellular and cell-matrix interactions, angiogenesis, wound healing and tissue remodeling. It can be assumed that the tyrosinase domain in p48 plays the role of the phenoloxidase enzyme, and TSP1 provides a link between the extracellular matrix and cell surface receptors, and may also be responsible for the repair of the tunic. The results obtained are consistent with experimental data on p48. The domain organization of protein suggests that p48 is an enzyme involved in the tunic tunning and is an important regulator of the organization of the extracellular matrix.

Keywords: ascidian, p48, thrombospondin, tyrosinase, tunic, tunning

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Authors: Alec Chabwinja, Cannan Tawonezvi, Jerneja Vidmar, Constance Chingwaru, Walter Chingwaru

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Objective: To evaluate the potential of commercial fermented / probiotic products available in Zimbabwe or internationally, and strains of Lactobacillus plantarum (L. plantarum) as prophylaxis and therapy against diarrhoeal and sexually transmitted infections. Methods: The antimicrobial potential of cultures of lactobacilli enriched from 4 Zimbabwean commercial food/beverage products, namely Dairibord Lacto sour milk (DLSM), Probrand sour milk (PSM), Kefalos Vuka cheese (KVC) and Chibuku opaque beer (COB); three probiotic products obtainable in Europe and internationally; and four strains of L. plantarum obtained from Balkan traditional cheeses and Zimbabwean foods against clinical strains of Escherichia coli (E. coli) and non-clinical strains of Candida albicans and Rhodotorula spp. was assayed using the well diffusion method. Three commercial Agar diffusion assay and a competitive exclusion assay were carried out on Mueller-Hinton agar. Results: Crude cultures of putative lactobacillus strains obtained from Zimbabwean dairy products (Probrand sour milk, Kefalos Vuka vuka cheese and Chibuku opaque beer) exhibited significantly greater antimicrobial activities against clinical strains of E. coli than strains of L. plantarum isolated from Balkan cheeses (CLP1, CLP2 or CLP3) or crude microbial cultures from commercial paediatric probiotic products (BG, PJ and PL) of a culture of Lactobacillus rhamnosus LGG (p < 0.05). Furthermore, the following has high antifungal activities against the two yeasts: supernatant-free microbial pellet (SFMP) from an extract of M. azedarach leaves (27mm ± 2.5) > cell-free culture supernatants (CFCS) from Maaz Dairy sour milk and Mnandi sour milk (approximately 26mm ± 1.8) > CFCS and SFMP from Amansi hodzeko (25mm ± 1.5) > CFCS from Parinari curatellifolia fruit (24mm ± 1.5), SFMP from P. curatellifolia fruit (24mm ± 1.4) and SFMP from mahewu (20mm ± 1.5). These cultures also showed high tolerance to acidic conditions (~pH4). Conclusions: The putative lactobacilli from four commercial Zimbabwean dairy products (Probrand sour milk, Kefalos Vuka vuka cheese and Chibuku opaque beer), and three strains of L. plantarum from Balkan cheeses (CLP1, CLP2 or CLP3) exhibited high antibacterial activities, while Maaz Dairy sour-, Mnandi sour- and Amansi hodzeko milk products had high antifungal activities. Our selection of Zimbabwean probiotic products has potential for further development into probiotic products for use in the control diarrhea caused by pathogenic strains of E. coli or yeast infections. Studies to characterise the probiotic potential of the live cultures in the products are underway.

Keywords: lactic acid bacteria, Staphylococcus aureus, Streptococcus spp, yeast, inhibition, acid tolerance

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Authors: K. Cartelier, D. Aime, V. Vernoud, J. Buitink, J. M. Prosperi, K. Gallardo, C. Le Signor

