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The excitation-dependent emission properties of carbon dots (Cdots) are extensively reported, but their red emission is often weak, limiting their wider application. Here we introduce ethidium bromide, as a functional precursor with red emission, to enhance the red emission for Cdots, with comparable intensity at a broad wavelength range to multi-emission Cdots (M-Cdots). We found that Cdots prepared with ethidium bromide/ethylenediamine exhibited strong blue and red emission at 440 and 615 nm, with optimal excitation at 360 and 470 nm as M-Cdots, respectively, but the Cdots from single ethidium bromide (EB-Cdots) possessed weak red emission. M-Cdots exhibited a broad absorption band at 478 nm, but a band blue-shifted to 425 nm was observed for EB-Cdots, while no absorption was observed at 478–425 nm for the Cdots prepared with citric acid and ethylenediamine. Thus, we proposed that C=O and C=N formed a π-conjugation structure as the absorption band at 478 nm for the red emission of M-Cdots, as also confirmed with the excitation at 470 nm. Moreover, the π-conjugation structure is fragile and sensitive to harsh conditions, so red emission was difficult to observe for the Cdots prepared with citric acid/ethylenediamine or single ethidium bromide. M-Cdots possess two centers for blue and red emission with different structures. The dual emission was therefore used for ratiometric sensing with dichromate (Cr2O72−) and formaldehyde (HCHO) as the targets using the intensity ratio of the emissions at 615 and 440 nm. Due to the comparable intensity at a broad wavelength range, we designed encryption codes with five excitations at 360, 400, 420, 450, and 470 nm as the inputs, and the emission colors were used for information decoding. Thus, we determined why red emission was difficult to realize for Cdots, and our results could motivate the design of red-emission Cdots for extensive applications. [ABSTRACT FROM AUTHOR]

The interaction of methylene blue (MB) and ethidium bromide (EB) intercalators with synthetic single-strand (ss) polynucleotides poly(rA) and poly(rU) and the double-strand (ds) poly(rA)–poly(rU) formed as a result of their hybridization was investigated in 0.04 mole solution in the range of phosphate/ligand ratios r = 0 ≤ r ≤ 10. The absorption spectra of complexes of the ligands with the polynucleotides were obtained, and adsorption isotherms in Scatchard coordinates were constructed on the basis of changes in the absorption spectra. It was found that MB interacts cooperatively with poly(rA) and also with ds-poly(rA)–poly(rU), while cooperation is not detected in the case of poly(rU). There is also no cooperation in the interaction of EB with ss- and ds-polynucleotides. The binding parameters for the interaction of these ligands with the polynucleotides (the association constants K and the number of bases n per binding site and also the cooperation factor) in the interaction of MS with poly(rA) and ds-poly(rA)- poly(rU) were determined from the adsorption isotherms. Both ligands were found to bind to polynucleotides by at least two methods, and MB here exhibits much higher affinity to poly(rA) and EB to ds-poly(rA)-poly(rU). [ABSTRACT FROM AUTHOR]

Valencene and nootkatone are aromatic sesquiterpenes with known biological activities, such as antimicrobial, antioxidant, anti-inflammatory, and antitumor. Given the evidence that encapsulation into nanosystems, such as liposomes, could improve the properties of several compounds, the present study aimed to evaluate the activity of these sesquiterpenes in their isolated state or in liposomal formulations against strains of Staphylococcus aureus carrying efflux pumps. The broth microdilution method evaluated the antibiotic-enhancing activity associated with antibiotics and ethidium bromide (EtBr). The minimum inhibitory concentration was assessed in strains of S. aureus 1199B, IS-58, and RN4220, which carry the efflux proteins NorA, Tet(K), and MsrA. In tests with strain 1199B, valencene reduced the MIC of norfloxacin and EtBr by 50%, while the liposomal formulation of this compound did not show a significant effect. Regarding the strain IS-58, valencene, and its nanoformulation reduced norfloxacin MIC by 60.3% and 50%, respectively. In the non-liposomal form, the sesquiterpene reduced the MIC of EtBr by 90%. Against the RN4220 strain, valencene reduced the MIC of the antibiotic and EtBr by 99% and 93.7%, respectively. Nootkatone and its nanoformulation showed significant activity against the 1199B strain, reducing the EtBr MIC by 21.9%. Against the IS-58 strain, isolated nootkatone reduced the EtBr MIC by 20%. The results indicate that valencene and nootkatone potentiate the action of antibiotics and efflux inhibitors in strains carrying NorA, Tet(K), and MsrA proteins, which suggests that these sesquiterpenes act as efflux pump inhibitors in S. aureus. Therefore, further studies are needed to assess the impact of incorporation into liposomes on the activity of these compounds in vivo. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus can exhibit resistance to various antibiotics. Among its resistance mechanisms, the active efflux of antibiotics can be seen as relevant. This study aimed to evaluate the ability of resveratrol to modulate norfloxacin resistance in S. aureus. The antimicrobial activity of resveratrol was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC). Then, the modulatory effect of resveratrol was evaluated using the MIC determination for the antibiotic or ethidium bromide in the presence and absence of resveratrol at a sub-MIC level. The MIC of norfloxacin against S. aureus SA1199B (NorA-overexpressing strain) decreased 16-fold when in the presence of resveratrol, with a similar behavior being observed for ethidium bromide. An evaluation of the ethidium bromide accumulation was also performed, showing that in the presence of resveratrol, the SA1199B strain had augmented fluorescence due to the accumulation of ethidium bromide. Altogether, the results suggested that resveratrol may act by inhibiting NorA. These in vitro data were supported by docking results, with interactions between resveratrol and the NorA efflux pump predicted to be favorable. Our findings demonstrated that resveratrol may modulate norfloxacin resistance through the inhibition of NorA, increasing the effectiveness of this antibiotic against S. aureus. [ABSTRACT FROM AUTHOR]

