Neuroprotective effects of Embelin in an ethidium bromide-induced multiple sclerosis in rats: Modulation of p38 MAPK signaling pathway.

BACKGROUND: Multiple sclerosis (MS) is a debilitating inflammatory disease characterized by demyelination, varied remyelination conservation, and partial axonal retention in central nervous system (CNS) lesions. The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in the pathophysiology of MS. Embelin (EMB), derived from the Embelia ribes plant, possesses diverse biological activities, including anti-inflammatory properties. OBJECTIVE: This study aimed to investigate the neuroprotective effects of EMB in an ethidium bromide (EB)-induced model of MS in Wistar rats. METHODS: Wistar rats were randomly divided into five groups (n = 8). MS-like manifestations were induced by injecting EB (0.1 %/10 µl) into the intracerebropeduncle (ICP) region of the rat brain for seven consecutive days. EMB was administered at doses of 1.25, 2.5, and 5 mg/kg. Behavioral assessments, neuroinflammatory cytokine analysis like tumor necrosis factor-α, interleukin-1-ß, interleukin-6 (TNF-α, IL-1ß, IL-6), oxidative stress marker measurements malondialdehyde, reduced glutathione, superoxide dismutase (MDA, GSH, SOD), and nitrite (NO), Acetylcholinesterase enzyme (AchE), and neurotransmitter level analysis, dopamine, serotonin, and norepinephrine (DA, 5-HT, and NE) were conducted. RESULTS: The study assessed behavioral, neurochemical, biochemical, and neuroinflammatory parameters, along with the modulation of p38 MAPK signaling. EMB administration significantly ameliorated neurological consequences induced by EB, improving motor coordination and gait abnormalities in rats. Furthermore, EMB effectively reduced neuroinflammatory cytokines (TNF-α, IL-1ß, IL-6) and oxidative stress markers (AchE, SOD, MDA, GSH, nitrite). Notably, EMB exhibited a modulatory effect on neurotransmitter levels, increasing GABA, DA, and 5-HT, while reducing glutamate in EB-treated groups. CONCLUSION: This study demonstrates the neuroprotective potential of EMB against the EB-induced model of MS in rats. EMB administration mitigated neurological impairments, attenuated neuroinflammation, alleviated oxidative stress, and restored neurotransmitter balance. These findings highlight the promise of EMB as a therapeutic candidate for MS treatment, providing insights into its potential mechanism of action involving the modulation of p38 MAPK signaling.

Synthesis, spectroscopic characterization, and antibacterial activity of chalcone (2E)-1-(3'-aminophenyl)-3-(4-dimethylaminophenyl)-prop-2-en-1-one against multiresistant Staphylococcus aureus carrier of efflux pump mechanisms and ß-lactamase.

BACKGROUND: The bacterium Staphylococcus aureus has stood out for presenting a high adaptability, acquiring resistance to multiple drugs. The search for natural or synthetic compounds with antibacterial properties capable of reversing the resistance of S. aureus is the main challenge to be overcome today. Natural products such as chalcones are substances present in the secondary metabolism of plants, presenting important biological activities such as antitumor, antidiabetic, and antimicrobial activity. OBJECTIVES: In this context, the aim of this work was to synthesize the chalcone (2E)-1-(3'-aminophenyl)-3-(4-dimethylaminophenyl)-prop-2-en-1-one with nomenclature CMADMA, confirm its structure by nuclear magnetic resonance (NMR), and evaluate its antibacterial properties. METHODS: The synthesis methodology used was that of Claisen-Schmidt, and spectroscopic characterization was performed by NMR. For microbiological assays, the broth microdilution methodology was adopted in order to analyze the antibacterial potential of chalcones and to analyze their ability to act as a possible inhibitor of ß-lactamase and efflux pump resistance mechanisms, present in S. aureus strain K4100. RESULTS: The results obtained show that CMADMA does not show direct antibacterial activity, expressing a MIC of ≥1024 µg/mL, or on the enzymatic mechanism of ß-lactamase; however, when associated with ethidium bromide in efflux pump inhibition assays, CMADMA showed promising activity by reducing the MIC of the bromide from 64 to 32 µg/mL. CONCLUSION: We conclude that the chalcone synthesized in this study is a promising substance to combat bacterial resistance, possibly acting in the inhibition of the QacC efflux pump present in S. aureus strain K4100, as evidenced by the reduction in the MIC of ethidium bromide.