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Legumes can produce protein-rich seeds without nitrogen fertilizer through root symbiosis with nitrogen-fixing rhizobia. Rich in lysine, these proteins are used for human nutrition and animal feed. However, the instability of seed protein yield and quality due to environmental fluctuations limits the wider use of legumes such as pea. Breeding efforts are needed to optimize and stabilize seed nutritional value, which requires to identify the genetic determinism of seed protein plasticity in response to the environment. Towards this goal, we have studied the plasticity of protein content and composition of seeds from a collection of 200 Medicago truncatula ecotypes grown under four controlled conditions (optimal, drought, and winter/spring sowing). A quantitative analysis of one-dimensional protein profiles of these mature seeds was performed and plasticity indices were calculated from each abundant protein band. Genome-Wide Association Studies (GWAS) from these data identified major GWAS hotspots, from which a list of candidate genes was obtained. A Gene Ontology Enrichment Analysis revealed an over-representation of genes involved in several amino acid metabolic pathways. This led us to propose that environmental variations are likely to modulate amino acid balance, thus impacting seed protein composition. The selection of candidate genes for controlling the plasticity of seed protein composition was refined using transcriptomics data from developing Medicago truncatula seeds. The pea orthologs of key genes were identified for functional studies by mean of TILLING (Targeting Induced Local Lesions in Genomes) lines in this crop. We will present how this study highlighted mechanisms that could govern seed protein plasticity, providing new cues towards the stabilization of legume seed quality.

Keywords: GWAS, Medicago truncatula, plasticity, seed, storage proteins

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Authors: Atena Sadat Hosseini, Mohammadhossein Habibi

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Acute myeloid leukemia (AML) is the most typically diagnosed leukemia. In older adults, AML imposes a dismal outcome. AML originates with a dominant mutation, then adds collaborative, transformative mutations leading to myeloid transformation and clinical/biological heterogeneity. Several chemotherapeutic drugs are used for this cancer. These drugs are naturally associated with several side effects, and finding a more accurate molecular mechanism of these drugs can have a significant impact on the selection and better candidate of drugs for treatment. In this study, we evaluated bortezomibin myeloma cells using bioinformatics analysis and evaluation of RNA-Seq data. Then investigated the molecular pathways proteins- proteins interactions associated with this chemotherapy drug. A total of 658upregulated genes and 548 downregulated genes were sorted.AUF1 (hnRNP D0) binds and destabilizes mRNA, degradation of GLI2 by the proteasome, the role of GTSE1 in G2/M progression after G2 checkpoint, TCF dependent signaling in response to WNT demonstrated in upregulated genes. Besides insulin resistance, AKT phosphorylates targets in the nucleus, cytosine methylation, Longevity regulating pathway, and Signal Transduction of S1P Receptor were related to low expression genes. With respect to this results, HIST2H2AA3, RP11-96O20.4, ZED9, PRDX1, and DOK2, according to node degrees and betweenness elements candidates from upregulated genes. in the opposite side, PYURF, NRSN1, FGF23, UPK3BL, and STAG3 were a prominent role in downregulated genes. Sum up, Using in silico analysis in the present study, we conducted a precise study ofbortezomib molecular mechanisms in myeloma cells. so that we could take further evaluation to discovermolecular cancer therapy. Naturally, more additional experimental and clinical procedures are needed in this survey.

Keywords: myeloma cells, acute myeloid leukemia, bioinformatics analysis, bortezomib

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Authors: Suneeta Gireesh Panicker

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Aim: Aminopeptidase P (APP) is an enzyme that has specificity for proline, can specifically cleave Xaa-Proline peptides and is a metallo-aminopeptidase. The bonds nearby to the imino acid proline are tough to cleave by many peptidases, but APP can specifically break peptide bonds engaged with proline. Membrane-bound form and a cytosolic form are the two forms in which this enzyme exists. The exact physiological function of APP remains unclear and hence the present work attempts to determine it. Methods: In the present study, the expression pattern of cytosolic Aminopeptidase P (DAP) was determined in all the embryonic stages and larval stages of wild-type Drosophila by using polyclonal monospecific antibodies. To show the presence of DAP RNA in embryonic and larval stages, RNA in situ hybridization was performed. DAP promoter-LacZ fusion reporter gene vector was used to construct transgenic embryos to study the regulation pattern of DAP. To study the DAP expression profile, a transgenic fly consisting of a DAP promoter with β-gal and GFP reporter genes in front of it was constructed. Results: DAP protein expression was observed in neuroectodermal cells, posterior midgut primordium, proctodeum, ventral neuroblast and primordial stomatogastric nervous system. It was observed in the ventral cord and midgut in stage 12. The completely developed embryos showed the intense occurrence of it in the ventral cord and gut region. The eye-antennal disc, wing disc and leg disc also showed the presence of DAP protein. LacZ expression in transgenic embryos also showed the same pattern. Conclusion: Similar to various known multiple-functional proteins, DAP could be one with different functions at different stages and in different cells. Data presented here designates DAP functions in the early embryonic and imaginal dics differentiation and development, suggesting that it may be required for the metabolism of proteins like neuropeptides and tachykinins.