Brazilian flora are highly diverse and have great potential for research into new medicines of plant origin. Platonia insignis Mart. is a fruit tree from the Amazon region with several potential pharmacological applications already demonstrated. The present study aimed to investigate the protective effect of a mixture of the biflavones volkensiflavone and moreloflavone obtained from P. insignis flowers against the lethality induced by ethidium bromide in efflux pump overproducer strains of Staphylococcus aureus. Microdilution tests were performed to determine the minimum inhibitory concentration of ethidium bromide in the absence or presence of biflavones. The addition of biflavones to the growth medium increased the minimum inhibitory concentration of ethidium bromide against all strains tested, indicating that biflavones protected the bacterial cells against cell damage induced by this biocide agent. Comparative analysis of the infrared absorption spectra of each isolated compound, as well as of the associated compounds, demonstrated the complexation reaction, indicating that there was a preferential interaction between the H2N- groups of primary amines of ethidium bromide with the carbonyls of biflavones that formed imines -C=NH. Such complexation could reduce the amount of free ethidium bromide molecules available for DNA intercalation, resulting in the observed protective effect. These results suggest a possible technological application of the biflavones from P. insignis as protective agents against cell damage induced by ethidium bromide. [ABSTRACT FROM AUTHOR]

The bacterial species Staphylococcus aureus presents a variety of resistance mechanisms, among which the expression of β-lactamases and efflux pumps stand out for providing a significant degree of resistance to clinically relevant antibiotics. The 1,8-naphthyridines are nitrogen heterocycles with a broad spectrum of biological activities and, as such, are promising research targets. However, the potential roles of these compounds on bacterial resistance management remain to be better investigated. Therefore, the present study evaluated the antibacterial activity of 1,8-naphthyridine sulfonamides, addressing their ability to act as inhibitors of β-lactamases and efflux pump (QacA/B and QacC) against the strains SA-K4414 and SA-K4100 of S. aureus. All substances were prepared at an initial concentration of 1024 μg/mL, and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Subsequently, their effects on β-lactamase- and efflux pump-mediated antibiotic resistance was evaluated from the reduction of the MIC of ethidium bromide (EtBr) and β-lactam antibiotics, respectively. The 1,8-naphthyridines did not present direct antibacterial activity against the strains SA-K4414 and SA-K4100 of S. aureus. On the other hand, when associated with antibiotics against both strains, the compounds reduced the MIC of EtBr and β-lactam antibiotics, suggesting that they may act by inhibiting β-lactamases and efflux pumps such as QacC and QacA/B. However, further research is required to elucidate the molecular mechanisms underlying these observed effects. [ABSTRACT FROM AUTHOR]

Ethidium bromide (EtBr) is a chemical agent commonly used to identify and visualize the bonding of DNA and RNA in molecular biology research. However, ethidium bromide itself is mutagenic and toxic. Ethidium bromide also has carcinogenic properties to human cells, teratogenic, and difficult to decompose. Therefore, a research for the treatment of EtBr waste is very important. There are several materials that can be used to reduce EtBr waste, but there is also needed a material which is cheap and widely available in the market. One of the methods to treat ethidium bromide waste is using activated charcoal. Activated charcoal has strong adsorption capacity due to its high surface area and porous structure. Another advantage of activated charcoal as an adsorbent is economical, effective, and environmentally friendly. In this study, EtBr waste was mixed with activated charcoal then homogenized for one hour. The mixing of EtBr waste (0.5 mg/mL concentration of EtBr) and activated charcoal was varied with a ratios weight by volume of 1:1, 1:2, and 2:1. The mixture of the solution was filtered using Whatmann filter paper No.1 to take the filtrate. The filtrate was further analyzed. The more activated charcoal was added, the more EtBr molecules were adsorbed. The pH in the filtration results for each variation was pH 7.0 - 8.0, dissolved oxygen with range 5.0 - 9.0 mg/L, biochemical oxygen demand (BOD) with range 25.06 - 78.2 mg/L, and chemical oxygen demand (COD) with range 302.56 - 310.98 mg/L. The concentration range was obtained as follows, for a 1:1 ratio of 0.045 - 0.060 mg/mL, for a 1:2 ratio of 0.028 - 0.032 mg/mL, while a 2:1 ratio of 0.081 - 0.090 mg/mL. In addition, the results showed that activated charcoal was able to bind Ethidium bromide due to bond interactions that occur between the EtBr molecule and activated charcoal. [ABSTRACT FROM AUTHOR]

The distance between nodes of Ranvier, referred to as internode length, positively correlates with axon diameter, and is optimized during development to ensure maximal neuronal conduction velocity. Following myelin loss, internode length is reestablished through remyelination. However, remyelination results in short internode lengths and reduced conduction rates. We analyzed the potential role of neurofilament phosphorylation in regulating internode length during remyelination and myelination. Following ethidium bromide induced demyelination, levels of neurofilament medium (NF-M) and heavy (NF-H) phosphorylation were unaffected. Preventing NF-M lysine-serine-proline (KSP) repeat phosphorylation increased internode length by 30% after remyelination. To further analyze the role of NF-M phosphorylation in regulating internode length, gene replacement was used to produce mice in which all KSP serine residues were replaced with glutamate to mimic constitutive phosphorylation. Mimicking constitutive KSP phosphorylation reduced internode length by 16% during myelination and motor nerve conduction velocity by ~27% without altering sensory nerve structure or function. Our results suggest that NF-M KSP phosphorylation is part of a cooperative mechanism between axons and Schwann cells that together determine internode length, and suggest motor and sensory axons utilize different mechanisms to establish internode length.