Comparative Antibacterial and Efflux Pump Inhibitory Activity of Isolated Nerolidol, Farnesol, and α-Bisabolol Sesquiterpenes and Their Liposomal Nanoformulations.

The efflux systems are considered important mechanisms of bacterial resistance due to their ability to extrude various antibiotics. Several naturally occurring compounds, such as sesquiterpenes, have demonstrated antibacterial activity and the ability to inhibit efflux pumps in resistant strains. Therefore, the objective of this research was to analyze the antibacterial and inhibitory activity of the efflux systems NorA, Tet(K), MsrA, and MepA by sesquiterpenes nerolidol, farnesol, and α-bisabolol, used either individually or in liposomal nanoformulation, against multi-resistant Staphylococcus aureus strains. The methodology consisted of in vitro testing of the ability of sesquiterpenes to reduce the Minimum Inhibitory Concentration (MIC) and enhance the action of antibiotics and ethidium bromide (EtBr) in broth microdilution assays. The following strains were used: S. aureus 1199B carrying the NorA efflux pump, resistant to norfloxacin; IS-58 strain carrying Tet(K), resistant to tetracyclines; RN4220 carrying MsrA, conferring resistance to erythromycin. For the EtBr fluorescence measurement test, K2068 carrying MepA was used. It was observed the individual sesquiterpenes exhibited better antibacterial activity as well as efflux pump inhibition. Farnesol showed the lowest MIC of 16.5 µg/mL against the S. aureus RN4220 strain. Isolated nerolidol stood out for reducing the MIC of EtBr to 5 µg/mL in the 1199B strain, yielding better results than the positive control CCCP, indicating strong evidence of NorA inhibition. The liposome formulations did not show promising results, except for liposome/farnesol, which reduced the MIC of EtBr against 1199B and RN4220. Further research is needed to evaluate the mechanisms of action involved in the inhibition of resistance mechanisms by the tested compounds.

Effect of piperine on the inhibitory potential of MexAB-OprM efflux pump and imipenem resistance in carbapenem-resistant Pseudomonas aeruginosa.

The escalating prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant threat to global public health through the spread of its 'high-risk' clones. Immediate and decisive research into antimicrobial agents against CRPA is crucial for the development of effective measures and interventions. Overexpression of the MexAB-OprM efflux pump is one of the major mechanisms of CRPA. Since the active efflux of antibacterial agents plays a significant role in mediating drug resistance in CRPA, the inhibition of efflux pumps has become a promising strategy to restore antibacterial potency. Piperine (PIP) has been proven to be a promising efflux pump inhibitor in some bacteria. However, there are no studies on whether PIP can act as a potential efflux pump inhibitor in CRPA. The present study aimed to identify the antibacterial activity of PIP against CRPA and to evaluate the effect on the MexAB-OprM efflux pump. Molecular docking was used to analyze the possible interaction of PIP with the proteins of the MexAB-OprM efflux pump in CRPA. The effect of PIP on the expression of the MexAB-OprM efflux pump was investigated by real-time quantitative PCR (qPCR) and ethidium bromide accumulation efflux assay. The effect of PIP on CRPA imipenem (IPM) resistance was investigated by the checkerboard dilution method. The results demonstrated that PIP exhibited the lowest binding affinity of -9.1 kcal towards efflux pump proteins. A synergistic effect between PIP and IPM on CRPA was observed. More importantly, PIP effectively hindered the efflux of ethidium bromide and IPM by up-regulating MexR gene expression while down-regulating MexA, MexB, and OprM gene expressions. In conclusion, PIP could enhance the antibacterial activity of IPM by inhibiting the MexAB-OprM efflux pump. Our work proved that PIP had the potential to be an efflux pump inhibitor of CRPA.

Deoxyribonucleoside treatment rescues EtBr-induced mtDNA depletion in iPSC-derived neural stem cells with POLG mutations.