Keywords: aminopeptidase P, in situ hybridization, transgenic fly, embryonic stages

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Authors: Andrea Parisi, Samantha Vilkins, Luis Furuya-Kanamori, John A. Crump, Benjamin P. Howden, Darren Gray, Kathryn Glass, Martyn Kirk

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Objectives: Salmonella is a leading cause of foodborne enterocolitis worldwide. Nontyphoidal Salmonella (NTS) infections that are Multi-Drug Resistant (MDR) (non-susceptible to ≥1 agent in ≥3 antimicrobial categories) may result in more severe outcomes, although these effects have not been systematically examined. We conducted a systematic review and meta-analysis to examine impacts of MDR NTS on health in high-income settings. Methods: We systematically reviewed the literature from scientific databases, including PubMed, Scopus and grey literature sources, using PRISMA guidelines. We searched for data from case-control studies, cohorts, outbreaks, reports and theses, imposing no language restriction. We included only publications from January 1990 to September 2016 from high income countries as classified by World Bank. We extracted data from papers on duration of illness, hospitalisation rates, morbidity and mortality for MDR and non-MDR NTS strains. Results: After removing duplicates, the initial search revealed 4258 articles. After further screening, we identified 16 eligible studies for the systematic review, and 9 of these were included in meta-analysis. NTS serotypes differed among the reported studies but serotype Typhimurium, Enteritidis, Newport and Heidelberg were among the most often reported as MDR pathogens. Salmonella infections that were MDR were associated with excess bloodstream infections (OR 1.63; 95%CI 1.18-2.26), excess hospitalisations (OR 2.77; 95%CI 1.47-5.21) and higher mortality (OR 3.54; 95%CI 1.10-11.40). Conclusions: MDR NTS infections are a serious public health concern. With the emergence of MDR Salmonella strains in the high-income countries, it is crucial to restrict the use of antimicrobials both in animals and humans, and intervene to prevent foodborne infections.

Keywords: Antimicrobial Resistance, Bloodstream Infection, Health Outcomes, Hospitalisation, Invasive Disease, Multi-Drug Resistance (MDR), Mortality, Nontyphoidal Salmonella

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Authors: V. Konjik, J. Schwartz, R. Sandhoff, M. Mack

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The rising number of multi-resistant pathogens demands the development of new antibiotics in order to reduce the lethal risk of infections. Here, we investigate roseoflavin, a vitamin B2 analogue which is produced by Streptomyces davawensis and Streptomyces cinnabarinus. We consider roseoflavin to be a 'Trojan horse' compound. Its chemical structure is very similar to riboflavin but in fact it is a toxin. Furthermore, it is a clever strategy with regard to the delivery of an antibiotic to its site of action but also with regard to the production of this chemical: The producer cell has only to convert a vitamin (which is already present in the cytoplasm) into a vitamin analog. Roseoflavin inhibits the activity of Flavin depending proteins, which makes up to 3.5 % of predicted proteins in organisms sequenced so far. We sequentially knocked out gene clusters and later on single genes in order to find the ones which are involved in the roseoflavin biosynthesis. Consequently, we identified the gene rosB, coding for the protein carrying out the first step of roseoflavin biosynthesis, starting form Flavin mononucleotide. Here we show, that the protein RosB has so far unknown features. It is per se an oxidoreductase, a decarboxylase and an aminotransferase, all rolled into one enzyme. A screen of cofactors revealed needs of oxygen, NAD+, thiamine and glutamic acid to carry out its function. Surprisingly, thiamine is not only needed for the decaboxylation step, but also for the oxidation of 8-demethyl-8-formyl Flavin mononucleotide. We had managed to isolate three different Flavin intermediates with different oxidation states, which gave us a mechanistic insight of RosB functionality. Our work points to a so far new function of thiamine in Streptomyces davawensis. Additionally, RosB could be extremely useful for chemical synthesis. Careful engineering of RosB may allow the site-specific replacement of methyl groups by amino groups in polyaromatic compounds of commercial interest. Finally, the complete clarification of the roseoflavin biosynthesis opens the possibility of engineering cost-effective roseoflavin producing strains.