Chemotherapy-inhibiting tumor cell evolution to drug-resistance is potentially suppressed by using a drug delivery vehicle (DDV) that has gating. Gating would be used to increase tumor-selectivity of delivery of DDV packaged drug. Tumor-selectivity increase would make possible increase in tumor-delivered drug dose, which would suppress opportunities to evolve drug resistance. Currently used DDVs do not have gating but gating is a natural feature of some bacteriophages (phages). Phage T4, which has recently been found highly persistent in murine blood, is a potential gated DDV. Thus, here, we proceed towards a T4-DDV by developing (1) improved procedure for generating high concentrations and amounts of phage T4, (2) elevated temperature-driven gate-opening and ethidium- and bleomycin-loading, and (3) purification of loaded T4 by rate zonal centrifugation. We test for loading by native agarose gel electrophoresis (AGE) with fluorescence detection. We observe loading in both phage T4 and T4 (tail-free) heads. The loaded particles have an openable, closed gate. Stored, mature T4 phages and phage heads do not release ethidium during at least a month at 4 °C and 6 days at 37 and 42 °C. Tumor-specific T4 phage delivery is projected via both the EPR effect and high T4 persistence. [ABSTRACT FROM AUTHOR]

With the recent stringent criteria for antibiotic susceptibility in probiotics, the presence of antibiotic resistance genes and plasmids associated with their transfer has become a limiting factor in the approval of probiotics. The need to remove genes related to antibiotic resistance and virulence through plasmid curing for the authorization of probiotics is increasing. In this study, we investigated the curing efficiency of ethidium bromide, acridine orange, and novobiocin at different concentrations and durations in five strains of plasmid-bearing lactic acid bacteria and examined the curing characteristics in each strain. Limosibacillus reuteri and Lacticaseibacillus paracasei exhibited curing efficiencies ranging from 5% to 44% following treatment with ethidium bromide (10-50 μg/ml) for 24-72 h, while Lactobacillus gasseri showed the highest efficiency at 14% following treatment with 10 μg/ml novobiocin for 24 h. Lactiplantibacillus plantarum , which harbors two or more plasmids, demonstrated curing efficiencies ranging from 1% to 8% after an additional 72-h treatment of partially cured strains with 10 μg/ml novobiocin. Plasmid curing in strains with larger plasmids exhibited lower efficiencies and required longer durations. In strains harboring two or more plasmids, a relatively low curing efficiency with a single treatment and a high frequency of false positives, wherein recovery occurred after curing, were observed. Although certain strains exhibited altered susceptibilities to specific antibiotics after curing, these outcomes could not be attributed to the loss of antibiotic resistance genes. Furthermore, the genomic data from the cured strains revealed minimal changes throughout the genome that did not lead to gene mutations.

Multiple sclerosis (MS) is a chronic neurodegenerative disease marked by oligodendrocyte loss, which results in central neuronal demyelination. AC/cAMP/CREB signaling dysregulation is involved in the progression of MS, including mitochondrial dysfunctions, reduction in nerve growth factors, neuronal inflammation, apoptosis, and white matter degeneration. Our previous research has shown that Forskolin (FSK), a naturally occurring direct adenylyl cyclase (AC)/cAMP/CREB activator, has neuroprotective potential to alleviate pathogenic factors linked with numerous neurological abnormalities. The current study intends to explore the neuroprotective potential of FSK at doses of 40 mg/kg and 60 mg/kg alone, as well as in combination with conventional medicines, such as Fingolimod (FNG), Donepezil (DON), Memantine (MEM), and Simvastatin (SIM) in EB-induced demyelinated experimental MS rats. Adult Wistar rats were divided into nine groups, and EB was infused stereotaxically in the rat brain's intracerebropeduncle (ICP) area. Chronic gliotoxin EB treatment results in demyelination as well as motor and cognitive dysfunctions. FSK, combined with standard medications, improves behavioral dysfunctions, such as neuromuscular and motor deficits and memory and cognitive abnormalities. Following pharmacological treatments improved remyelination by enhancing myelin basic protein and increasing AC, cAMP, and CREB levels in brain homogenates. Furthermore, FSK therapy restored brain mitochondrial-ETC complex enzymes and neurotransmitter levels while decreasing inflammatory cytokines and oxidative stress markers. The Luxol fast blue (LFB) stain results further indicate FSK's neuroprotective potential in preventing oligodendrocyte death. Therefore, the results of these studies contribute to a better understanding of the possible role that natural phytochemicals FSK could have in preventing motor neuron diseases, such as multiple sclerosis. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is a severe immune-mediated neurological disease characterized by neuroinflammation, demyelination, and axonal degeneration in the central nervous system (CNS). This is frequently linked to motor abnormalities and cognitive impairments. The pathophysiological hallmarks of MS include inflammatory demyelination, axonal injury, white matter degeneration, and the development of CNS lesions that result in severe neuronal degeneration. Several studies suggested downregulation of nuclear factor erythroid-2-related factor-2 (Nrf2)/Heme oxygenase-1 (HO-1) signaling is a causative factor for MS pathogenesis. Acetyl-11-keto-β-boswellic acid (AKBA) is an active pentacyclictriterpenoid obtained from Boswellia serrata, possessing antioxidant and anti-inflammatory properties. The present study explores the protective potential of AKBA on behavioral, molecular, neurochemical, and gross pathological abnormalitiesandhistopathological alterations by H&E and LFB staining techniques in an experimental model of multiple sclerosis, emphasizing the increase inNrf2/HO-1 levels in the brain. Moreover, we also examine the effect of AKBA on the intensity of myelin basic protein (MBP) in CSF and rat brain homogenate. Specific apoptotic markers (Bcl-2, Bax, andcaspase-3) were also estimated in rat brain homogenate. Neuro behavioralabnormalities in rats were examined using an actophotometer, rotarod test, beam crossing task (BCT),and Morris water maze (MWM). AKBA 50 mg/kg and 100 mg/kg were given orally from day 8 to 35 to alleviate MS symptoms in the EB-injected rats. Furthermore, cellular, molecular, neurotransmitter, neuroinflammatory cytokine, and oxidative stress markers in rat whole brain homogenate, blood plasma, and cerebral spinal fluid were investigated. This study shows that AKBA upregulates the level of antioxidant proteins such as Nrf2 and HO-1 in the rat brain. AKBA restores altered neurochemical levels, potentially preventing gross pathological abnormalities during MS progression. [ABSTRACT FROM AUTHOR]