Mutations in POLG, the gene encoding the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (Pol-γ), lead to diseases driven by defective mtDNA maintenance. Despite being the most prevalent cause of mitochondrial disease, treatments for POLG-related disorders remain elusive. In this study, we used POLG patient-induced pluripotent stem cell (iPSC)-derived neural stem cells (iNSCs), one homozygous for the POLG mutation c.2243G>C and one compound heterozygous with c.2243G>C and c.1399G>A, and treated these iNSCs with ethidium bromide (EtBr) to study the rate of depletion and repopulation of mtDNA. In addition, we investigated the effect of deoxyribonucleoside (dNs) supplementation on mtDNA maintenance during EtBr treatment and post-treatment repopulation in the same cells. EtBr-induced mtDNA depletion occurred at a similar rate in both patient and control iNSCs, however, restoration of mtDNA levels was significantly delayed in iNSCs carrying the compound heterozygous POLG mutations. In contrast, iNSC with the homozygous POLG mutation recovered their mtDNA at a rate similar to controls. When we treated cells with dNs, we found that this reduced EtBr-induced mtDNA depletion and significantly increased repopulation rates in both patient iNSCs. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation also within intact neural lineage cells and suggest that those with compound heterozygous mutation have a more severe defect of mtDNA synthesis. Our findings further highlight the potential for dNs to improve mtDNA replication in the presence of POLG mutations, suggesting that this may offer a new therapeutic modality for mitochondrial diseases caused by disturbed mtDNA homeostasis.

1, 4-naphthoquinone efficiently facilitates the disintegration of pre-existing biofilm of Staphylococcus aureus through eDNA intercalation.

1, 4-naphthoquinone, a plant-based quinone derivative, has gained much attention for its effectiveness against several biofilm-linked diseases. The biofilm inhibitory effect of 1, 4-naphthoquinone against Staphylococcus aureus has already been reported in our previous study. We observed that the extracellular DNA (eDNA) could play an important role in holding the structural integrity of the biofilm. Hence, in this study, efforts have been directed to examine the possible interactions between 1, 4-naphthoquinone and DNA. An in silico analysis indicated that 1, 4-naphthoquinone could interact with DNA through intercalation. To validate the same, UV-Vis spectrophotometric analysis was performed in which a hypochromic shift was observed when the said molecule was titrated with calf-thymus DNA (CT-DNA). Thermal denaturation studies revealed a change of 8℃ in the melting temperature (Tm) of CT-DNA when complexed with 1, 4-naphthoquinone. The isothermal calorimetric titration (ITC) assay revealed a spontaneous intercalation between CT-DNA and 1, 4-naphthoquinone with a binding constant of 0.95 ± 0.12 × 108. Furthermore, DNA was run through an agarose gel electrophoresis with a fixed concentration of ethidium bromide and increasing concentrations of 1, 4-naphthoquinone. The result showed that the intensity of ethidium bromide-stained DNA got reduced concomitantly with the gradual increase of 1, 4-naphthoquinone suggesting its intercalating nature. To gain further confidence, the pre-existing biofilm was challenged with ethidium bromide wherein we observed that it could also show biofilm disintegration. Therefore, the results suggested that 1, 4-naphthoquinone could exhibit disintegration of the pre-existing biofilm of Staphylococcus aureus through eDNA intercalation.

Toxoplasma gondii attenuates the ethidium bromide induced demyelination lesions in multiple sclerosis model rats.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease. Since the modulation of the immune system by parasites has been proven, and there have been reports of a reduction in the clinical symptoms of MS in people with toxoplasmosis, this study aimed to investigate the effect of toxoplasmosis on MS in an animal model. MS model was induced by the ethidium bromide injection in the areas specified in the Rat's brain in the stereotaxic device and Toxoplasma gondii RH strain injection of the rat's peritoneal for creation of toxoplasmosis. The effect of acute and chronic toxoplasmosis on the MS model was evaluated by examining the development of clinical symptoms of MS, body weight, changes in the levels of inflammatory cytokines, inflammatory cell infiltration, cell density, and spongy tissue in the brain. The body weight in the acute toxoplasmosis with MS was the same as the MS group, and a significant decrease was observed, but no weight loss was observed in the chronic toxoplasmosis with MS. In the chronic toxoplasmosis, the progress of clinical signs such as Immobility of limbs, including tail, hands, and feet, was observed less compared to other groups. The histology results in the group of chronic toxoplasmosis showed high cell density and inhibition of spongy tissue formation, and the infiltration of inflammatory cells in this group was less. TNF-α and INF-γ decreased in MS with chronic toxoplasmosis compared to the MS group. Our findings showed that chronic toxoplasmosis with inhibition of spongy tissue formation and prevention of cell infiltration and. As a result, the reduction of inflammatory cytokines could reduce clinical symptoms in MS in the animal model.

Inhibition of NorA efflux pump of Staphylococcus aureus by anacardic acids isolated from the cashew nutshell liquid of Anacardium occidentale L.