Keywords: antibiotic, flavin analogue, roseoflavin biosynthesis, vitamin B2

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Authors: Matej Buzgo, Erico Himawan, Ksenija JašIna, Aiva Simaite

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Electrospinning is a versatile and efficient technology for producing nanofibers for biomedical applications. One of the most common polymers used for the preparation of nanofibers for regenerative medicine and drug delivery applications is polycaprolactone (PCL). PCL is a biocompatible and bioabsorbable material that can be used to stimulate the regeneration of various tissues. It is also a common material used for the development of drug delivery systems by blending the polymer with small active molecules. However, for many drug delivery applications, e.g. cancer immunotherapy, PCL biodegradation rate that may exceed 9 months is too long, and faster nanofiber dissolution is needed. In this paper, we investigate the dissolution and small molecule release rates of PCL blends with two hydrophilic polymers: polyethylene oxide (PEO) or polyvinylpyrrolidone (PVP). We show that adding hydrophilic polymer to the PCL reduces the water contact angle, increases the dissolution rate, and strengthens the interactions between the hydrophilic drug and polymer matrix that further sustain its release. Finally using this method, we were also able to increase the nanofiber degradation rate when PCL-PEO and PCL-PVP were used as a shell in the electrospun core-shell nanofibers and spread up the release of active proteins from their core. Electrospinning can be used for the preparation of the core-shell nanofibers, where active ingredients are encapsulated in the core and their release rate is regulated by the shell. However, such fibers are usually prepared by coaxial electrospinning that is an extremely low-throughput technique. An alternative is emulsion electrospinning that could be upscaled using needleless blades. In this work, we investigate the possibility of using emulsion electrospinning for encapsulation and sustained release of the growth factors for the development of the organotypic skin models. The core-shell nanofibers were prepared using the optimized formulation and the release rate of proteins from the fibers was investigated for 2 weeks – typical cell culture conditions.

Keywords: electrospinning, polycaprolactone (PCL), polyethylene oxide (PEO), polyvinylpyrrolidone (PVP)

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Authors: Lorena Coelho, David Ramada, Catarina Nobre, Joaquim Gaião, Juliana Duarte

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The development of processes that allows the valorization of waste and by-products generated by industries is crucial to promote symbiotic relationships between different sectors and is mandatory to “close the loop” in the circular economy paradigm. In recent years, by-products and waste from agro-food and forestry sector have attracted attention due to their potential application and technical characteristics. The extraction of bio-based active compounds to be reused is in line with the circular bioeconomy concept trends, combining the use of renewable resources with the process’s circularity, aiming the waste reduction and encouraging reuse and recycling. Among different types of bio-based materials, which are being explored and can be extracted, proteins fractions are becoming an attractive new raw material. Within this context, BioTrace4Leather project, a collaboration between two Technological Centres – CeNTI and CTIC, and a company of Tanning and Finishing of Leather – Curtumes Aveneda, aims to develop innovative and biologically sustainable solutions for leather industry and accomplish the market circularity trends. Specifically, it aims to the valorisation of waste and by-products from the tannery industry through proteins extraction and the development of an innovative and biologically sustainable materials. The achieved results show that keratin, gelatine, and collagen fractions can be successfully extracted from hair and leather bovine waste. These products could be reintegrated into the industrial manufacturing process to attain innovative and functional textile and leather substrates. ACKNOWLEDGEMENT This work has been developed under BioTrace4Leather scope, a project co-funded by Operational Program for Competitiveness and Internationalization (COMPETE) of PORTUGAL2020, through the European Regional Development Fund (ERDF), under grant agreement Nº POCI-01-0247-FEDER-039867.

