Dye Ethidium Bromide Research Articles

OPTIMIZATION OF CONCENTRATION AND SOAKING TIME OF HARRIS HEMATOXYLIN IN DNA BAND EXAMINATION USING ELECTROPHORESIS

Background: Deoxyribonucleic acid (DNA) molecules can be effectively visualized when stained and observed under ultraviolet light during the electrophoresis process. The commonly used dye Ethidium bromide (EtBr) is considered hazardous due to its potential to cause mutations, cancer, and congenital disabilities. Various alternative dyes have been reported, one of which is hematoxylin. Hematoxylin compounds do not have mutagenic potential and are easier to apply than EtBr. However, there is no optimal variation in concentration and duration of staining for clear and effective visualization of DNA bands. Purpose: To find the concentration and staining time of harris hematoxylin staining for DNA bands from agarose gel electrophoresis. Method: An experimental method with a group comparison statistical design. The amplified DNA I6S rRNA gene from Escherichia coli (584 bp), which had undergone electrophoresis, was stained using harris hematoxylin dye at 0.01%; 0.02%; 0.03%; 0.04%; and 0.05% concentrations and immersion times soaking times of 10, 15, 20, and 30 minutes variations. The intensity of the DNA bands was analyzed using ImageJ. The staining power of the experimental groups was compared to the intensity of control dye and given a grading score of 1 - 4. The experiment was repeated twice, and the mean grading score was calculated. The highest mean value was considered the most optimal value. Result: A concentration of 0.02% showed relatively constant staining intensity for each soaking time. A mean value of 3.5 was obtained for a 0.01% concentration for 15 minutes. A 0.03% and 0.04% concentrations for 20 minutes. Conclusion: The highest mean value of 4 was obtained for Harris hematoxylin at 0.05% for 15 minutes.

Green synthesis of silver chloride nanoparticles derived from Artemisia sieberi extract (AgClNPs-ASE) induces cell cycle arrest and triggers apoptosis in human breast (MDA-MB-231) cancer cells

In recent years, metallic nanoparticles have attracted tremendous attention in nanomedicine. Here, we report green synthesis and determination of silver chloride nanoparticles synthesized using Artemisia sieberi extract (AgClNPs-ASE) and its anticancer properties against human breast cancer cells. Numerous techniques, such as X-ray diffraction (XRD), UV-visible spectra, transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive spectra (SEM–EDS), and dynamic light scattering (DLS) analysis determined the morphology characteristics and size of the obtained nanoparticles. Cytotoxic properties of AgClNPs-ASE were carried out toward human breast cancer MDA-MB-231 cells and nontumorigenic HEK293 cells. AgClNPs-ASE revealed better efficacy toward breast cancer cells and was relatively less cytotoxic to nontumorigenic cells. Induction of apoptosis was further revealed by Hoechst 33258 dye and dual acridine orange and ethidium bromide (AO/EB) staining and in cells along with upregulation of caspase-8 and 9 proteins. AgClNPs-ASE could trigger the apoptosis by upregulation of early (1.93%) and late (58.86%) apoptosis followed by fluorescein isothiocyanate (FITC)‐Annexin‐V/PI staining and enhanced cell cycle population (sub-G1). AgClNPs-ASE significantly increased NO production in cancer cells. Moreover, qRT-PCR study showed that AgClNPs-ASE treated cells induce increased expression of the LncRNA GAS5 gene. These findings suggest the remarkable apoptosis in cancer cells triggered by AgClNPs-ASE in vitro.

Neuronal Panx1 drives peripheral sensitization in experimental plantar inflammatory pain

BackgroundThe channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood.MethodsWild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund’s adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and β-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. β-galactosidase (β-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice.ResultsGlobal or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/β-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca2+ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/β-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of β-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG.ConclusionsThe present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.

Induction of apoptosis in B16-BL6 melanoma cells following exposure to electromagnetic fields modeled after intercellular calcium waves.