The efficient removal of Ethidium Bromide (EB), a toxic yet widely used chemical in biochemical industries and laboratories, is crucial to avoid its detrimental effect on human health, aquatic life, and the environment. One effective method is adsorption onto inert surfaces, but the choice of adsorbents is critical for the success of the removal protocols, primarily because of the limitations such as low adsorption capacity, high cost, limited availability, and a lack of understanding of the adsorption mechanism. In this work, Pyrophyllite Nanoclay (PNC), a naturally abundant aluminosilicate is explored as a promising adsorbent for EB. The adsorption process follows the Freundlich isotherm and displays pseudo-second-order kinetics. Under the optimized condition, PNC demonstrates nearly quantitative adsorption of EB and the maximum adsorption efficiency obtained from experiment was 6.53 mg/g (0.017 mmol/g) at room temperature under optimized condition. Through thermodynamic, kinetic analysis coupled with characterization techniques, the mechanism of adsorption is proposed. The adsorbent proves to be reusable, maintaining its performance over three cycles. The utility of the process is further validated and optimized using Response Surface Methodology (RSM). The present work focuses on the performance of PNC as adsorbent and elucidates distinctive surface adsorption mechanism (in comparison to other nanoclays), thus emphasizing the significance of structural, experimental, mechanistic and validation studies in choosing the right adsorbent for the removal toxic chemicals. [Display omitted] • Adsorption of Ethidium bromide on Pyrophyllite nanoclay (PNC) is investigated for the first time. • Kinetic and thermodynamic studies supported by characterization techniques reveal surface adsorption mechanism. • PNC is recyclable for at least three times. • Response Surface Methodology (RSM) optimization is conducted to validate the process. [ABSTRACT FROM AUTHOR]

Bitter leaf (Vernonia amygdalina Del.) plant is a tree species that is highly cultivated in Nigeria for its nutritive and therapeutic values. This study aimed to determine the antioxidant and antigenotoxicity effects (in vitro) of 52 accessions of V. amygdalina collected from six geopolitical zones of Nigeria (North East, North West, North Central, South South, East and West) by evaluating the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO- ) scavenging antioxidant activities, flavonoid and phenolic contents as well as ethidium bromide-induced DNA (deoxyribonucleic acid) damage of bitter leaf. The results showed that accessions MN628016 (Oshimili South, Delta State) and MN628010 (Akoko Edo, Edo State), respectively had higher amounts of flavonoid (287.19 mg/g/FW) and phenolic (307.90 mg/g/FW) contents. The accessions MN627984 (Ikpoba Okha, Edo State) and MN627975 (Owerri, Imo State) had the highest nitric oxide (85.73%) and DPPH radical scavenging inhibitory effect (98.92%). The highest percentage fragmented DNA (45.05%), was observed in Allium cepa roots homogenised and mixed with ethidium bromide followed by the A. cepa roots homogenised and mixed with the leaf extract of V. amygdalina accession MN627977 (36.12%). However, V. amygdalina accessions MN628008 (457.62%, Warri North, Delta State) had the highest percentage increase of fragmented DNA followed by MN628024 (395.04%, Oshimili North, Delta State), MN628015 (345.54%; Aniocha North, Delta State) and MN627984 (342.04%; Ikpoba Okha, Edo State) while accession MN628010 (7.32%; Akoko Edo, Edo State) had the lowest. Accession MN628010 which possessed the highest amount of phenolic content had the lowest percentage increase of fragmented DNA and accession MN627984 which possessed the highest nitric oxide radical scavenging inhibitory effect was among the accessions with the highest percentage increase of fragmented DNA. The findings of this study suggest that the observed lowest percentage of fragmented DNA of A. cepa roots growth induced with the solution of ethidium bromide and treated with V. amygdalina accessions MN628010 extracts (antigenotoxic) could be as a result of the high antioxidant activities in the V. amygdalina accessions. In summary, the findings of this study showed that the 52 V. amygdalina accessions obtained from different locations of Nigeria will help to combat ethidium bromide induced genoxicities and any other genotoxicant that may lead to different complications in plant (A. cepa roots), as all the accessions possessed antioxidant and antigenotoxic properties, as such, possessed comparable amount of natural antioxidant activities and antigenotoxicity. [ABSTRACT FROM AUTHOR]

Ethidium bromide is extensively used in many biochemical laboratories. Due to its high toxicity, its removal from the wastewater before being disposed becoming an emerging issue. Swelling clay minerals, due to their inexpensive material cost, high efficiency, and vast reserves, have been evaluated for the removal of different types of contaminants. In this study, the removal of ethidium bromide, particularly its mechanism of removal was assessed, using Ca-montmorillonite (SAz-1) as a representative swelling clay minerals. The ethidium bromide removal could reach equilibrium within 1 h and a capacity of 1,276 mmol kg-1, same magnitude as the cation exchange capacity of SAz-1. The total amount of metal cations desorbed from SAz-1 to the amount of ethidium removed was linearly correlated with a slope of 0.7, suggesting cation exchange as the dominant mechanism. In addition, the less-than-unity desorption/adsorption ratio could be attributed to the removal of ethidium dimers bridged with bromide counterions. An increase of the basal spacing to 18.3 Å for SAz-1 after adsorption implied that intercalation of flat-lying ethidium dimers into the clay interlayer, which increased its thermal stability, and active participation of the amine groups of ethidium molecules in the adsorptive interaction was evidenced from Fourier transform infrared band shifts. The results suggested that Ca-montmorillonite is an excellent candidate for fast and effective removal of ethidium bromide from aqueous solution. As such, it could serve as an ideal packing material for kits to be manufactured and used for ethidium bromide removal in case of spill. [ABSTRACT FROM AUTHOR]