Keywords: leather by-products, circular economy, sustainability, protein fractions

Procedia PDF Downloads 137

Authors: Malgorzata Krysiak, Anna Wegrzyn, Maciej Garstka, Radoslaw Mazur

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Under constantly fluctuating environmental conditions, the thylakoid membrane protein network evolved the ability to dynamically respond to changing biotic and abiotic factors. One of the most important protective mechanism is rearrangement of the chlorophyll-protein (CP) complexes, induced by protein phosphorylation. In a temperate climate, low temperature is one of the abiotic stresses that heavily affect plant growth and productivity. The aim of this study was to determine the role of LHCII antenna complex phosphorylation in the dark-chilling response. The study included an experimental model based on dark-chilling at 4 °C of detached chilling sensitive (CS) runner bean (Phaseolus coccineus L.) and chilling tolerant (CT) garden pea (Pisum sativum L.) leaves. This model is well described in the literature as used for the analysis of chilling impact without any additional effects caused by light. We examined changes in thylakoid membrane protein phosphorylation, interactions between phosphorylated LHCII (P-LHCII) and CP complexes, and their impact on the dynamics of photosystem II (PSII) under dark-chilling conditions. Our results showed that the dark-chilling treatment of CS bean leaves induced a substantial increase of phosphorylation of LHCII proteins, as well as changes in CP complexes composition and their interaction with P-LHCII. The PSII photochemical efficiency measurements showed that in bean, PSII is overloaded with light energy, which is not compensated by CP complexes rearrangements. On the contrary, no significant changes in PSII photochemical efficiency, phosphorylation pattern and CP complexes interactions were observed in CT pea. In conclusion, our results indicate that different responses of the LHCII phosphorylation to chilling stress take place in CT and CS plants, and that kinetics of LHCII phosphorylation and interactions of P-LHCII with photosynthetic complexes may be crucial to chilling stress response. Acknowledgments: presented work was financed by the National Science Centre, Poland grant No.: 2016/23/D/NZ3/01276

Keywords: LHCII, phosphorylation, chilling stress, pea, runner bean

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Authors: Mohamed Trigui, Fatma Masmoudi, Imen Zouari

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Massive utilizations of chemical fertilizer and chemical pesticides in agriculture sector to improve the farming productivity have created increasing environmental damages. Then, agriculture must become sustainable, focusing on production systems that respect the environment and help to reduce climate change. Isolation and microbial identification of new bacterial strains from naturally saline habitats and compost extracts could be a prominent way in pest management and crop production under saline conditions. In this study, potential mechanisms involved in plant growth promotion and suppressive activity against fungal diseases of a compost extract produced from poultry manure/olive husk compost and halotolerant and halophilic bacterial strains under saline stress were investigated. On the basis of the antimicrobial tests, different strains isolated from Sfax solar saltern (Tunisia) and from compost extracts were selected and tested for their plant growth promoting traits, such as siderophores production, nitrogen fixation, phosphate solubilization and the production of extracellular hydrolytic enzymes (protease and lipase) under in-vitro conditions. Among 450 isolated bacterial strains, 16 isolates showed potent antifungal activity against the tested plant pathogenic fungi. Their identification based on 16S rRNA gene sequence revealed they belonged to different species. Some of these strains were also characterized for their plant growth promoting capacities. Obtained results showed the ability of four strains belonging to Bacillus genesis to ameliorate germination rate and root elongation compared to the untreated positive controls. Combinatorial capacity of halotolerant bacteria with antimicrobial activity and plant growth promoting traits could be promising sources of interesting bioactive substances under saline stress.