The rising of diseases caused by multidrug-resistant bacteria has encouraged researchers to explore more antimicrobial substances, as well as chemicals capable of potentiating the action of existing ones against multidrug-resistant bacteria. Anacardium occidentale produces a fruit known as cashew nut, filled with a dark, almost black, caustic, and flammable liquid called cashew nutshell liquid (CNSL). The goal of the study was to evaluate the intrinsic antimicrobial activity of the major compounds present in CNSL, called anacardic acids (AA), as well as their possible modulatory action as an adjuvant of Norfloxacin against a Staphylococcus aureus strain overproducing the NorA efflux pump (SA1199B). Microdilution assays were performed to determine the minimum inhibitory concentration (MIC) of AA against different microbial species. Norfloxacin and Ethidium Bromide (EtBr) resistance modulation assays were performed in the presence or absence of AA against SA1199-B. AA showed antimicrobial activity against Gram-positive bacterial strains tested but no activity against Gram-negative bacteria or yeast strains. At subinhibitory concentration, AA reduced the MIC values for Norfloxacin and EtBr against the SA1199-B strain. Furthermore, AA increased the intracellular accumulation of EtBr in this NorA overproducer strain, indicating that AA are NorA inhibitors. Docking analysis showed that AA probably modulates Norfloxacin efflux by spatial impediment at the same binding site of NorA.

Evaluation of efflux pump inhibitory activity of some plant extracts and using them as adjuvants to potentiate the inhibitory activity of some antibiotics against Staphylococcus aureus.

Background: Antibiotic-resistant pathogens became a real global threat to human and animal health. This needs to concentrate the efforts to minimize and control these organisms. Efflux pumps are considered one of the important strategies used by bacteria to exclude harmful materials from the cell. Inhibition of these pumps can be an active strategy against multidrug resistance pathogens. There are two sources of efflux pump inhibitors that can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin is characterized by a wide margin of safety for the host cell. Aim: In this study, the ability of some plant extracts like (propolis show rosemary, clove, capsaicin, and cumin) to potentiate the inhibitory activity of some antibiotics such as (ciprofloxacin, erythromycin, gentamycin, tetracycline, and ampicillin) against Staphylococcus aureus pathogen were tested. Methods: Efflux pump inhibitory activity of the selected plant extracts was tested using an ethidium bromide (EtBr) accumulation assay. Results: The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin, and gentamycin. While it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in a combination of the minimum inhibitory concentration for the selected plant extracts (rosemary, clove, capsaicin, and cumin) with any of the tested antibiotics. Interestingly, according to the results of the EtBr accumulation assay, Propolis has potent inhibitory activity against the S. aureus (MRS usa300) pump system. Conclusion: This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin, and gentamycin against S. aureus strains, in case of the efflux-mediated antimicrobial resistance mechanisms.

ADMET study, spectroscopic characterization and effect of synthetic nitro chalcone in combination with norfloxacin, ciprofloxacin, and ethidium bromide against Staphylococcus aureus efflux pumps.

Chalcones are present in a wide variety of plants, having in their structure two aromatic rings that are linked together by a chain composed of three carbon atoms with α, ß-unsaturated to carbonyl system. Bacteria have several drug resistance mechanisms, among them the efflux pump; this mechanism, when active, is able to expel different compounds from inside bacterial cells. Several efflux pumps have already been identified for Staphylococcus aureus bacteria, including MepA and NorA. Many chalcones have been isolated and identified with various activities, such as antimicrobial. In view of this, this article aimed to evaluate the antibiotic modifying effect of chalcone (E)-1-(2-hydroxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one against S. aureus carrier of NorA and MepA efflux pump. Regarding the antibiotic, there was a synergism when associated with ciprofloxacin in SA-K2068 strain, showing this chalcone as an alternative to reverse the resistance to this medicine. The physicochemical properties calculated were fundamental in the description of the predicted pharmacokinetic properties. Despite the mutagenic risk caused by the metabolic activation of nitrochalcone, it is possible to notice a pharmacological principle in a longer half-life for the performance of biological activities. The compound has a good bioavailability, as it is highly absorbed in the intestine and easily transported by plasma proteins, in addition to not presenting neurotoxic, hepatotoxic, and cardiotoxic damage.

The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation.