Ethidium bromide (EtBr) is widely used in most laboratories to detect nucleic acids in gel electrophoresis applications. It is a well-known carcinogenic and mutagenic agent, which can affect biotic components of the place in which it is disposed. Usually the gel-waste is either buried in the ground or incinerated, whereas the liquid waste is disposed of down the sink following the recommended methods of treatment. The recommended methods do not involve biological potential, but rather make use of chemicals, which may further deteriorate soil and water quality. The present study identifies and characterizes the EtBr-degrading bacterial isolates BR3 and BR4. A bibliographic review of the risk status of using these isolates for the treatment of lab waste in laboratory settings is also presented. BR3 was identified as Proteus terrae N5/687 (LN680103) and BR4 as Morganella morganii subsp. morganii ATCC 25830 (AJ301681) with 99.9% and 99.48% similarity, respectively, using an EzBioCloud microbial identifier. The literature revealed the bacterium Proteus terrae as a non-pathogenic and natural microflora of humans, but Morganella morganii as an opportunistic pathogen. These organisms belong to risk group II. Screening the sensitivity of these isolates to antibiotics revealed a sufficient number of antibiotics, which can be used to control them, if required. BR3 and BR4 exhibited resistance to individual antibiotics, ampicillin and vancomycin, whereas only BR3 was resistant to tetracycline. The current investigation, along with earlier reported work on these isolates, identifies BR3 as a useful isolate in the industrial application for the degradation of EtBr. Identical and related microorganisms, which are available in the culture collection repositories, can also be explored for such potential to formulate a microbial consortium for the bioremediation of ethidium bromide prior to its disposal. [ABSTRACT FROM AUTHOR]

Molecular and cellular mechanisms underlying the peripheral conditioning lesion remain unsolved. We show here that injection of a chemical demyelinating agent, ethidium bromide, into the sciatic nerve induces a similar set of regeneration-associated genes and promotes a 2.7-fold greater extent of sensory axon regeneration in the spinal cord than sciatic nerve crush. We found that more severe peripheral demyelination correlates with more severe functional and electrophysiological deficits, but more robust central regeneration. Ethidium bromide injection does not activate macrophages at the demyelinated sciatic nerve site, as observed after nerve crush, but briefly activates macrophages in the dorsal root ganglion. This study provides a new method for investigating the underlying mechanisms of the conditioning response and suggests that loss of the peripheral myelin may be a major signal to change the intrinsic growth state of adult sensory neurons and promote regeneration.

Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host.

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role.

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role.

The interaction of (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium (PVP) dyes with calf-thymus DNA was examined by spectrophotometric UV-vis absorption/emission titration and competitive ethidium bromide displacement. The association constant values were situated in 106 M-1 range, typical to cationic dyes containing the monocyanine chromophore system. The variation of the counterion of the cationic PVP dye from iodide to tetrafluoroborate produced dissimilarities in the interaction pattern with DNA. The PVP iodide competitively displaced the ethidium bromide (EB) from the EB-DNA system, thus suggesting the intercalation ability of the dye, while the PVP tetrafluoroborate did not supported the intercalation of the dye. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the antibacterial and inhibitory action of NorA, Tet(K), MsrA and MepA efflux pumps in S. aureus strains using the sesquiterpenes named trans-caryophyllene and caryophyllene oxide, both isolated and encapsulated in liposomes. The antibacterial and inhibitory action of these efflux pumps was evaluated through the serial microdilution test in 96-well microplates. Each sesquiterpene and liposome/sesquiterpene was combined with antibiotics and ethidium bromide (EtBr). The antibiotics named norfloxacin, tetracycline and erythromycin were used. The 1199 B, IS-58, RN4220 and K2068 S. aureus strains carrying NorA, Tet(K), MsrA and MepA, respectively, were tested. In the fluorescence measurement test, K2068 S. aureus was incubated with the sesquiterpenes and EtBr, and the fluorescence emission by EtBr was measured. The tested substances did not show direct antibacterial activity, with MIC >1024 μg/mL. Nonetheless, the isolated trans-caryophyllene and caryophyllene oxide reduced the MIC of antibiotics and EtBr, indicating inhibition of NorA, Tet(K) and MsrA. In the fluorescence test, these same sesquiterpenes increased fluorescence emission, indicating inhibition of MepA. Therefore, the sesquiterpenes named trans-caryophyllene and caryophyllene oxide did not show direct antibacterial action; however, in their isolated form, they showed possible inhibitory action on NorA, Tet(K), MsrA and MepA efflux pumps. They may also act in antibiotic potentiation. Further studies are needed to identify the mechanisms involved in antibiotic potentiation and efflux pump inhibitory action., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn-DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.