Keywords: abiotic stress, biofertilizer, biotic stress, compost extract, halobacteria, plant growth promoting (PGP), soil fertility

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Authors: Muñiz-Lino Marcos Agustín, Vazquez Borbolla Jessica, Licéaga-Escalera Carlos

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The ectomesenchymal tissues involved in tooth development and their remnants are the origin of different odontogenic lesions, including tumors and cysts of the jaws, with a wide range of clinical behaviors. Dentigerous cyst (DC) represents approximately 20% of all cases of odontogenic cysts, and it has been demonstrated that it can develop benign and malignant odontogenic tumors. DC is characterized by bone destruction of the area surrounding the crown of a tooth which has not erupted and it contain is liquid. The treatment of odontogenic tumors and cysts usually are partial or total removal of the jaw, causing important secondary co-morbidities. However, molecules implicated in DC pathogenesis as well in its development to odontogenic tumors remains unknown. A cellular model may be useful to study these molecules, but that model has not been established yet. Here, we reported the establishment of a cell culture derived from a dentigerous cyst. This cell line was named DeCy-1. In spite of its ectomesenchymal morphology, DeCy-1 cells express epithelial markers such as cytokeratins 5, 6, and 8. Furthermore, these cells express the ODAM protein, which is present in odontogenesis and in dental follicle, indicating that DeCy-1 cells derived from odontogenic epithelium. Analysis by electron microscopy of this cell line showed that it has a high vesicular activity, suggesting that DeCy-1 could secrete molecules that may be involved in DC pathogenesis. Thus, secreted proteins were analyzed by PAGE-SDS, where we observed approximately 11 bands. In addition, the capacity of these secretions to degrade proteins was analyzed by gelatin substrate zymography. A degradation band of about 62 kDa was found in these assays. Western blot assays suggested that the matrix metalloproteinase 2 (MMP-2) is responsible of this protease activity. Thus, our results indicate that the establishment of a cell line derived from DC is a useful in vitro model to study the biology of this odontogenic lesion and its participation in the development of odontogenic tumors.

Keywords: dentigerous cyst, MMP20, cancer, cell culture

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Authors: Barun K. De

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Acquired immunodeficiency syndrome (AIDS) is caused by two types of human immunodeficiency viruses, collectively designated HIV. HIV infection is spreading globally particularly in developing countries. Before an individual is diagnosed with HIV, the disease goes through different phases. First there is an acute early phase that is followed by an established or chronic phase. Subsequently, there is a latency period after which the individual becomes immunodeficient. It is in the acute phase that an individual is highly infectious due to a high viral load. Presently, HIV diagnosis involves use of tests that do not detect the acute phase infection during which both the viral RNA and p24 antigen are expressed. Instead, these less sensitive tests detect antibodies to viral antigens which are typically sero-converted later in the disease process following acute infection. These antibodies are detected in both asymptomatic HIV-infected individuals as well as AIDS patients. Studies indicate that early diagnosis and treatment of HIV infection can reduce medical costs, improve survival, and reduce spreading of infection to new uninfected partners. Newer 4th generation combination antigen/antibody tests are highly sensitive and specific for detection of acute and established HIV infection (HIV1 and HIV2) enabling immediate linkage to care. The CDC (Center of Disease Control, USA) recently recommended an algorithm involving three different tests to screen and diagnose acute and established infections of HIV-1 and HIV-2 in a general population. Initially a 4th generation combo test detects a viral antigen p24 and specific antibodies against HIV -1 and HIV-2 envelope proteins. If the test is positive it is followed by a second test known as a differentiation assay which detects antibodies against specific HIV-1 and HIV-2 envelope proteins confirming established infection of HIV-1 or HIV-2. However if it is negative then another test is performed that measures viral load confirming an acute HIV-1 infection. Screening results of a Phoenix area population detected 0.3% new HIV infections among which 32.4% were acute cases. Studies in the U.S. indicate that this algorithm effectively reduces HIV infection through immediate treatment and education following diagnosis.

Keywords: new algorithm, HIV, diagnosis, infection

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Authors: Kuladip Jana

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol Karim Sheikhi, Mehdi Hedayati

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Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.

