Altered Membrane Permeability Research Articles

Kaempferia galanga Linn. Extract – A potential antibacterial agent for preservation of poultry products

Poultry is one of the most widely consumed animal-based foods in the world. Understanding the characteristics of bacterial communities present during processing and the targeted design of natural preservatives could improve the quality and safety concerns of poultry industry. In this study, pathogens and spoilage bacteria arising from the processing of poultry products were identified using high-throughput 16S rRNA sequencing and traditional culture screening. Ten dominant pathogenic and spoilage bacteria were isolated, including 8 g-positive and 2 g-negative bacteria. The mechanism and antibacterial activity of Kaempferia galanga Linn. extract (KGE), a spice added to the poultry products, against the targeted pathogenic bacteria were analyzed further. KGE exhibited significant antibacterial activity against all tested pathogenic bacteria. The antibacterial mechanism of KGE involved alteration of cell integrity and cell membrane permeability, resulting in an outflow of intracellular substances, such as nucleic acids, proteins, and alkaline phosphatase. Scanning electron microscopy further confirmed KGE-mediated cell membrane damage and cytoplasmic leakage. The chemical characterization indicated that the large amounts of acetic acid present in KGE might account for its excellent antibacterial activity. Taken together, the study confirms the excellent antibacterial activity of KGE and its potential use as a natural preservative for poultry products.

Improvement of Oxidative Stress and Mitochondrial Dysfunction by β-Caryophyllene: A Focus on the Nervous System.

Mitochondrial dysfunction results in a series of defective cellular events, including decreased adenosine triphosphate (ATP) production, enhanced reactive oxygen species (ROS) output, and altered proteastasis and cellular quality control. An enhanced output of ROS may damage mitochondrial components, such as mitochondrial DNA and elements of the electron transport chain, resulting in the loss of proper electrochemical gradient across the mitochondrial inner membrane and an ensuing shutdown of mitochondrial energy production. Neurons have an increased demand for ATP and oxygen, and thus are more prone to damage induced by mitochondrial dysfunction. Mitochondrial dysfunction, damaged electron transport chains, altered membrane permeability and Ca2+ homeostasis, and impaired mitochondrial defense systems induced by oxidative stress, are pathological changes involved in neurodegenerative disorders. A growing body of evidence suggests that the use of antioxidants could stabilize mitochondria and thus may be suitable for preventing neuronal loss. Numerous natural products exhibit the potential to counter oxidative stress and mitochondrial dysfunction; however, science is still looking for a breakthrough in the treatment of neurodegenerative disorders. β-caryophyllene is a bicyclic sesquiterpene, and an active principle of essential oils derived from a large number of spices and food plants. As a selective cannabinoid receptor 2 (CB2) agonist, several studies have reported it as possessing numerous pharmacological activities such as antibacterial (e.g., Helicobacter pylori), antioxidant, anti-inflammatory, analgesic (e.g., neuropathic pain), anti-neurodegenerative and anticancer properties. The present review mainly focuses on the potential of β-caryophyllene in reducing oxidative stress and mitochondrial dysfunction, and its possible links with neuroprotection.

Mitochondrial Dysfunction and Oxidative Stress Caused by Cryopreservation in Reproductive Cells.

Mitochondria, fundamental organelles in cell metabolism, and ATP synthesis are responsible for generating reactive oxygen species (ROS), calcium homeostasis, and cell death. Mitochondria produce most ROS, and when levels exceed the antioxidant defenses, oxidative stress (OS) is generated. These changes may eventually impair the electron transport chain, resulting in decreased ATP synthesis, increased ROS production, altered mitochondrial membrane permeability, and disruption of calcium homeostasis. Mitochondria play a key role in the gamete competence to facilitate normal embryo development. However, iatrogenic factors in assisted reproductive technologies (ART) may affect their functional competence, leading to an abnormal reproductive outcome. Cryopreservation, a fundamental technology in ART, may compromise mitochondrial function leading to elevated intracellular OS that decreases sperm and oocytes’ competence and the dynamics of fertilization and embryo development. This article aims to review the role played by mitochondria and ROS in sperm and oocyte function and the close, biunivocal relationships between mitochondrial damage and ROS generation during cryopreservation of gametes and gonadal tissues in different species. Based on current literature, we propose tentative hypothesis of mechanisms involved in cryopreservation-associated mitochondrial dysfunction in gametes, and discuss the role played by antioxidants and other agents to retain the competence of cryopreserved reproductive cells and tissues.