Background and objective: One of the main causes of bacterial resistance to antimicrobials is multidrug resistance induced by the increased efficiency of the efflux pumps. In this study we analyzed how the conditions of assay affect the efflux of indicator substrates ethidium (Et+) and tetraphenylphosphonium (TPP+) in Salmonella enterica ser. Typhimurium cells. Impact of the outer membrane permeability barrier, composition and temperature of the medium on accumulation of the indicator compounds also was analyzed. Materials and methods: The fluorescence of Et+ and Nile Red was measured using 96-well plates and a plate reader. In parallel to traditional studies of fluorescence we applied a constructed selective electrode to follow the accumulation of Et+ in S. enterica cells. Simultaneously with monitoring of Et+ concentration in the cell incubation medium, electrochemical measurements of TPP+ accumulation were performed. Furthermore, Et+ and TPP+ were used within the same sample as agents competing for the interaction with the efflux pumps. An inhibitor phenylalanyl-arginyl-β-naphtylamide (PAβN) was applied to evaluate the input of RND-family pumps in the total efflux of these indicator compounds. Results: S. enterica cells with the intact outer membrane (OM) bound very low amounts of Et+ or TPP+. Cells with the permeabilized OM accumulate considerably higher amounts of the indicator compounds at pH 8.0, but only Et+ was considerably accumulated at pH 6.5. At conditions of electrochemical monitoring accumulation of Et+ by the permeabilized cells at 37 °C was considerably faster than at 23 °C, but at the higher temperature most of the cell-accumulated Et+ was extruded back to the medium. The fluorescence of Et+ in suspension of cells incubated in 400 mmol/L Tris buffer was about twice higher compared to 100 mmol/L one. The inhibitory action of TPP+ on Et+ efflux was evident only in 400 mmol/L Tris although PAβN effectively increased Et+ fluorescence at both buffer concentrations. Conclusions: Results of our experiments indicate that ionic strength of the incubation medium influence the selectivity, the medium temperature and the assay conditions impact the kinetics of efflux. The lower accumulated amount and the weaker fluorescence of Et+ registered in slightly acidic medium indicate that ΔΨ plays a role in the accumulation of this indicator cation. The bound amount of Et+ to the de-energized or permeabilized cells considerably varies depending on the conditions and methods of de-energization or permeabilization of cells. Tris/EDTA permeabilization of the cells does not inhibit the efflux.

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-β-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. The results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measurements of Et+ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably decreases in the presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.

In Listeria monocytogenes, it has been proposed that the VirSR twocomponent signal transduction systems (TCSs) and two ATP-binding cassette (ABC) transporters, VirAB and AnrAB, constitute a complex TCS/ABC transporter system which has been recognized as a unique resistance mode. The role of the putative VirAB-VirSR-AnrAB system in antimicrobial resistance and the respective contributions of the members of the system to resistance were investigated in this study. We constructed gene deletion mutants of L. monocytogenes EGD-e and complemented strains of the mutants and determined MICs of antimicrobial agents against these strains against using the agar dilution method. We assessed the relative expression levels of target genes by reverse transcription-quantitative PCR (RT-qPCR) and measured promoter activity levels by β-galactosidase assays. Our results showed that the VirAB-VirSR-AnrAB system mediates not only nisin and bacitracin resistance but also resistance to cephalosporins, ethidium bromide (EtBr), and benzalkonium chloride (BC). In this system, two ABC transporters, VirAB and AnrAB, play distinct roles in cefotaxime resistance: the former is responsible only for antimicrobial sensing and signaling by VirSR, while the latter contributes to transportation of antimicrobials. Notably, VirAB itself, rather than the VirAB-VirSR-AnrAB system as a whole, contributes to kanamycin and tetracycline resistance. On the basis of the results obtained from this study, we speculate that VirAB acts as a sensor for VirSR in response to cephalosporins, bacitracin, nisin, EtBr, and BC, while VirAB itself plays a role in response to kanamycin and tetracycline in L. monocytogenes EGD-e. [ABSTRACT FROM AUTHOR]

Gram-negative infections are increasingly difficult to treat because of their impermeable outer membranes (OM) and efflux pumps which maintain a low intracellular accumulation of antibiotics within cells. Historically, measurement of accumulation of drugs or dyes within Gram-negative cells has concentrated on analyzing whole bacterial populations. Here, we have developed a method to measure the intracellular accumulation of ethidium bromide, a fluorescent DNA intercalating dye, in single cells using flow cytometry. Bacterial cells were stained with SYTOTM 84 to easily separate cells from background cell debris. Ethidium bromide fluorescence was then measured within the SYTOTM 84 positive population to measure accumulation. In S. Typhimurium SL1344, ethidium bromide accumulation was low, however, in a number of efflux mutants, accumulation of ethidium bromide increased more than twofold, comparable to previous whole population analysis of accumulation. We demonstrate simultaneous measurement of ethidium bromide accumulation and GFP allowing quantification of gene expression or other facets of phenotype in single cells. In addition, we show here that this assay can be adapted for use with efflux inhibitors, with both Gram-negative and Gram-positive bacteria, and with other fluorescent substrates with different fluorescence spectra. [ABSTRACT FROM AUTHOR]

Intermittent fasting diet (IF) as a restrictive regimen prevents neural degeneration and stimulates overexpression of various neurotropic factors in the hippocampus of animal models. This study evaluates the potential effect of the IF in the prevention of learning and memory dysfunction and improving the alterations in the number and volume of neurons in an ethidium bromide (EB) induced mouse model of demyelination. Mice were randomly assigned into N group (Normal Diet and normal saline injection), F group (IF and normal saline injection), EBN group (Normal Diet and EB injection), EBF group (IF and EB injection). The hidden platform test was carried out based on path length, escape latency and swim speeds of mice. Stereological studies were determined by the Cavalieri and the Optical Dissector technique. Maintenance of mice on the IF results in significantly decreased the body weight and biochemical parameters, increased total number of neurons and volume of the hippocampus, and improved learning and memory parameters of adult male mice. However, IF in EBF group did not show as excellently as F group. The EBF group displayed significantly spatial memory improvement than that in EBN group. There were no statistically significant differences between EBF and EBN groups in stereological and learning parameters, though the EBF group displayed faster escape latencies, and swam faster and shorter path lengths than the EBN group in these parameters. Therefore as a conclusion, The IF fairly improved some adverse effects of EB in experimental demyelination models. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Efflux pump inhibitors are able to reverse antibiotic resistance by stopping efflux. • Abundant capsaicinoids are more potent than standard efflux pump inhibitors. • Synthetical capsinoids of promising antimycobacterial nature. Abstract Capsaicinoids are reported to have a bunch of promising pharmacological activities, among them antibacterial effects against various strains of bacteria. In this study the effect on efflux pumps of mycobacteria was investigated. The importance of efflux pumps, and the inhibition of these, is rising due to their involvement in antibiotic resistance development. In order to draw structure and activity relationships we tested natural and synthetical capsaicinoids as well as synthetical capsinoids. In an accumulation assay these compounds were evaluated for their ability to accumulate ethidium bromide into mycobacterial cells, a well-known substrate for efflux pumps. Capsaicin and dihydrocapsaicin, the two most abundant capsaicinoids in Capsicum species, proved to be superior efflux pump inhibitors compared to the standard verapamil. A dilution series showed dose dependency of both compounds. The compound class of less pungent capsinoids qualified for further investigation as antibacterials against Mycobacterium smegmatis. [ABSTRACT FROM AUTHOR]