Exposure to time-varying electromagnetic fields (EMF) has the capacity to influence biological systems. Our results demonstrate that exposure to time-varying EMF modeled after the physiological firing frequency of intercellular calcium waves can inhibit proliferation and induce apoptosis in malignant cells. Single exposure of B16-BL6 cells to a Ca2+ EMF for 40 min reduced the number of viable cells by 50.3%. Cell imaging with acridine orange and ethidium bromide dye revealed substantial cellular apoptosis, preapoptotic cells, nuclear fragmentation, and large spacing between cells in the Ca2+ EMF condition when compared to the control condition. The ability of Ca2+ EMF to influence the proliferation and survival of malignant cells suggests that exposure to specific EMF may function as a potential anticancer therapy.

Multifunctional silk fibroin and cellulose acetate composite nanofibers incorporated with palladium and platinum nanoparticles for enhanced wound healing: Comprehensive characterization and in vivo assessment

Wound healing is a complex and dynamic process that relies on various factors for optimal tissue regeneration. Current wound management methods often lack the efficiency needed to address these challenges. In this study, we prepared and conducted a comprehensive analysis of silk fibroin and cellulose acetate composite nanofibers incorporated with palladium (Pd) and platinum (Pt) nanoparticles, emphasizing their potential as advanced materials for wound healing. Electron microscopy reveals nanofibrous scaffolds with interconnected fibers, an average diameter of 168.95 ± 135.79 nm, and Pd and Pt nanoparticles of 10.64 nm and 9.21 nm, with thicknesses ranging from 1.48 nm to 2.13 nm. The analysis of swelling, degradation, porosity, and mechanical strength highlights their capacity to absorb exudates from wound surfaces, promote cell adhesion, and support tissue regeneration. In vitro evaluation demonstrates significant antibacterial activity against Escherichia coli and Staphylococcus aureus, with inhibition zone values of 16 mm and 18 mm, respectively. Cell migration, proliferation, and biocompatibility are assessed using the L929 fibroblast cell line with the ethidium bromide (EtBr) dye. In vivo studies on Sprague-Dawley rats indicate SF/CA/Pd-Pt composite nanofibers have exceptional wound healing efficiency, reaching 99.31% compared to the SF/CA matrix. High hemocompatibility, with erythrocyte percentages ranging from 1.6% to 4.1%, further supports these nanofibers wound healing properties. Histopathological analysis, employing hematoxylin and eosin staining, provides valuable insights into tissue regeneration, assessing reepithelialization, granulation, angiogenesis, and reduced inflammation. These findings emphasize the potential of Pd and Pt nanoparticles incorporated into SF/CA composite nanofiber scaffolds as advanced biomedical materials, particularly for wound healing applications.

The role of IL-4 −590 (C&gt;T) Gene as diagnostic biomarker of Hashimoto thyroiditis disease patients in AL-Najaf provenance /Iraq

The study of IL-4 genotyping in Hashimoto patients is considered one of the challenges of sustainable development .Hashimoto disease is an autoimmune disease that causes the immune system to attack the cells of the thyroid gland, causing the death of the cells that produce thyroid hormones, and thus leads to chronic hypothyroidism. The aim of current study is to determine the genotyping of IL-4 -590C&gt;T in the DNA of Hashimoto patients and compared with healthy subjects .A 54 blood samples were collected from people with Hashimoto's disease and 25 samples from healthy people during period from March to September of the year 2022 , DNA was extracted from blood using Favorprep Kit, then, using a specific primers for the C and T alleles of interleukin 4-590 C/T, then doubled using Arms PCR. Then, they were transported in agarose gel with the ethidium bromide dye and detected by the gel documentation system , ultraviolet rays use to determine the type of alleles present in each sample and compared with control group. The current study, which includes 50/54 female and 4/54 male with Hashimoto thyroiditis with ages ranged from 20-59 years old, the results show up that the age group 40-49 years were the most affected, Also the body mass index was had increases among patients. CT genotype present in 74% of Hashimoto patients while 16% of control. C allele was found as high rate in both patients and control with percentage 100% and 96% respectively. in conclusion: The Hashimoto patients have more frequency of CT genotyping of IL-4 -590 C&gt;T than control ,while C Allele was high in both Hashimoto patients and controls. The -590 CC genotype in the IL4 gene may consider to be a strong predictive factor for the development of hypothyroidism in HD.

