Evaluation of insecticidal potential of organochemicals on SF9 cell line

Background Organophosphates and Pyrethroids are the most widely used pesticides worldwide and are known to have significant toxicity on the nervous system of the target pest. Assessment for combined toxicity of Organophosphate and Pyrethroid on Sf9 (Spodoptera frugiperda) cells is less explored. The present study demonstrates and compares the two organochemicals whose trade names are Ammo and Profex, for its cytotoxic potential on the insect Sf9 cells. Ammo and Profex were selected as the test chemicals as toxicity of these insecticides at molecular and cellular level is poorly understood. Results The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that Ammo and Profex exhibited significant cytotoxicity to Sf9 cells in a time- and dose-dependent manner. In our study, the IC50 value was obtained by MTT assay and the sub-lethal concentrations (IC50/20-17.5 µg/ml, IC50/10-35 µg/ml, and IC50/5–70 µg/ml for Ammo and IC50/20-20 µg/ml, IC50/10-40 µg/ml, and IC50/5-80 µg/ml for Profex) were selected for further tests. Acridine orange/ethidium bromide staining proved the apoptotic cell death on exposure of both the insecticides confirming its toxic potential. Furthermore, antioxidant status was assessed using DCF-DA staining and both the insecticides resulted into an increased reactive oxygen species (ROS) generation. A dose- and time-dependent significant (p < 0.05) alterations in lipid peroxidase (LPO), glutathione (GSH) and catalase (CAT) activity were observed. Conclusion The results showed that both Ammo and Profex triggered apoptosis in Sf9 cells through an intrinsic mitochondrial pathway via the generation of ROS. Of the two insecticides, Ammo was found to be more toxic compared to Profex. The present study is important to evaluate the environmental safety and risk factors of Organochemicals’ exposure to crops and livestock.

MODIFIKASI METODE ISOLASI DNA CETYL TRIMETHYLAMMONIUM BROMIDE (CTAB) UNTUK SAMPEL EPITEL PIPI MANUSIA

Isolation of deoxyribonucleotide (DNA) is an important step in molecular analysis. In this process, DNA must be obtained in sufficient quantities and in good quality for any further analysis. The Cetyl Trimethylammonium Bromide (CTAB) method is commonly used in DNA isolation of plant or fungal. This method is an alternative in DNA isolation since it is easy and inexpensive. This study aims to modify the CTAB method for DNA isolation from human cheek epithelium for any molecular analysis. Epithelial cells were taken from the oral cavity of the researcher. The isolation protocol included cell lysis step with CTAB buffer and proteinase-K, purification step with the addition of chloroform:isoamylalcohol (24:1), precipitation step with isopropanol. The results of the ratio analysis of DNA spectrophotometer at wavelengths of 260 and 280 nm in the range of 1.73-1.85. The quality of DNA isolation was observed by agarose gel electrophoresis and a firm band was obtained after Ethidium Bromide staining. The DNA concentration in both methods ranged from 400-480 mg/mL. The time required for both methods ranges from 2.5-3 hours. The modified CTAB method DNA isolation protocol produces DNA that has good quality and quantity for molecular analysis processes, such as Polymerase Chain Reaction (PCR).

Antineoplastic Activity of Products Derived From Cellulose-containing Materials: Study of Levoglucosenone and Structurally-related Analogous as New Alternatives for Breast Cancer Treatment