We have set-up an electrochemical advanced oxidation process for ethidium bromide (1), based on the Eu-doped MnWO 4 (Eu:MnWO 4), obtained through a template-driven synthesis, along with developing a suitable monitoring method. Under galvanostatic conditions, Eu:MnWO 4 -coated graphite electrode serves as anode, applicable for removal of 1. To go further and augment the catalytic method, we have applied a modified carbon paste electrode for the monitoring of 1 with the limit of detection (LOD) of 54 nM. Enhancement of the hydrogen evolution reaction is an indication of electrocatalytic properties of the material, whereby developed method emerges as a candidate for straightforward application in electrochemical advanced oxidation processes (EAOPs). We have enriched experimental data with theoretical insights, provided by Density Functional Theory (DFT), and proposed oxidation mechanism of 1. Based on obtained results, we propose the new nanomaterial as a potent electrochemical modifier, suitable for catalytic treatment and process monitoring of the 1 -polluted waters. [Display omitted] • Eu:MnWO 4 -based nanomaterial for ethidium bromide sensing and control. • A novel analytical approach for both ethidium bromide degradation and concentration monitoring. • Ethidium bromide degradation mechanism was studied theoretically. • The practical feasibility of this system was tested by monitoring the treated water parameters. [ABSTRACT FROM AUTHOR]

Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility in Escherichia coli and other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump in E. coli and other Enterobacteriaceae . However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility in E. coli . By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility in E. coli by directly repressing transcription of the flhDC operon, but not the other flagellum genes/operons tested. flhDC encodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to the flhDC promoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed in E. coli strains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility in E. coli to maintain homeostasis and escape hazards. IMPORTANCE Efflux and motility play a major role in bacterial growth, colonization, and survival. In Escherichia coli , the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824-1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of the flhDC master regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress in E. coli ., Competing Interests: The authors declare no conflict of interest.

Association of ethidium bromide and caffeine in aqueous solution was studied using spectrophotometry. The results were analyzed using a molecular dimer heteroassociation model. The molecular association equilibrium constants were determined. The experimental absorption spectrum of a mixture of heterocyclic molecules was deconvoluted into individual components using a regularization method. Quantitative information on the structure of the heterocomplex was obtained using data for the individual spectral components. The distance and angle between point transition dipole moments of the heterogeneous molecules were 0.3 nm and 16o. A calculation of the relative contents of different types of associates in a mixed solution showed that heteroassociation of the molecules decreased the effective concentration of ethidium bromide and, accordingly, the mutagenic activity of the dye. [ABSTRACT FROM AUTHOR]

The article looks at a study which evaluate ion-motive force of LmrA is a bacterial ATP-binding cassette (ABC) multidrug exporter that uses metabolic energy to transport ions, cytotoxic drugs, and lipids. It mentions sodium-motive force can drive this electrogenic ion exchange mechanism in cells under physiological conditions and ion-coupled ethidium transport requires ATP binding and hydrolysis. It also mentions transport membrane-soluble substrates from thephospholipid bilayer.

The interaction between lomefloxacin (LMF) and a ctDNA-H 1 complex was investigated by various spectroscopies, as well as viscometry and molecular modeling techniques. The RLS results pointed at different intensities of the ctDNA, H 1 and ctDNA-H 1 complex systems in the presence of LMF. The fluorescence intensity measurements of the ctDNA, H 1 and ctDNA-H 1 complex in the presence of LMF as binary and ternary systems at three temperatures exhibited regular quenching of the fluorescence emission curves signifying a static behavior of the binary and ternary systems. Stern-Volmer constant values of ctDNA-LMF complex formation were 7.94 × 10 7 , 4.94 × 10 7 and 3.54 × 10 7 M −1 at 298, 303 and 308 K respectively. In the presence of H1, the Stern-Volmer constant values had been changed to 1.41 × 10 8 , 0.84 × 10 8 and 0.21 × 10 8 M −1 at 298, 303 and 308 K respectively. The Stern-Volmer constant values of binary and ternary systems clearly revealed static quenching behavior of ctDNA upon interaction with LMF in absence and presence of H1. Thermodynamic parameters obtained from Van't Hoff plots revealed the different essence of interaction between LMF with ctDNA, H 1 and ctDNA complex systems. The interaction between LMF with ctDNA and the ctDNA-H 1 complex in the presence of ethidium bromide (EB) and acridine orange (AO) as intercalator probes showed that a competitive ness occurred between EB and AO with LMF, indicating that the LMF bound to ctDNA as an intercalator. Circular dichroism (CD) curves in far UV-CD were used to determine the enhancement of ellipticity in ctDNA upon interaction with LMF in the absence and presence of H 1 . The specific viscosity of ctDNA in the absence and presence of H 1 increased with an enhancement of the LMF concentration which was another reason for LMF binding to ctDNA as an intercalator. Molecular modeling data confirmed the experimental results pertaining to the interaction behavior in the ctDNA-LMF complex in the absence and presence of H 1 . [ABSTRACT FROM AUTHOR]