Effect of high levels of CO2 and O2 on membrane fatty acid profile and membrane physiology of meat spoilage bacteria

The membrane is the major protective barrier separating the cell from the environment and is thus important for bacteria to survive environmental stress. This study investigates changes in membrane lipid compositions and membrane physiology of meat spoiling bacteria in response to high CO2 (30%) and O2 (70%) concentrations, as commonly used for modified atmosphere packaging of meat. Therefore, the fatty acid profile as well as membrane fluidity, permeability and cell surface were determined and correlated to the genomic settings of five meat spoiling bacteria Brochothrix (B.) thermosphacta, Carnobacterium (C.) divergens, C. maltaromaticum, Leuconostoc (L.) gelidum subsp. gelidum and L. gelidum subsp. gasicomitatum cultivated under different gas atmospheres. We identified different genomic potentials for fatty acid adaptations, which were in accordance with actual measured changes in the fatty acid composition for each species in response to CO2 and/or O2, e.g., an increase in saturated, iso and cyclopropane fatty acids. Even though fatty acid changes were species-specific, the general physiological responses were similar, comprising a decreased membrane permeability and fluidity. Thus, we concluded that meat spoiling bacteria facilitate a change in membrane fatty acids upon exposure to O2 and CO2, what leads to alteration of membrane fluidity and permeability. The observed adaptations might contribute to the resistance of meat spoilers against detrimental effects of the gases O2 and CO2 and thus help to explain their ability to grow under different modified atmospheres. Furthermore, this study provides fundamental knowledge regarding the impact of fatty acid changes on important membrane properties of bacteria.

Overexpression of WssgtL3.1 gene from Withania somnifera confers salt stress tolerance in Arabidopsis.

Overexpression of Withania somnifera SGT gene (WssgtL3.1) in transgenic Arabidopsis improves various agronomic and physiological traits and alters conjugated sterol levels to mitigate the effect of salt stress. Sterols are essential constituents of cell membranes that are involved in several biological functions, including response to various biotic and abiotic stresses by altering membrane permeability and signaling pathways. Sterol glycosyltransferases (SGTs) are enzymes that are involved in sterol modification by converting sterols into sterol-conjugates to play essential roles in adaptive responses. However, their roles under abiotic stresses are lesser-known. Among abiotic stresses, salinity imposes serious threat to crop yield worldwide, hence the present study intends to investigate the role of WssgtL3.1-overexpressed Arabidopsis plants under salt stress indicating the crosstalk between SGT gene and salinity to develop improved crop varieties with better stress tolerance ability. The findings revealed that overexpression of WssgtL3.1 gene in A. thaliana improved the resistance against salt stress in the overexpressing lines. Transgenic lines showed significantly higher germination rate, increased plant growth with less chlorophyll damage compared to wild-type (WT) control plants. Moreover, better tolerance also correlated with enhanced osmolytes (proline and soluble sugar), better membrane integrity, decreased H2O2 production and lesser MDA accumulation and Na+/K+ ratio with more negative osmotic potential in overexpressed lines. Additionally, in sterol profiling, significant enhancement in stigmasterol was also observed in transgenic lines than WT plants. Furthermore, in expression profiling, salt responsive genes LEA 4-5, sucrose synthase, and transporter of monosaccharide (ERD) significantly upregulated in overexpressing lines as compared to WT. Thus our data strongly support the defensive role of Withania somnifera SGT gene (WssgtL3.1) against salt stress and contribute to improved salinity tolerance in plants through sterol modulation.