Purpose: Breast cancer is the leading cause of cancer death among women worldwide. For this reason, the development of new therapies is still essential. In this work we have analyzed the antitumor potential of levoglucosenone, a chiral building block derived from glucose, and three structurally related analogues obtained from soybean hulls pyrolysis.Methods: Employing human and murine mammary cancer models, we have evaluated the effect of our compounds on cell viability through MTS assay, apoptosis induction by acridine orange / ethidium bromide staining and/or flow cytometry and the loss of mitochondrial potential by tetramethylrhodamine methyl ester staining. Autophagy and senescence induction were also evaluated by Western blot and β-galactosidase activity respectively. Secreted metalloproteases activity was determined by quantitative zymography. Migratory capacity was assessed by wound healing assays while invasive potential was analyzed using Matrigel-coated transwell chambers. In vivo studies were also performed to evaluate subcutaneous tumor growth and experimental lung colonization.Results: Apoptosis was identified as the main mechanism responsible for the reduction of monolayer cell content induced by the compounds without detecting modulations of autophagy or senescence processes. Two of the four compounds were able to modulate in vitro events associated with tumor progression, such as migratory potential, invasiveness, and proteases secretion. Furthermore, tumor volume and metastatic spread were significantly reduced in vivo after treatment with the compounds.Conclusion: We could obtain from soybean hulls, a material with almost no commercial value, a variety of chemical compounds useful for breast cancer treatment.

Restoring chemo-sensitivity to temozolomide via targeted inhibition of poly (ADP-ribose) polymerase-1 by naringin in glioblastoma

Inclining mortality with a constant plummet in the survival rates associated with glioblastoma still stands as an inveterate predicament. The only promising therapy with temozolomide (TMZ) is now side-lined due to escalated resistance mediated by Poly (ADP-ribose) Polymerase-1 (PARP-1). In the light of this, the very study was designed to evaluate the potential of an active phyto component named naringin, in inhibiting PARP-1, using in silico and in vitro methods. Under in silico settings, inhibitor bound crystal structure of PARP-1, i.e., 4UND was retrieved and molecular docking studies were performed against naringin using Schrodinger software. In vitro cytotoxicity and apoptotic detection assay were performed using C6 glioma cells. Docking studies revealed high affinity and low binding energy at the inhibition site with good stability. An increase in cytotoxicity to C6 cells was observed with TMZ and naringin combination when compared to TMZ alone. Isobologram plot confirmed the synergistic effect of the drug combination. A significant increase in the number of apoptotic cells with combination drugs, as evaluated by acridine orange and ethidium bromide staining reassured the reversal of resistance. In conclusion, chemosensitivity to TMZ was restored by successful inhibition of PARP-1 using naringin and the drug combination was hence proven effective in reversing TMZ resistance.

Evaluation of the Effect of Hyaluronic Acid–Based Biomaterial Enriched With Tenascin-C on the Behavior of the Neural Stem Cells

One of the most important natural extracellular constituents is hyaluronic acid (HA) with the potential to develop a highly organized microenvironment. In the present study, we enriched HA hydrogel with tenascin-C (TN-C) and examined the viability and survival of mouse neural stem cells (NSCs) using different biological assays. Following NSCs isolation and expansion, their phenotype was identified using flow cytometry analysis. Cell survival was measured using MTT assay and DAPI staining after exposure to various concentrations of 50, 100, 200, and 400 nM TN-C. Using acridine orange/ethidium bromide staining, we measured the number of live and necrotic cells after exposure to the combination of HA and TN-C. MTT assay revealed the highest NSCs viability rate in the group exposed to 100 nM TN-C compared to other groups, and a combination of 1% HA + 100 nM TN-C increased the viability of NSCs compared to the HA group after 24 hours. Electron scanning microscopy revealed the higher attachment of these cells to the HA + 100 nM TN-C substrate relative to the HA substrate. Epifluorescence imaging and DAPI staining of loaded cells on HA + 100 nM TN-C substrate significantly increased the number of NSCs per field over 72 hours compared to the HA group ( P < 0.05). Live and dead assay revealed that the number of live NSCs significantly increased in the HA + 100 TN-C group compared to HA and control groups. The enrichment of HA substrate with TN-C promoted viability and survival of NSCs and could be applied in neural tissue engineering approaches and regenerative medicine.