Purpose: It was reported that mitochondrial DNA (mtDNA) was significantly increased in aneuploid human embryos compared to euploid embryos and was also associated with maternal age. In this study, we further established the mouse model of mtDNA quantitation in reproductive samples based on whole-genome amplification (WGA) and next-generation sequencing (NGS). Methods: WGA followed by NGS-based mtDNA quantitation was first performed on 6 single- and 100-cell samples from a tumor-derived mouse cell line, which was exposed to ethidium bromide to reduce mtDNA content. The relative mtDNA content was normalized to nuclear DNA. This method was then applied to mouse reproductive samples, including 40 pairs of oocytes and polar bodies from 8 CD-1 female mice of advanced reproductive age and 171 blastocysts derived via in vitro maturation (IVM) or in vivo maturation (IVO) from young (6-9 weeks) and reproductively aged (13.5 months) female CF-1 mice. Results: Exposure to ethidium bromide for 3 and 6 days decreased mtDNA levels in both the single- and 100-cell samples as expected. Results demonstrated that the first polar body contained an average of 0.9% of mtDNA relative to oocytes. Compared to the cells in blastocysts, oocytes contained about 180 times as much mtDNA per cell. mtDNA levels were compared among blastocysts from reproductively young and old female mice that had either been produced by IVM or IVO. Cells in blastocysts from younger mice contained significantly lower amounts of mtDNA compared to aged mice ( P < 0.0001). Cells in blastocysts produced via IVO had higher mtDNA content than IVM-derived blastocysts ( P = 0.0001). Cells in aneuploid blastocysts were found to have significantly higher (1.74-fold) levels of mtDNA compared to euploid blastocysts ( P = 0.0006). Conclusion: A reliable method for assessing mtDNA content in mouse gametes and embryos was established. Relative mtDNA levels were elevated in aneuploid embryos relative to euploid embryos, were higher in blastocysts from reproductively old mice relative to young mice, and were lower in embryos derived from IVM compared to IVO. [ABSTRACT FROM AUTHOR]

The aim of this study was to modify the growth, reproductive and yield parameters of Praecitrullus fistulosus by mutagenesis. The seeds of the plant were treated with chemical mutagens including ethidium bromide and colchicine in concentration of (0.05 and 0.10%) and (0.01 and 0.02%), respectively. Seeds were also treated with UV rays at periods of 1 and 2 h and X-rays for 75 KeV at periods of 5 and 10 sec. The growth features were observed at an interval of one week till the 11th week of their growth period. The data showed that seed germination value and lethality (%) of plants were 80 and 75%, respectively and highest in control plants. The time of seed germination (1.6 days) was least in plants treated with X-rays (10 sec). The mutation frequency (80%) was found to be highest in X-rays (10 sec) and colchicine 0.02%. The vegetative growth parameters such as stem length (77 cm), length of leaves (6.6 cm), average number of leaves (36) and leaf surface area (49.6 cm2) was highest against ethidium bromide 0.01%, colchicine 0.02%, ethidium bromide 0.10% and UV rays 1 h, respectively. While the average diameter of stem of control plants was highest (49.6 cm2). The minimum flowering time (31 days) and fruiting time (42.5 days) were observed in ethidium bromide 0.05% and colchicine 0.01% treated plants. The highest number of fruits (4) was observed in colchicine 0.01% treated plants. In conclusion, seeds of P. fistulosus treated with ethidium bromide and colchicine caused positive impact on growth, reproduction and yield attributes as compared to UV and X-rays treatments. [ABSTRACT FROM AUTHOR]

The use of natural products is crucial to suppress the development of these micro-organisms and to reduce the concentration necessary to inhibit these microrganisms, reducing the toxicity risks also. In this study, the essential oil from Chenopodium ambrosioides Leaves and its main constituent α-Terpinene were used in the antibacterial and potentiating activity of antibiotics and ethidium bromide assays, against the bacterial strains Staphylococcus aureus IS-58, carriers of efflux pumps. The Minimum Inhibitory Concentration (MIC) was determined using a microdilution method. The capacity of the aforementioned was also tested in combination with tetracycline and ethidium bromide, with the aim of improving the activity of the antibacterials. The MIC of the C. ambrosioides L. essential oil and of α-Terpinene were above 1024 μg/mL, comprising a clinically irrelevant value. However, when associated with the antibiotics, the C. ambrosioides L. essential oil, significantly decreased the MIC of tetracycline and ethidium bromide. The efflux pump is the only mechanism the bacteria possesses to reduce the toxicity of ethidium bromide, and thus this reduction in the MIC demonstrates that the C. ambrosioides L. essential oil is an effective option in the inhibition of the efflux pump present in these micro-organisms. [ABSTRACT FROM AUTHOR]

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 µM. The study concluded that boeravinone B not only inhibits the NorAmediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the anti-proliferative activity of free and nanoencapsulated gedunin against human non-small-cell lung cancer (NCI-H292) cells. Methods: Gedunin-loaded nanoliposomes (LG) were prepared using thin-film hydration method. Antiproliferative effects of free and LD were evaluated by sulforhodamine B (SRB) assay. Apoptotic effects of gedunin-loaded liposomes were assessed by evaluating expressions of p53, Bax and survivin genes, caspase 3/7 activities, DNA fragmentation and morphological changes after staining with Hoechst 33342 and acridine orange/ethidium bromide (AO/EB). Results: Cell proliferation data and microscopic visualization demonstrated a higher anti-proliferative activity for LG than the encapsulant (liposomes) alone. LG exhibited dose- and time-dependent 10-fold anti-proliferative activity compared to the free drug, while displaying tolerable belligerence towards normal human lung fibroblast (MRC-5) cells. Apoptosis detection assays and gene expression analysis revealed the transcriptional modulation of the apoptosis-related genes (p53, survivin and Bax), increased activity of caspase 3/7 and the condensation of nuclear chromatin, implying the induction of apoptosis by the nano-formulation in NCI-H292 cells. Conclusion: LG may therefore be considered as a potential nano-formulation which can target nonsmall-cell lung cancer. [ABSTRACT FROM AUTHOR]