STUDY OF THE INTERACTION OF SINGLE-STRANDED POLYRIBOADENYLIC ACID WITH ETHIDIUM BROMIDE AND METHYLENE BLUE

The study of the interaction of phenothiazine dye methylene blue (MB) and phenantridine dye ethidium bromide (EtBr) with synthetic single-stranded (ss)-poly(rA) has been carried out at the ionic strength of the solution 0.1 mol, in wide interval of ligand/phosphate ratio change – P/D, by the method of absorption spectroscopy. The binding curves of MB and EtBr with ss-poly(rA) were obtained. MB was shown to interact with this polynucleotide cooperatively, while for EtBr the cooperativity was not revealed. The binding parameters of MB and EtBr with ss-poly(rA) were determined – association constant K and base number n per binding site. It was shown that both ligands bind to ss-poly(rA) at least by two modes, though the affinity of MB to the polynucleotide is higher by an order, than that of EtBr.

Mycobacterium tuberculosis ketol-acid reductoisomerase down-regulation affects its ability to persist, and its survival in macrophages and in mice

Branched-chain amino acids (BCAAs) leucine, isoleucine and valine biosynthetic pathways have been reported from plants, fungi and bacteria including Mycobacterium tuberculosis (Mtb) but are absent in animals. This makes interventions with BCAAs biosynthesis an attractive proposition for antimycobacterial drug discovery. In the present study, Mycobacterium tuberculosis H37Ra (Mtb-Ra) ketol-acid reductoisomerase encoding ORF MRA_3031 was studied to establish its role in Mtb-Ra growth and survival. Recombinant knockdown (KD) and complemented (KDC) strains along with wild-type (WT) Mtb-Ra were studied under in-vitro and ex-vivo conditions. KD was defective for survival inside macrophages and showed time dependent decrease in its colony forming unit (CFU) counts, while, WT and KDC showed time dependent increase in CFUs, after macrophage infection. Also, KD showed reduced ability to form persister cells, had altered membrane permeability against ethidium bromide and nile red dyes, and had reduced biofilm maturation, compared to WT and KDC. The in-vivo studies showed that KD infected mice had lower CFU counts in lungs, compared to WT. In summary Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation.

Significance of Fluorescent Spectroscopy in Screening Oral Potentially Malignant Disorders and Oral Cancer by Characterization of Salivary DNA Using Ethidium Bromide-A Comparative Study.

Sangeetha RamamoorthyBackground Optical diagnosis is one of the upcoming methods in screening and diagnosing oral cancer at the earlier stage. Currently, DNA-based diagnosis is used along with light-based imaging methods to diagnose oral cancer rapidly. Aim The aim of this study was to discriminate oral cancer and oral potentially malignant disorders from normal patient with fluorescence spectroscopic characterization of salivary DNA using ethidium bromide dye. Materials and Methods A total of 40 patients with average age of 20 to 60 years in both the genders were selected and divided into three groups. Group A included clinically proven cases of oral cancer, group B1 included clinically diagnosed cases of leukoplakia, group B2 included clinically diagnosed cases of oral submucous fibrosis, and group C included controls. Salivary DNA fluorescence spectrum obtained after adding ethidium bromide was analyzed using FluoroLog spectrophotometer at 480 nm wavelength. Results The discriminant analysis of fluorescent emission of salivary DNA shows predictive accuracy of 90% between group C and group A, 95% between group C and group B1, and 65% observed between group C and group B2. Conclusion From this study, screening of oral cancer can be done at the earliest with the help of fluorescence spectroscopic characterization of salivary DNA. This method can be done rapidly and noninvasively.