Synthesis of novel thiourea-/urea-benzimidazole derivatives as anticancer agents

A new series of urea and thiourea derivatives containing benzimidazole group as potential anticancer agents have been designed and synthesized. The structures of the synthesized compounds were characterized and confirmed by spectroscopic techniques such as 1H NMR, 13C NMR, and mass spectrometry. In vitro anticancer assay against two breast cancer (BC) cell lines, MDA-MB-231ER(−)/PR(−) and MCF-7ER(+)/PR(+), revealed that the cytotoxicity of 1-(2-(1H-benzo[d]imidazol-2-ylamino)ethyl)-3-p-tolylthiourea (7b) and 4-(1H-benzo[d]imidazol-2-yl)-N-(3-chlorophenyl)piperazine-1-carboxamide (5d) were higher in MCF-7 with IC50 values of 25.8 and 48.3 µM, respectively, as compared with MDA-MB-231 cells. Furthermore, 7b and 5d were assessed for their apoptotic potential using 4′,6-diamidino-2-phenylindole, acridine orange/ethidium bromide staining, and Caspase-3/7. After incubation with MCF-7, the compounds 7b and 5d induced apoptosis through caspase-3/7 activation. In conclusion, the compounds 7b and 5d are potential candidates for inducing apoptosis in different genotypic BC cell lines.

SCREENING OF BIOACTIVE COMPONENTS BY GC-MS ANALYSIS AND THE STUDY ON IN VITRO ANTICANCER EFFICACY OF MICROPORUS AFFINIS (BLUME & T. NEES) KUNT.

Natural compounds with biological activity are normally present in plants, mushrooms and their natural sources. Applied mycology is one of the most stimulating and rapidly evolving areas of the biological sciences. Hence the present study focussed on exploring Microporus affinis (Blume & T. Nees) Kunt., the least explored and edible bracket fungus. Chemical characterization by GC-MS analysis resulted in the presence of 47 bioactive compounds. 9, 12- Octadecadienoic acid (Z,Z)- methyl ester, Ergosterol, Monolinolein, Thiacremonone, Stellasterol, n- Hexadecanoic acid, Ribitol, Maltol etc., were the leading compounds. Because of the presence of various bioactive compounds which have been already reported to possess antitumor, antioxidant and anticancer activities, M. affinis extract has been tested for in vitro anticancer efficacy on DLD1 cell lines (cultured in DMEM medium) using MTT assay. It resulted in the decrease of percentage of viability as the increase in concentration of the extract. Apoptosis was determined by using Acridine orange and Ethidium bromide staining. Thus, the taxa, M. affinis can be recommended for further anticancer assays for validation.

Microsatellite marker-based variation in the growth hormone genes of Nile tilapia (Oreochromis niloticus)

Polymorphisms in growth hormone genes have been found to cause variation in growth performance of fish. The objective of the study was to reveal variations in microsatellite loci located in the growth hormone genes of Nile tilapia (Oreochromis niloticus). Five microsatellite loci namely GH-MS01, IGFII, IGFII-MS01, IGFII-MS03, and STR were analyzed to assess the genetic variation in the growth hormone genes of four stocks of O. niloticus viz. FBG-Mini Hatchery, FM-Mini hatchery, Eon Aquaculture Ltd. and BFRI. The microsatellite markers were amplified by polymerase chain reaction, separated by polyacrylamide gel electrophoresis and visualized through ethidium bromide staining. All the five loci were found to be polymorphic. The average number of alleles of FM-Mini hatchery stock (3.8) was found to be highest and that of the FBG-Mini hatchery (2.8) and Eon Aquaculture stocks was found to be lowest. The average observed heterozygosity (Ho) value of the FM-Mini hatchery stock was the highest (0.140) and that of FBG-Mini hatchery stock was the lowest (0.040). On the other hand, the average expected heterozygosity (He) was highest in the BFRI stock (0.660) and lowest in the FM-Mini hatchery and FBG-Mini hatchery stock (0.432). The fixation index (1 - (Ho / He) values were positive in all the loci (except locus GH-MS01 in Eon Aquaculture stock), which means these stocks (O. niloticus) were deficient in heterozygosity. Deviation from Hardy-Weinberg expectation at STR locus in FBG-Mini hatchery and Eon Aquaculture stocks were not significant but in all other cases the deviations were found to be significant. The results provide evidence that genetic variation exists within the growth hormone genes in all four stocks of O. niloticus. The polymorphisms that have been detected in the present study can be used to study association with growth and thus selection of fast growing Nile tilapia in Bangladesh.