Correlation between corrected serum calcium and serum albumin in children with idiopathic nephrotic syndrome in North central, Nigeria

Background: Nephrotic syndrome is a clinical condition caused by alteration of glomerular membrane permeability resulting in a net loss of protein, and vitamin D binding proteins in urine leading to hypoalbuminaemia and hypocalcaemia. A positive correlation between serum albumin and ionized calcium in childhood nephrotic syndrome has been described but the correlation between total serum calcium or corrected serum calcium and serum albumin has not been extensively described.Methods: This study was carried out at Dalhatu Araf Specialist Hospital, Lafia Nigeria. Fifteen children with idiopathic nephrotic syndrome were recruited consecutively as the cases, 15 age and gender matched healthy children were recruited as the controls. Total serum calcium and albumin was assayed in all these children. Corrected serum calcium was calculated for the cases. Tests of correlation was carried out to see if there was any relationship between corrected or total serum calcium and serum albumin.Results: The mean total serum calcium and serum corrected calcium levels in the cases was 2.04±0.34 mmol/l and 2.5 mmol/l respectively. The mean total serum calcium was 2.12±0.32 mmol/l for the controls. The mean serum albumin level was 14.7±4.1 g/l and 34.6±2.7 mmol/l for the cases and controls respectively. A negative and weak correlation was found between serum albumin and corrected serum calcium and a similar negative correlation between serum albumin and total serum calcium.Conclusions: The common reports of a positive correlation between serum ionized calcium and serum albumin cannot be applied to total or corrected serum calcium and serum albumin.

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts.

Background Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.

Impact of metal-oxide nanoparticles on growth, physiology and yield of tomato (Solanum lycopersicum L.) modulated by Azotobacter salinestris strain ASM.

The current study for the first time demonstrates the interference of a free-living, N2-fixing, and nanoparticle (NP) tolerant Azotobacter salinestris strain ASM recovered from metal-polluted soil with tomato plant-metal oxide NPs (ZnO, CuO, Al2O3, and TiO2) interactions in a sandy clay loam soil system with bulk materials as control. Tomato plants were grown till full maturity in soils amended with 20-2000mgkg-1 of each metal-oxide NP with and without seed biopriming and root-inoculation of A.salinestris. A.salinestris was found metabolically active, producing considerably high amounts of bioactive indole-3-acetic-acid, morphologically unaffected, and with low alteration of cell membrane permeability under 125-1500 μgml-1 of NPs. However, ZnO-NPs slightly alter bacterial membrane permeability. Besides, A.salinestris secreted significantly higher amounts of extracellular polymeric substance (EPS) even under NP exposure, which could entrap the NPs and form metal-EPS complex as revealed and quantified by SEM-EDX. NPs were also found adsorbed on bacterial biomass. EPS stabilized the NPs and provided negative zeta potential to NPs. Following soil application, A.salinestris improved the plant performance and augmented the yield of tomato fruits and lycopene content even in NPs stressed soils. Interestingly, A.salinestris inoculation enhanced photosynthetic pigment formation, flower attributes, plant and fruit biomass, and reduced proline level. Bacterial inoculation also reduced the NP's uptake and accumulation significantly in vegetative organs and fruits. The organ wise order of NP's internalization was roots>shoots>fruits. Conclusively, A.salinestris inoculation could be an alternative to increase the production of tomato in metal-oxide NPs contaminated soils.

Transport processes in equine oocytes and ovarian tissue during loading with cryoprotective solutions

BackgroundRational design of cryopreservation strategies for oocytes and ovarian cortex tissue requires insights in the rate at which cryoprotective agents (CPA) permeate and concomitant water transport takes place. The aim of the current study was to investigate possible differences in permeation kinetics of different CPAs (i.e., glycerol/GLY, ethylene glycol/EG, dimethyl sulfoxide/DMSO, and propylene glycol/PG), in equine oocytes as well as ovarian tissue. MethodsMembrane permeability of oocytes to water (Lp) and to CPAs (Ps) was inferred from video microscopic imaging of oocyte volume responses during perfusion with anisotonic and CPA solutions. CPA diffusion into ovarian tissue and tissue dehydration was monitored during incubation, using osmometer and weight measurements, to derive CPA diffusion coefficients (D). ResultsMembrane permeability of oocytes towards CPAs was found to increase in the order GLY < EG < DMSO<PG. Permeability towards water in anisotonic solutions was determined to be higher than in CPA solutions, indicating CPAs alter membrane permeability properties. CPA diffusion in ovarian tissue increased in the order GLY,PG < EG,DMSO. Tissue dehydration was found to increase with exposure to increasing CPA concentrations, which inversely correlated with CPA diffusivity. ConclusionsIn conclusion, it is shown here that the rate of CPA movement across membrane bilayers is determined by different physical barrier factors than those determining CPA movement in tissues. General significanceThe parameters presented in this study can be applied in models describing solute and water transport in cells and tissues, as well as in cryopreservation protocols.