A key mechanism driving antimicrobial resistance (AMR) stems from the ability of bacteria to up-regulate efflux pumps upon exposure to drugs. The resistance gained by this up-regulation is pliable because of the tight regulation of efflux pump levels. This leads to temporary enhancement in survivability of bacteria due to higher efflux pump levels in the presence of antibiotics, which can be reversed when the cells are no longer exposed to the drug. Knowledge of the extent of resistance thus gained would inform intervention strategies aimed at mitigating AMR. Here, we combine mathematical modeling and experiments to quantify the maximum extent of resistance that efflux pump up-regulation can confer via phenotypic induction in the presence of drugs and genotypic abrogation of regulation. Our model describes the dynamics of drug transport in and out of cells coupled with the associated regulation of efflux pump levels and predicts the increase in the minimum inhibitory concentration (MIC) of drugs due to such regulation. To test the model, we measured the uptake and efflux as well as the MIC of the compound ethidium bromide (EtBr), a substrate of the efflux pump LfrA, in wild-type Mycobacterium smegmatis mc2155, as well as in two laboratory-generated strains. Our model captured the observed EtBr levels and MIC fold-changes quantitatively. Further, the model identified key parameters associated with the resulting resistance, variations in which could underlie the extent to which such resistance arises across different drug-bacteria combinations, potentially offering tunable handles to optimize interventions aimed at minimizing AMR.

Inhibition of Schwann cell pannexin 1 attenuates neuropathic pain through the suppression of inflammatory responses.

BACKGROUND: Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain. METHODS: A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake. RESULTS: CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression. CONCLUSION: In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.

Evaluation of the antibacterial and inhibitory activity of NorA and MepA efflux pumps from Staphylococcus aureus by diosgenin.

The increase in bacterial resistance to available antibiotics has driven several researchers to search for new agents with therapeutic properties. Diosgenin is a naturally occurring steroidal saponin that has demonstrated several pharmacological properties. In the present study, we report the antimicrobial activity of diosgenin against the standard and multidrug-resistant bacteria of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, in addition to the efflux pump inhibitory activity against Staphylococcus aureus strains carrying NorA and MepA pumps. For this purpose, the broth microdilution method was used, from which the value of the Minimum Inhibitory Concentration (MIC) was obtained, and this was associated with subinhibitory concentration (MIC/8) with antibiotic of clinical use and ethidium bromide for strains carrier by efflux pump. Diosgenin showed antimicrobial activity for standard S. aureus bacteria and potentiating activity in association with gentamicin and ampicillin for P. aeruginosa multidrug-resistant bacteria, it also showed potentiation in association with norfloxacin against the E. coli strain and gentamicin against the S. aureus strain. Antimicrobial activity against efflux pump-bearing strains revealed that saponin did not interfere with the efflux pump mechanism or intervened antagonistically. Thus, saponin has shown to be very promising against bacterial resistance in association with aminoglycoside, fluoroquinolones and beta-lactam, however additional studies should be carried out to better elucidate the mechanism of action of diosgenin.

Evaluation of ellagic acid and gallic acid as efflux pump inhibitors in strains of Staphylococcus aureus.

The Gram-positive bacterium Staphylococcus aureus is responsible for a number of infections and has been described to exhibit resistance to antibacterial drugs through different resistance mechanisms. Among these, active efflux has been shown to be one of the main mechanisms of bacterial resistance associated with S. aureus. In this sense, the aim of the present study was to evaluate the ability of ellagic acid and gallic acid to reverse resistance by inhibiting the efflux pumps present in S. aureus strains IS-58 and K2068, which express the TetK and MepA flux pumps, respectively. In addition, the toxicity of both compounds was verified in Drosophila melanogaster. Broth microdilution assays were performed to obtain the minimum inhibitory concentration (MIC) values of ellagic acid and gallic acid, whereas efflux pump inhibition was tested using a subinhibitory concentration of standard efflux pump inhibitors, gallic acid and ellagic acid (MIC/8), where the ability of these compounds to decrease the MIC of ethidium bromide (EtBr) and antibiotics was verified. Toxicity was evaluated by mortality and negative geotaxis assays in D. melanogaster. Ellagic acid and gallic acid showed no direct antibacterial activity on S. aureus strains carrying the efflux pumps TetK and MepA. However, when we looked at the results for the TetK pump, we saw that when the two acids were associated with the antibiotic tetracycline, a potentiation of the antibacterial effect occurred; this behavior was also observed for the antibiotic ciprofloxacin in the MepA strain. For the efflux pump inhibition results, only the compound gallic acid showed potentiating effect on antibacterial activity when associated with the substrate EtBr for the IS-58 strain carrying the TetK efflux pump. Ellagic acid and gallic acid showed no toxicity on the model arthropod D. melanogaster. These results indicate the possible use of gallic acid as an adjuvant in antibiotic therapy against multidrug-resistant bacteria.