Keywords: hTSH, bioinformatics, protein expression, cross reactivity

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Authors: Hasan Demiroğlu, Uğur Avcıbaşı, Serhan Sakarya, Perihan Ünak

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Implanted devices are progressively practiced in innovative medicine to relieve pain or improve a compromised function. Implant-associated infections represent an emerging complication, caused by organisms which adhere to the implant surface and grow embedded in a protective extracellular polymeric matrix, known as a biofilm. In addition, the microorganisms within biofilms enter a stationary growth phase and become phenotypically resistant to most antimicrobials, frequently causing treatment failure. In such cases, surgical removal of the implant is often required, causing high morbidity and substantial healthcare costs. Staphylococcus aureus is the most common pathogen causing implant-associated infections. Successful treatment of these infections includes early surgical intervention and antimicrobial treatment with bactericidal drugs that also act on the surface-adhering microorganisms. Linezolid is a promising anti-microbial with ant-staphylococcal activity, used for the treatment of MRSA infections. Linezolid is a synthetic antimicrobial and member of oxazolidinoni group, with a bacteriostatic or bactericidal dose-dependent antimicrobial mechanism against gram-positive bacteria. Intensive use of antibiotics, have emerged multi-resistant organisms over the years and major problems have begun to be experienced in the treatment of infections occurred with them. While new drugs have been developed worldwide, on the other hand infections formed with microorganisms which gained resistance against these drugs were reported and the scale of the problem increases gradually. Scientific studies about the production of bacterial biofilm increased in recent years. For this purpose, we investigated the activity of Lin, Lin radiolabeled with 131I (131I-Lin) and cold iodinated Lin (127I-Lin) against clinical strains of Staphylococcus aureus DSM 4910 in biofilm. In the first stage, radio and cold labeling studies were performed. Quality-control studies of Lin and iodo (radio and cold) Lin derivatives were carried out by using TLC (Thin Layer Radiochromatography) and HPLC (High Pressure Liquid Chromatography). In this context, it was found that the binding yield was obtained to be about 86±2 % for 131I-Lin. The minimal inhibitory concentration (MIC) of Lin, 127I-Lin and 131I-Lin for Staphylococcus aureus DSM 4910 strain were found to be 1µg/mL. In time-kill studies of Lin, 127I-Lin and 131I-Lin were producing ≥ 3 log10 decreases in viable counts (cfu/ml) within 6 h at 2 and 4 fold of MIC respectively. No viable bacteria were observed within the 24 h of the experiments. Biofilm eradication of S. aureus started with 64 µg/mL of Lin, 127I-Lin and 131I-Lin, and OD630 was 0.507±0.0.092, 0.589±0.058 and 0.266±0.047, respectively. The media control of biofilm producing Staphylococcus was 1.675±0,01 (OD630). 131I and 127I did not have any effects on biofilms. Lin and 127I-Lin were found less effectively than 131I-Lin at killing cells in biofilm and biofilm eradication. Our results demonstrate that the 131I-Lin have potent anti-biofilm activity against S. aureus compare to Lin, 127I-Lin and media control. This is suggested that, 131I may have harmful effect on biofilm structure.

Keywords: iodine-131, linezolid, radiolabeling, slime layer, Staphylococcus

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Authors: Fatemeh Haghiralsadat, Mohadese Hashemi, Elham Akhoundi Kharanaghi, Mojgan Yazdani, Mahboobe Sharafodini, Omid Javani

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Niosomes are colloidal particles formed from the self-assembly of non-ionic surfactants in aqueous medium resulting in closed bilayer structures. As a consequence of this hydrophilic and hydrophobic structure, niosomes have the capacity to entrap compounds of different solubilities. Niosomes are promising vehicle for drug delivery which protect sensitive drugs and improve the therapeutic index of drugs by restricting their action to target cells. Essential oils are complex mixtures of volatile compounds such as terpenoids, phenol-derived aromatic components that have been used for many biological properties including bactericidal, fungicidal, insecticidal, antioxidant, anti-tyrosinase and other medicinal properties. Encapsulation of essential oils in niosomes can be an attractive method to overcome their limitation such as volatility, easily decomposition by heat, humidity, light, or oxygen. Cuminum cyminum L. (Cumin) is an aromatic plant included in the Apiaceae family and is used to flavor foods, added to fragrances, and for medical preparations which is indigenous to Egypt, the Mediterranean region, Iran and India. The major components of the Cumin oil were reported as cuminaldehyde, γ -terpinene, β-pinene, p-cymene, p-mentha-1, 3-dien-7-al, and p-mentha-1, 4-dien-7-al which provide the antimicrobial and antioxidant activity. The aim of this work was to formulate Cumin essential oil-loaded niosomes to improve water solubility of natural product and evaluate its physico-chemical features and stability. Cumin oil was obtained through steam distillation using a clevenger-type apparatus and GC/MS was applied to identify the main components of the essential oil. Niosomes were prepared by using thin film hydration method and nanoparticles were characterized for particle size, dispersity index, zeta potential, encapsulation efficiency, in vitro release, and morphology.