Surface fabrication of magnetic core-shell silica nanoparticles with perylene diimide as a fluorescent dye for nucleic acid visualization

• Design and synthesis of non-toxic and economic magnetic nanoparticle colorant dye for nucleic acid visualization. • Development dual-functional nanoparticle have strong magnetic properties and intense fluorescent light. • Evaluation of particle adsorption loading efficiency and their release of nucleic acid. • The magnetic dye can be easy recovered and controlled their movement by applying external magnetic. Up to date most traditional fluorescent dyes that serve to detect nucleic acids in a selective manner face growing environmental and economic challenges due to their high toxicity, expensiveness, the long staining times required and the low sensitivities. Further attention went to replace the current widely used ethidium bromide (EB) by more safe and stable dyes but still some of the above drawbacks persist. In this work an innovative fluorescent dye has been designed and synthesized via impregnating perylene diimide into the functionalized surface of magnetic core–shell silica nanoparticles. One of the free hydroxyl groups on the perylene diimide enables the colored magnetic nanoparticles to easily disperse in water and their movement can be controlled with magnetism which allow the separation after their used. These particles are of low toxicity, environmentally friendly and have high sensitivity. Fluorescent properties and molecular interaction were studied. The designed particles show high DNA binding capacity which makes the developed particle promising for DNA extraction, delivery and fluorescent labeling. Theoretical studies confirm that the dye is placed between two DNA base pair similar with pervious observation with EB and the distance was less than 6 Å which is much less than the distance of EB dye interaction (15 Å) as reported before [1] .

Construction and transfer of PEI-miRNA-126/210 polyplex into Human Umbilical Vein Endothelial Cells with investigation of its effect on islets survival and function

Background:Type 1 diabetes is an autoimmune disorder characterized by the loss of pancreatic islets. Islet allotransplantation is a potentially beneficial therapeutic approach for diabetes. Islets suffer a variety of cellular insults including ischemia and partial vascular loss during isolation, resulting in a significant reduction in viability prior to transplantation. The present study aimed to investigate the effect of angiogenic microRNA (miRNA)-126 and -210 on islet function and viability in an indirect way. Methods:Poly Ethylenimine (PEI)-miRNA-126 and -210 polyplexes were constructed at various Nitrogen/Phosphate (N/P) ratios. After confirmation by gel retardation and ethidium bromide dye exclusion assay, its cytotoxicity and transfection efficiency were analyzed by MTT and fluorescent assays, respectively. After that, the selected polyplexes were used to transfect Human Umbilical Vein Endothelial Cells (HUVECs) in vitro and were indirectly co-cultured with islet cells for three days. Real-time polymerase chain reaction and enzyme-linked immunoassay were conducted to verify the regulation of target genes and the functionality of the islets. Results:The findings showed that PEI could condense miRNAs at N/P=5. The viability of the HUVECs was decreased by increasing the amount of PEI. Additionally, ployplex-126 and -210 led to a decrease in the expressions of target genes, phosphoinositol-3 kinase regulatory subunit 2, sprouty-related EVH1 domain-containing protein 1, and ephrin-A3 in the islets. Moreover, the expressions of Bax and Bcl2 and their ratio in the treated groups by polyplex-126 and -210 led to better survival and function of the islets, with a higher expression of insulin and response to glucose stimulations. Furthermore, polyplex-210 could downregulate the anti-angiogenic protein, thrombospondin 1, compared to the other groups. Finally, the secretion of C-peptide was higher in polyplex-210 than in the other groups, adjusted for insulin secretion. Conclusion:The results indicated that angiogenic miRNAs could promote the survival and function of islet cells by interacting with their targets.

