Nova Biologica Reperta

Anticancer effect of Artemisia extract and cisplatin on induction of apoptosis and inhibition of proliferation in A2780 human ovarian cancer

Javad Baharara

Cisplatin, as a chemotherapy drug, causes serious side effects in the advanced stages of the cancer. Recently, Artemisia has been considered for its bioactive compounds, anti-proliferative and anti-inflammatory effects. The aim of this study was to evaluate the anti-cancer and anti-metastatic effects of the methanolic extract of aerial organs of Artemisia and cisplatin, either alone or in combination, in human ovarian cancer cell line A2780. The viability of A2780 cells after treatment with Artemisia extract, cisplatin and their combination was evaluated by MTT assay and the alterations in the morphology of the cell nuclei were examined by DAPI staining. The induction of apoptosis was assessed by Annexin V test, cell migration and changes in expression levels of apoptotic genes (Bax and P53) and metastasis (MMP2 and MMP9) using real-time PCR. MTT test data showed that Artemisia extract, cisplatin and their combination decreased the viability of ovarian cancer cells. DAPI and Annexin V indicated the DNA fragmentation and increased percentage of cellular apoptosis in comparison with the control group. The migration and real-time PCR data showed a decline in thr cell invasion and expression of genes involved in metastasis (MMP2 and MMP9) in cancer cells while the expression of apoptotic genes (Bax and P53) was increased in the treated groups. The results of this study showed that while both Artemisia extract and cisplatin posses anti-proliferative effect, apoptotic and suitable anti-metastatic effects on their own in A2780 cell line, their combination have synergic effects and posses those desired properties in lower concentration of cisplatin, which can reduce the side effects of cisplatin in cancer treatment.

Frequency of PGP and MRPA efflux pump genes in drug resistance in clinical isolates of Leishmania tropica and L. major

Maede Parishan

This study aimed to identify PGP and MRPA genes in clinical isolates of Leishmania. The genes of pgpa (MRPA) and mdr1 (PGP) are involved in the drug resistance, their products act as dependent transporters of ATP (ABC Transporter) in the reflux of drugs from the cytosol to the outer space of the cell. Hence, 40 volunteers with leishmaniasis were randomly selected. Firstly, Amastigotes were examined under a light microscope, then inoculated into NNN-specific biphasic culture medium. Deoxy ribonucleic acids were extracted by phenol-chloroform method and were determined by ITS-specific primers. Then the frequency of two pumps involved in "drug resistance" was investigated by PCR. In this study, the mdr1 gene, which had previously been shown to be present in the in vitro resistant strains, was shown to have a higher frequency of pgpas, which could be due to the presence of MDR. It transports the drug from the inner layers of the lipid bilayer membrane to the outer layers, reducing the concentration of the drug inside the cell and causing drug resistance, while the MRPA pump is in the membrane of the cell organelles.

Optimization of S-layer protein purification from Deinococcus radiodurans strain R1

Mahdi Alijanianzadeh

S-layer proteins of Deinococcus radiodurans are the best self-assemble systems among other proteins that have an essential role in the fabrication of nanowires. Therefore, the purification of these proteins is necessary. The purpose of this research was to optimize the purification of s-layer protein from D. radiodurans with the response surface method. The three factors of SDS concentration, incubation time and mass percent in five levels were considered, and 20 runs were designed by Design-Expert software with a central composite method. Each run includes microbe culture, mass cell preparation, microbe incubation in specific SDS concentration and time and mass percent, separation of the bacteria from detergent with a centrifuge at 5000g, sedimentation of s-layer proteins from detergent solution with a centrifuge at 20000g, determination of protein concentration, and protein purity by Bradford and SDS-PAGE methods, respectively. Finally, the data obtained were analyzed. Analysis of the results demonstrated that at the 95% confidence level, the effect of the detergent concentration factor on the purified protein percent was more than other factors. The optimization results of factors are 5.64% SDS concentration, 7.33% mass percent, and 3 hours incubation time. At optimized conditions the protein concentration and purity percent were obtained 0.584 mg/ml and 47.61% respectively.

Isolation of some bacteria for halophilic microbiome of Urmia Lake

Reza Khakvar

Urmia Lake is the largest lake in the Iranian plateau and the second largest Salt Lake in the world. This study was conducted to identify hypersaline bacteria in the lake through the screening with molecular markers. For the molecular study of the bacterial microbiome of the lake, samples were collected from water, sludge and soil of the different parts of the lake during different seasons of 2018 and 2019, and then transferred to the laboratory under standard conditions. Bacterial isolates were purified from the samples using universal culture media. ERIC molecular marker was used to study the species diversity. After clustering analysis of the species on the basis of their genetic markers, one bacterium from each cluster was selected as the representative of each cluster and then identified by DNA barcoding method using the 16srDNA. Biochemical tests were performed to confirm the molecular results. In total, 102 bacterial isolates were isolated and purified from the samples, of which only 29 isolates were extremely-halophilic. The molecular diversity of isolates, based on ERIC molecular marker, showed that isolates can be assigned to five different clusters. Five isolates selected from each cluster were selected and their 16SrDNA region were amplified and sequenced with 16SrDNA-specific primers. The results showed that the five selected isolates with 99% similarity belonged to the species Microbulbifer halophilus, Halomonas salina, Bacillus sonorensis, Salinivibrio costicola and Bacillus aquimaris. The results of molecular identification were consistent with the results of biochemical tests.