Keywords: Cuminum cyminum L., Cumin, niosome, essential oil, encapsulation

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Authors: Nicholas C. Rose, Christopher D. Spicer

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The regeneration of damaged or diseased tissues would provide an invaluable therapeutic tool in biological research and medicine. Cells must be provided with a number of different biochemical signals in order to form mature tissue through complex signaling networks that are difficult to recreate in synthetic materials. The ability to attach and detach bioactive proteins from material in an iterative and dynamic manner would therefore present a powerful way to mimic natural biochemical signaling cascades for tissue growth. We propose to reversibly attach these bioactive proteins using ortho-boronoimine (oBI) linkages and related derivatives formed by the reaction of an ortho-boronobenzaldehyde with a nucleophilic amine derivative. To enable the use of oBIs for biomaterial modification, we have studied binding and cleavage processes with precise detail in the context of small molecule models. A panel of oBI complexes has been synthesized and screened using a novel Förster resonance energy transfer (FRET) assay, using a cyanine dye FRET pair (Cy3 and Cy5), to identify the most reactive boron-aldehyde/amine nucleophile pairs. Upon conjugation of the dyes, FRET occurs under Cy3 excitation and the resultant ratio of Cy3:Cy5 emission directly correlates to conversion. Reaction kinetics and equilibria can be accurately quantified for reactive pairs, with dissociation constants of oBI derivatives in water (KD) found to span 9-orders of magnitude (10⁻²-10⁻¹¹ M). These studies have provided us with a better understanding of oBI linkages that we hope to exploit to reversibly attach bioconjugates to materials. The long-term aim of the project is to develop a modular biomaterial platform that can be used to help combat chronic diseases such as osteoarthritis, heart disease, and chronic wounds by providing cells with potent biological stimuli for tissue engineering.

Keywords: dynamic, bioconjugation, bornoimine, rapid, physiological

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Authors: Shivankar Agrawal, Sunil Kumar Deshmukh, Colin Barrow, Alok Adholeya

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A variety of genetic and environmental factors cause various cosmetics and dermatological problems. There are already claimed drugs available in market for treating these problems. However, the challenge remains in finding more potent, environmental friendly, causing minimal side effects and economical cosmeceuticals. This leads to an increased demand for natural cosmeceutical products in the last few decades. Plant derived ingredients are limited because plants either contain toxic metabolites, grow too slow or seasonal harvesting is a problem. To identify new bioactive cosmetics ingredients of marine microbial bioresource, we screened 35 marine fungi isolated from marine samples collected from Andaman Island and west coast of India. Fungal crude extracts were investigated for their antityrosinase, antioxidant and antibacterial activities for the purpose of identifying anti-aging, skin-whitening and anti-acne biomolecule with the potential in cosmetics. In the tyrosinase inhibition and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays, two fungal extracts, including “P2”, Talaromyces stipitatus and “D4”, Aspergillus terreus showed high inhibitory activity at 1mg/mL for tyrosinase inhibition and 0.5mg/mL for DPPH scavenging. The in vitro antimicrobial activity was investigated by the agar well diffusion method. In the tyrosinase inhibition assay, 8 extracts showed significant antibacterial activity against bacteria causing skin and wound infection in humans. In the course of systematic screening program for bioactive marine fungi, strain “D5” was found to be most potent strain with MIC value of 1mg/mL, which was morphologically identified as Simplicillium lamellicola. The effects of the most active crude extracts against their susceptible test microorganisms were also investigated by SEM analysis. Further investigations will focus on purification and characterization major active components responsible for these activities.

Keywords: antioxidant, antimicrobial activity, tyrosinase, cosmeceuticals, marine fungi

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