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    Investigation of Anti-leishmanial Effects of Bee Products (Honey, Propolis) on Leishmania tropica Promastigotes
    (Ankara Microbiology Soc, 2020) Aksoy, Tulay; Sivcan, Eda; Dogan, Fatma; Cetin, Songul; Yar, Turkan Mutlu
    This study was aimed to investigate the anti-leishmanial effects of bee products (honey and propolis) by using the causative agent of cutaneous leishmaniasis Leishmania tropica promastigotes, in in vitro culture. In vitro anti-leishmanial efficacy of honey (pine, flower and chestnut) and propolis used in the study were evaluated using the microdilution method. Honey, which is a bee product, was dissolved with RPMI medium containing fetal calf serum (FCS) and diluted in the same medium, and serial dilutions were prepared in concentrations between 62.5-1000 mg/ml. Propolis, on the other hand, was dissolved with ethyl alcohol and only 2.5 mu l was used from all these concentrations since the alcohol content was more than 50% in these concentrations prepared and we thought that this rate would negatively effect the parasite development. Then, RPMI containing FCS was diluted in the medium and serial dilutions were prepared at concentrations between 50-800 mu g/ml. To the dilutions prepared, the promastigot suspension was added so that their final concentrations in the wells were 1 x 10(6) promastigot/ml and then the medium was incubated for 24 and 48 hours in 26 degrees C. After the incubation, promastigotes were determined microscopically for morphology, mobility and live parasite density, and cell viability was determined by MTS method and 50% inhibitor concentrations (IC50) were compared with control groups. Anti-leishmanial activity of propolis (50, 100, 200, 400 and 800 mu g/ml) and honey (62.5, 125, 250, 500 and 1000 mg/ml) on promastigotes was evaluated in vitro. In microscopic examinations, pine honey showed anti-leishmanial activity starting from 62.5 mg/ml, flower honey 250 mg/ml, and chestnut honey 125 mg/ml, and pine honey was more effective on promastigotes (p< 0.05), and propolis was effective from 100 mu g/ml concentration. It has been determined that very low concentrations of propolis caused changes in the morphological structure of the parasites and were more effective than the other bee products. The prevention of cell proliferation and decreasing of the IC50 values according with the time of pine honey (IC50= 109.28 mg/ml), flower honey (IC50= 248.07 mg/ml), chestnut honey (IC50= 147.65 mg/ml) and propolis (IC50= 82.98 pg/ml) applied on L.tropica promastigot cell culture was determined by MTS method. In this study, it was found that various concentrations of pine, flower, chestnut honey and propolis showed anti-leishmanial activity on L. tropica promastigotes. It has been observed that pine honey is more effective on promastigotes after 48 hours of incubation period, and propolis is more effective in both morphology and cell inhibition of the parasites even at very low concentrations. It is believed that these data can be used as an alternative treatment method against cutaneous leishmaniasis infections and further studies are required.
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    Molecular Characterization of Lucimycin Gene of Lucilia sericata
    (Ankara Microbiology Soc, 2020) Cetin, Songul; Aksoy, Tulay
    Lucilia sericata, a member of the Calliphoridae family, is one of the most common species in the genus Lucilia. Medical importance of L.serkata stems from its use in maggot debridement therapy (MDT). MDT is the name of L.sericata larvae being sterilized and used in the treatment of non-healing wounds. L.sericata maggots used in the treatment of chronic and non-healing wounds (decubitus ulcer, venous leg ulcer, diabetic foot ulcer, etc.) clean the wounds with the help of secreted proteolytic trypsin and lucimycin-like enzymes. The aim of the study was to determine the molecular characterization of lucimycin gene obtained from L.sericata larvae in MDT by using molecular methods and to contribute to the literature. In this study, continuous production of adult colonies of L.sericata species was carried out in insectarium unit where conditions such as light, humidity and temperature were formed. The life cycle of L.sericata was followed and the production of eggs, larvae, pupae, adult flies and fly colonies of the species were formed. In the third stage larvae obtained from adult flies in the insectarium unit, RNA was isolated and subsequently cDNA synthesis was performed by reverse transcription. Polymerase chain reaction (PCR) analysis of the synthesized cDNAs with the specific primers designed for the lucimycin gene of L.sericata was performed and the obtained amplicons were cloned into pJET1.2/blunt vector and the plasmid was purified. The recombinant plasmids were sequenced with vector-specific primers and target gene region sequences were obtained. After the molecular characterization of the isolate with nucleotide sequences was determined, it was registered to GenBank database with the accession number MF964229. The PCR product of 288 bp was obtained from the cDNA obtained from the larvae of L.sericata produced in the insectarium unit by PCR using lucimycin specific primers. The PCR product imaged on the gel was purified by transformation and subsequent colonies were screened to see whether they contained recombinant plasmids. Three of the colonies were identified as recombinant plasmids containing L.sericata lucimycin gene by PCR screening. From three colonies confirmed by PCR screening, recombinant plasmids containing L.sericata lucimycin gene were purified by miniprep. The recombinant plasmid product was confirmed to contain the L.sericata lucimycin gene by PCR from a total of 20 mu l of the recombinant plasmid miniprep product. DNA sequencing analysis was performed to confirm the plasmid after cloning. The 288 bp L.sericata lucimycin sequence was confirmed by DNA sequence analysis. The lucimycin gene isolated was confirmed by specific and pJET1.2 forward and reverse primers using Blastn algorithm as a result of species and/or subspecies using the Blastn algorithm and the related isolate was recorded in GenBank database with the MF964229 accessory number. The DNA sequence of the isolated sample was compared with other isolates found in GenBank by Pubmed/Blast program. KJ413251.1 was found to be 99% similar to the GenBank isolate. The 113th nucleotide was C (cytosine) in the sequence of our isolate, while the existence of G (guanine) in the sequence numbered KJ413251.1 GenBank revealed the difference between the two sequences. In this study the molecular characterization of lucimycin gene derived from L. sericata larvae were determined for the first time in Turkey, it is assumed that this molecule which has an antifungal property, can be used in the studies that will be carried out in the future, especially in microorganisms causing cutaneous infections. The study is important since the isolate is registered as a biological asset of Turkey in GenBank and also being the second study in the world.