Angiotensin-Converting Enzyme Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients

Objectives: The purpose of this study is to determine whether the angiotensin-converting enzyme activity and various biomarkers are used to investigate the severity of Covid-19 and to study the genetic variation occurs in angiotensin-converting enzyme-2 (ACE-2) receptor in severe Covid-19-related genes in the Iraqi population of Kerbala Province. Methods: This case-control study was conducted on 176 subjects who survived hospitalization and diagnosed by physician. Various biomarkers including ferritin, C-reactive protein, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, angiotensin-converting enzyme-2 activity levels were determined. Accordingly, they were divided into three groups: 59 of them were infected with severe Covid-19, 54 of them were infected with moderate Covid-19 and 63 of them were checked and obtained as apparently healthy control. Severe and moderate patients were collected from Al-Hayat tertiary center at Al-Hussein Medical City, Kerbala Health Directorates, Kerbala – Iraq during Oct., 2020- July, 2021 with matched age ranged between (23-88) years. Blood samples of apparently healthy and Covid-19 samples were subjected to genomic DNA extraction within 24-48 hours of aspiration. The genomic DNA extracted was subjected to electrophoresis through 1.5% of agarose gels which was detected by staining with the fluorescent dye ethidium bromide and then visualized by illumination with UV light to confirm the presence and integrity of the extracted DNA. Results: Genotyping of ACE-2 (I/D) polymorphism (rs4646994), which has a high prevalence, was performed by polymerase chain reaction assay. The amplification of an Alu repetitive element in an intron of the ACE-2 has shown three potential genotypes of I/I and D/D as homozygous, and I/D as heterozygous. Individuals with normal homozygous (DD) revealed band of (190 bp), while individuals with normal (II) revealed band of (490 bp), and the individuals with heterozygous (ID) revealed two bands (190, 490 bp) respectively. Every severe Covid-19 group carried (DD) allele genotype, moderate group carried (ID and II) alleles and finally the control group carried (DD, ID, II) alleles genotype. Conclusion: In the ACE-2 polymorphism, the D/D genotype allele is implicated as a risk factor for severe Covid-19 patients, in Iraqi population.

Assessment of biological activity of the caffeine‐derived Pt (II) and Pd (II) complexes

AbstractThe substitution reactions of [Pd (caffeine)2Cl2] and [Pt (caffeine)2Cl2] (caffeine = 1,3,7‐trimethylxanthine) complexes with bio‐molecules such as 9‐methylguanine (9‐MetGua) and guanosine‐5′‐monophosphate (5′‐GMP) were studied spectrophotometrically. Kinetic measurements were performed under the pseudo‐first‐order conditions at 37°C and pH = 7.2 (25‐mM Hepes buffer) with the addition of 50‐mM NaCl. All reactions were carried out in two reaction steps giving the [M (caffeine)2(Nu)2] (M = Pd (II) or Pt (II) and Nu = 5′‐GMP or 9‐MetGua) as the reaction product. The reaction mechanism was also confirmed by nuclear magnetic resonance (NMR) spectroscopy and density functional theory (DFT) calculations. Kinetically data revealed a much higher reactivity of [Pd (caffeine)2Cl2] complex compared with analog platinum‐complex, while 9‐MetGua reacted faster than 5′‐GMP. The interactions of [Pd (caffeine)2Cl2] and [Pt (caffeine)2Cl2] complexes with calf thymus‐DNA (CT‐DNA) and human serum albumin (HSA) were investigated as well. Competitive studies with DNA were performed in the presence of ethidium bromide (EB) and Hoechst dye 33258 (Hoe). The complexes interact with DNA via minor groove rather than by intercalation. High values of binding constants indicate a good binding affinity of complexes toward HSA (104 M−1). Additionally, the experimental results of binding studies between [Pt (caffeine)2Cl2] complex with DNA and HSA were compared by a molecular docking study. The cytotoxicity of [Pd (caffeine)2Cl2] and [Pt (caffeine)2Cl2] complexes was tested against the mouse breast cancer (4T1) and colon cancer (CT26) as well as the human breast cancer (MDA‐MB‐468) and colon cancer (HCT‐116)] cell lines. The results indicate that the [Pt (caffeine)2Cl2] complex has higher cytotoxic capacity compared with [Pd (caffeine)2Cl2] at concentrations higher than 2 μM.