Evaluation of the callogenesis of Centella asiatica, a medicinal plant

Fereshteh Heidargholinezhad

Centella asiatica, is well known to be a valuable medicinal plant for producing valuable compounds such as asiaticoside, asiatic acid, madcasoside and madcasic acid. The plant is believed to improve memory, lower blood pressure, be a strong antioxidant and anticancer. Therefore, it is important to optimize tissue culture methods in order to facilitate the extraction of medicinal compounds, gene transfer as well as improvement of medicinal properties of the plant. Calli prepared from various medicinal plants can be used to increase the amount of medicinal compounds in the cell suspension culture and gene transfer. The aim of this study was to investigate the effect of different concentrations of two hormones, BAP and NAA, on leaf explant for callus initiation, as one of the important sources of secondary metabolites production. For this purpose, leaf explants were treated with 6 different concentrations of BAP (0, 0.5, 1, 1.5, 2.5, 3.5 mg/L) and 5 different concentrations of NAA (0, 0.25, 0.5, 1, 2 mg/L). The results of this study showed that the best callus was obtained by the combination 1.5 mg/L of BAP and 0.5 mg/L of NAA, resulting in the leaf explants with callus induction of 100%, fresh weight of 1.457 gr and callus diameter of 1.459 cm. The results showed that two hormones of BAP and NAA have synergistic effects on the increase of the quality and quantity of the produced calli.

The effect of nano selenium and sodium selenate on biochemical, physiological, and growth parameters of Bell pepper (Capsicum anumm)

Alireza Iranbakhsh

Selenium, a non-essential element for plants, is essential for animals as well as human beings. Although the role of selenium in plants is yet to be properly understood, previous researches have shown that this element can affect plant growth and metabolism. In this study, the effect of foliar application of selenium nanoparticles (0, 5, 10, and 20 mg/L) and sodium selenate (0, 5, 10, and 20 mg/L) on the physiological and biochemical responses of bell pepper (Capsicum anumm L.) was investigated. The potential changes in various growth and biochemical indices were evaluated in response to the treatments. According to the results, selenium treatments at concentrations of 10 and 20 mg/L reduced the biomass accumulation in both roots and shoots. These treatments also increased the content of hydrogen peroxide and malondialdehyde. The foliar application of selenium led to the increase of the concentrations of soluble phenols, proline and thiols. The activity of antioxidant enzymes including catalase, peroxidase, ascorbate peroxidase, and polyphenol oxidase were increased in response to the selenium treatments. The protease activity displayed a similar upward trend following the selenium treatments.

Determining of optimal temperature for germination of lentil cultivars (Lens culinaris Medik) for autumn cultivation in Khuzestan province

Saeed Reza Hosseinzadeh

Successful germination is the main factor in proper establishment and seedling yield. Information on seed germination, in the face of temperature changes at the time of planting, is a way to identify more suitable cultivars for cultivation at the planting site. For this purpose, a factorial experiment was performed based on completely randomized design with 3 replications. This experiment was conducted to investigate the temperature changes on the germination and growth indicators of lentil cultivars at Khatam Al-Anbia University of Behbahan. The studied treatments included five lentil cultivars of Gachsaran, Kimia, Robat, Ziba and Bilehsevar, and the temperature levels were 20, 25 and 30 °C, similar to the autumn conditions of Khuzestan Province. The results showed that all the seedlings of investigated cultivars cultivated at temperatures of 20 and 30 °C had a significant decrease in all the investigated traits, i.e., germination percent, germination rate, germination vigor, seed vigor index, endosperm consumption, plumule length, radicle length, seedling length, plumule dry weight and radicle dry weight, in comparison with those seedlings cultivated in 25 °C. The study of the interaction of cultivar-temperature effects showed that Bilehsevar cultivar had the highest germination and growth indices at 25°C compared to other investigated cultivars. This increase in traits such as germination rate, germination vigor, seed vigor index, plumule dry weight and radicle length was more significant in Bilehsevar cultivar compared to other cultivars. The second rank of the highest germination and growth indices was assigned to Gachsaran cultivar under 25°C conditions, which had a significant increase compared to other cultivars (Kimia, Robat and Ziba). Considering the autumn cultivation of lentils and the changes of temperature in that season (temperature range of 20 to 30 °C) in the studied area, the use of Bilehsevar and Gachsaran cultivars under the temperature of 25 °C, due to their higher germination indicators compared to the other cultivars, i.e., Ziba, Kimia and Robat, could be recommended.