Forskolin, an Adenylcyclase/cAMP/CREB Signaling Activator Restoring Myelin-Associated Oligodendrocyte Destruction in Experimental Ethidium Bromide Model of Multiple Sclerosis.

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.

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA4 and LPA6 knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA2 knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2 S)-OMPT (LPA3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA4 and LPA6 knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

Genetic interaction between F-box motif encoding YDR131C and retrograde signaling-related RTG1 regulates the stress response and apoptosis in Saccharomyces cerevisiae.

The retrograde signaling pathway is well conserved from yeast to humans, which regulates cell adaptation during stress conditions and prevents cell death. One of its components, RTG1 encoded Rtg1p in association with Rtg3p communicates between mitochondria, nucleus, and peroxisome during stress for adaptation, by regulation of transcription. The F-box motif protein encoded by YDR131C  constitutes a part of SCF Ydr131c -E3 ligase complex, with unknown function; however, it is known that retrograde signaling is modulated by the E3 ligase complex. This study reports epistasis interaction between YDR131C and RTG1, which regulates cell growth, response to genotoxic stress, decreased apoptosis, resistance to petite mutation, and cell wall integrity. The cells of ydr131cΔrtg1Δ genetic background exhibits growth rate improvement however, sensitivity to hydroxyurea, itraconazole antifungal agent and synthetic indoloquinazoline-based alkaloid (8-fluorotryptanthrin, RK64), which disrupts the cell wall integrity in Saccharomyces cerevisiae. The epistatic interaction between YDR131C and RTG1 indicates a link between protein degradation and retrograde signaling pathways.

Carnosic Acid Attenuates an Early Increase in ROS Levels during Adipocyte Differentiation by Suppressing Translation of Nox4 and Inducing Translation of Antioxidant Enzymes.

The objective of this study was to investigate molecular mechanisms underlying the ability of carnosic acid to attenuate an early increase in reactive oxygen species (ROS) levels during MDI-induced adipocyte differentiation. The levels of superoxide anion and ROS were determined using dihydroethidium (DHE) and 2'-7'-dichlorofluorescin diacetate (DCFH-DA), respectively. Both superoxide anion and ROS levels peaked on the second day of differentiation. They were suppressed by carnosic acid. Carnosic acid attenuates the translation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4), p47phox, and p22phox, and the phosphorylation of nuclear factor-kappa B (NF-κB) and NF-κB inhibitor (IkBa). The translocation of NF-κB into the nucleus was also decreased by carnosic acid. In addition, carnosic acid increased the translation of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCSc), and glutathione S-transferase (GST) and both the translation and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, these results indicate that carnosic acid could down-regulate ROS level in an early stage of MPI-induced adipocyte differentiation by attenuating ROS generation through suppression of NF-κB-mediated translation of Nox4 enzyme and increasing ROS neutralization through induction of Nrf2-mediated translation of phase II antioxidant enzymes such as HO-1, γ-GCS, and GST, leading to its anti-adipogenetic effect.

Antibacterial and antibiotic modifying activity, ADMET study and molecular docking of synthetic chalcone (E)-1-(2-hydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)prop-2-en-1-one in strains of Staphylococcus aureus carrying NorA and MepA efflux pumps.

A large number of infections are caused by multi-resistant bacteria worldwide, adding up to a figure of around 700,000 deaths per year. Because of that many strategies are being developed in order to combat the resistance of microorganisms to drugs, in recent times, chalcones have been studied for this purpose. Chalcones are known as α, ß-unsaturated ketones, characterized by having the presence of two aromatic rings that are joined by a three-carbon chain, they are a class of compounds considered an exceptional model due to chemical simplicity and a wide variety of biological activities, which include anticancer, anti-inflammatory, antioxidants, antimicrobials, anti-tuberculosis, anti-HIV, antimalarial, anti-allergic, antifungal, antibacterial, and antileishmanial. The objective of this work was evaluate the antibacterial and antibiotic modifying activity of chalcone (E)-1-(2-hydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)prop-2-en-1-one against the bacteria Staphylococcus aureus carrying a NorA and MepA efflux pump. The results showed that chalcone was able to synergistically modulate the action of Norfloxacin and Ethidium Bromide against the bacteria Staphylococcus aureus 1199B and K2068, respectively. The theoretical physicochemical and pharmacokinetic properties of chalcone showed that the chalcone did not present a severe risk of toxicity such as genetic mutation or cardiotoxicity, constituting a good pharmacological active ingredient.

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.