Transmissive Antibiotic Resistance, Associated with the SXT Element, in Cholera Vibrios Isolated in the Territory of Russia

Aim. Detection of SXT elements in cholera vibrios O1 and nonO1/nonO139 serogroups and study of the effectiveness of their conjugative transmission to Escherichia coli cells.Materials and methods. In conjugation experiments, Vibrio cholerae O1 El Tor (3) and V. cholerae nonO1/ nonO139 (3) strains were used as donors. Donor strains, recipients, and transconjugants were tested in realtime PCR for sensitivity to antibiotics and for the presence of drug resistance genes and integrase gene (int). Electrophoresis was carried out on a 0.7% agarose gel with ethidium bromide staining.Results. Resistance to chloramphenicol, trimethoprim/sulfamethoxazole, streptomycin was transmitted in conjugation experiments with a frequency of 2.1 × 10–9–7.1 × 10–9. The genes int and dfrA1 (resistance to trimethoprim/sulfamethoxazole) were found in most V. cholerae strains, and were stably transmitted to E. coli QD Rif r cells and in reverse crosses of V. cholerae O1 El Tor 5879 Nalr .Conclusion. The detection of the SXT element in V. cholerae strains and its successful horizontal transfer emphasize the need to detect such mobile genetic elements to control the spread of antibiotic resistance in V. cholerae.

BPA toxicity during development of zebrafish embryo

Bisphenol A (BPA) is a monomer used in the production of polycarbonate, a polymer commonly found in plastics, epoxy resins and thermal papers. The presence of BPA in food, water, air and dust has been of great concern in recent years not only due to environmental and ecological issues but also because of its supposed risk to public health related to its mutagenic and carcinogenic potential. In this study we evaluated the toxicity of bisphenol A in zebrafish embryos (Danio rerio) and determined the 50% lethal concentration (LC50) of this chemical. BPA was used at concentrations ranging from 1 μM to 100 μM in E3 medium/0.5% dimethylsulfoxide (DMSO) from previously prepared stock solutions in 100% DMSO. Controls included embryos exposed only to E3 medium or supplemented with 0.5% DMSO. Camptothecin (CPT), a known inhibitor of cell proliferation was used as positive control at a concentration of 0.001 μM in E3 medium/0.5% DMSO. Adults zebrafish were placed for breeding a day before the experimental set up, then, viable embryos were collected and selected for use. Experiments were carried out in triplicates, according to specifications from Organization for Economic Cooperation and Development (OECD). One embryo/well (25 embryos per concentration) was distributed in 96 well microplates in presence or absence of the chemicals. The plates were kept in BOD incubators with a controlled temperature of 28.5 ºC and with photoperiod of 14 h light:10 h dark. After 24h, 48h, 72h and 96h exposure, the exposed embryos were evaluated according to the following parameters: mortality, coagulation, rate of heartbeat, hatching and presence of morphological abnormalities. Photography was obtained by photomicroscopy. Apoptosis was evaluated by DNA ladder assay. DNA was extracted by phenol:chloroform method and analyzed by 2% agarose gel electrophoresis. DNA fragments were visualized after ethidium bromide staining in ultraviolet transilluminator. The LC50 determined for BPA was 70 μM after 24 hours, 72 μM after 48 hours, 47 μM after 72 hours and 31 μM after 96 hours exposure. BPA induced morphological and physiological alterations such as yolk sac and pericardial edema, hatching delay or inhibition, spine deformation, decreasing in heartbeat rate and mortality. In conclusion, this study demonstrated that BPA induced marked malformations in zebrafish embryos at concentrations above 25 μM corroborating the current concerns related to the widespread presence of BPA in the air, food and water used by humans as well as in the bodily fluids and tissues.