RND Efflux Systems Contribute to Resistance and Virulence of Aliarcobacter butzleri.

Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, as well as virulence. Thus, this work aimed to evaluate the contribution of three RND efflux systems, AreABC, AreDEF and AreGHI, in the resistance and virulence of A. butzleri. Mutant strains were constructed by inactivation of the gene that encodes the inner membrane protein of these systems. The bacterial resistance profile of parental and mutant strains to several antimicrobials was assessed, as was the intracellular accumulation of the ethidium bromide dye. Regarding bacterial virulence, the role of these three efflux pumps on growth, strain fitness, motility, biofilm formation ability, survival in adverse conditions (oxidative stress and bile salts) and human serum and in vitro adhesion and invasion to Caco-2 cells was evaluated. We observed that the mutants from the three efflux pumps were more susceptible to several classes of antimicrobials than the parental strain and presented an increase in the accumulation of ethidium bromide, indicating a potential role of the efflux pumps in the extrusion of antimicrobials. The mutant strains had no bacterial growth defects; nonetheless, they presented a reduction in relative fitness. For the three mutants, an increase in the susceptibility to oxidative stress was observed, while only the mutant for AreGHI efflux pump showed a relevant role in bile stress survival. All the mutant strains showed an impairment in biofilm formation ability, were more susceptible to human serum and were less adherent to intestinal epithelial cells. Overall, the results support the contribution of the efflux pumps AreABC, AreDEF and AreGHI of A. butzleri to antimicrobial resistance, as well as to bacterial virulence.

Study of Colorimetric properties of Ethidium bromide dye-doped PVP/DNA film

The effects of deoxyribonucleic acid (biological polymer DNA) on full brightness, and color matching functions of Ethidium bromide dye doped PVP/DNA films were studied. Ethidium bromide dye doped PVP/DNA films were deposited on soda-lime glass substrates by the deep casting method. The variation of CIE chromaticity coordinates for PVP/DNA films with increasing Ethidium bromide dye concentration values are determined. The CIE 1931 color space move to the magenta region. The results indicate that Ethidium bromide dye doped PVP/DNA films have potential promising material for optical device applications and candidates used for LEDs pumped by UV chips and applied in many advanced technologies.

Ethidium bromide interactions with DNA: an exploration of a classic DNA-ligand complex with unbiased molecular dynamics simulations.

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)2 also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.

Ultrasonic-assisted biosynthesis of ZnO nanoparticles using Sonneratia alba leaf extract and investigation of its photocatalytic and biological activities

The present study focuses on the synthesis of ZnO nanoparticles (ZnO NPs) using Sonneratia alba (S. alba) leaf extract (LE) in the aqueous medium. The as-synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), UV–Vis spectroscopy (UV–Visible), Scanning electron microscopy (SEM), Energy dispersive X-ray analysis (EDAX), Transmission electron microscopy (TEM), Dynamic light scattering (DLS), Zeta potential and N2adsorption-desorption isotherm. The XRD pattern verifies the formation of the crystalline wurtzite structure of ZnO NPs. Further, the bandgap of ZnO NPs was calculated using Tauc’s plot and observed to be 3.2 eV. SEM analysis reveals the formation of spherical shape ZnO NPs. The as-synthesized ZnO NPs show excellent photocatalytic activity against Ethidium bromide (EtBr) and Brilliant green (BG) dye. Kinetic study shows degradation efficiency towards EtBr (97.07%) and BG (95.01%) with rate constant of 0.018 min−1 (EtBr, R2 = 0.98472) and 0.033 min−1 (BG, R2 = 0.9813), respectively. Moreover, the synthesized material shows excellent antibacterial activity against gram-positive and negative bacteria with excellent antioxidant activity and anti-inflammatory.

Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay.

The use of resistant (R) genes is the most effective strategy to manage bacterial leaf blight (BLB) disease of rice. Several attempts were made to incorporate R genes into susceptible rice cultivars using marker-assisted backcross breeding (MABB). However, MABB relies exclusively on PCR for foreground selection of R genes, which requires expensive equipment for thermo-cycling and visualization of results; hence, it is limited to sophisticated research facilities. Isothermal nucleic acid amplification techniques such as loop-mediated isothermal amplification (LAMP) assay do not require thermo-cycling during the assay. Therefore, it will be the best alternative to PCR-based genotyping. In this study, we have developed a LAMP assay for the specific and sensitive genotyping of seven BLB resistance (R) genes viz., Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa21 in rice. Gene-specific primers were designed for the LAMP assay. The LAMP assay was optimized for time, temperature, and template DNA concentration. For effective detection, incubation at 60°C for 30min was optimum for all seven R genes. A DNA intercalating dye ethidium bromide and a calorimetric dye hydroxynaphthol blue was used for result visualization. Further, sensitivity assay revealed that the LAMP assay could detect R genes at 100fg of template DNA compared to 1ng and 10pg, respectively, in conventional PCR and q-PCR assays. The LAMP assay developed in this study provides a simple, specific, sensitive, robust, and cost-effective method for foreground selection of R genes in the resistance breeding programs of resource-poor laboratory.

Application of luminescence spectral assay to evaluate the results of a scientific experiment

The method of characterization of functional state of epithelium of mucous membrane of alveolar glands of poultry proventriculus, using luminescence spectral assay with fluorescent dyes Ethidium bromide (EtBr) and 5-([4.6-Dichlorotriazin-2-yl]amino) fluorescein hydrochloride (DTAF) is offered. The dynamics of nucleic acids and proteins content in chickens’ cells under experimental klebsiellosis in antibiotic-treated poultry is established using this method. It is found that at the 2th – 7th days of treatment the markers of the cells functional status in the experimental group chickens are similar to those of poultry of the intact group.

Does Cholinergic Stimulation Affect the P2X7 Receptor-Mediated Dye Uptake in Mast Cells and Macrophages?

Background: Extracellular ATP is a powerful trigger of neuroinflammation by activating immune cells via P2X7 receptors. Acetylcholine and nicotinic agonists inhibit ATP-triggered proinflammatory cytokines via the so-called “cholinergic anti-inflammatory pathway” (CAP). However, it remains unclear as to what stage of ATP-induced signaling cholinergic agents provide this anti-inflammatory effect. Using the specific property of P2X7 receptor to open a pathway permeable to large molecules, associated with activation of inflammasome, we studied the action of cholinergic agents on this key event in CAP activation.Methods: Freshly isolated mouse peritoneal mast cells and primary human macrophages were used. To assess P2X7 channel opening, the permeability to the fluorescent dye YO-PRO1 or ethidium bromide (EtBr) was measured by flow cytometry. Expression of nicotinic receptors was probed in macrophages with the fluorescently labeled α-bungarotoxin or with patch-clamp recordings.Results: ATP opened P2X7 ion channels in mast cells and macrophages permeable to YO-PRO1 or EtBr, respectively. This stimulatory effect in mast cells was inhibited by the specific P2X7 antagonist A839977 confirming that YO-PRO1 uptake was mediated via ATP-gated P2X7 ion channels. Cholinergic agents also slightly induced dye uptake to mast cells but not in macrophages, which expressed functional α7 nicotinic receptors. However, both in mast cells and in macrophages, acetylcholine and nicotine failed to inhibit the stimulatory effect of ATP on dye uptake.Conclusion: These data suggest that in immune cells, cholinergic agents do not act on P2X7 receptor-coupled large pore formation but can mediate the anti-inflammatory effect underlying CAP downstream of ATP-driven signaling.