Copper toxicity affects phosphorus uptake mechanisms at molecular and physiological levels in Cucumis sativus plants

Due to the deliberate use of cupric fungicides in the last century for crop-defence programs, copper (Cu) has considerably accumulated in the soil. The concentrations of Cu often exceed the safety limits of risk assessment for Cu in soil and this may cause toxicity in plants. Copper toxicity induces nutritional imbalances in plants and constraints to plants growth. These aspects might be of paramount importance in the case of phosphorus (P), which is an essential plant macronutrient. In this work, hydroponically grown cucumber plants were used to investigate the influence of the exposure to different Cu concentrations (0.2, 5, 25 and 50 μM) on i) the phenotypic traits of plants, particularly at root level, ii) the nutrient content in both roots and shoots, and iii) the P uptake mechanisms, considering both the biochemical and molecular aspects. At high Cu concentrations (i.e. above 25 μM), the shoot and root growth resulted stunted and the P influx rate diminished. Furthermore, two P transporter genes (i.e. CsPT1.4 and CsPT1.9) were upregulated at the highest Cu concentration, albeit with different induction kinetics. Overall, these results confirm that high Cu concentrations can limit the root acquisition of P, most likely via a direct action on the uptake mechanisms (e.g. transporters). However, the alteration of root plasma membrane permeability induced by Cu toxicity might also play a pivotal role in the observed phenomenon.

Mechanisms of bactericidal action of Eugenol against Escherichia coli

Bioactive compounds exhibit antimicrobial activity against diverse microbial pathogens and can be an alternative to antibiotics to avoid the increasing microbial antibiotic resistance. To understand the mechanism underlying the antibacterial activity, the present study was undertaken using the bioactive compound, eugenol. A wide range of antibacterial activity was demonstrated by eugenol against the test pathogens with MIC ranging from 0.0312 to 8 μg/mL. The corresponding MBC of the compound was 2-4 fold higher. Time kill curve revealed eugenol to effectively reduce E. coli to undetectable levels within 4 h. The compound demonstrated disruptive action on the cytoplasmic membrane as measured by intracellular ATP. An increase in MIC value of the compound in the presence of divalent cations and increased membrane depolarization of the bacterium was observed. The morphological and physiological changes in E. coli were also recorded through SEM and TEM studies. The present study suggests eugenol alters membrane permeability resulting in leakage of intracellular contents. This may further allow the drug to interact with the intracellular sites causing cell damage.

Antibacterial Activity of Lupinifolin from Derris reticulata and Its Effect on Cytoplasmic Membrane of Methicillin Resistant

Lupinifolin from Derris reticulata Craib. was extracted with hexane by Soxhlet extractor and purified by crystallization. The yellow needle-shaped lupinifolin crystals were identified and confirmed by nuclear magnetic resonance (NMR) spectra and Liquid chromatography mass spectrometry (LC/MS). The lupinifolin showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 8 and 16 µg/ml against Methicillin resistant S. aureus (MRSA), respectively. The flow cytometry (FCM) was performed to determine the alteration of cytoplasmic membrane (CM) permeability of MRSA by using Propidium iodide (PI) 5 µg/ml as an indicator for bacterial membrane integrity. It was found that the bacterial CM permeability was effected by lupinifolin with the MIC of 8 µg/ml comparable to the control when investigated by Propidium iodide intensity. Additionally, DNA laddering assay was carried out to evaluate apoptosis in bacterial cells. It was shown that the lupinifolin has no effect on DNA fragmentation.

Fungicide-Tolerant Plant Growth-Promoting Rhizobacteria Mitigate Physiological Disruption of White Radish Caused by Fungicides Used in the Field Cultivation.

Excessive use of fungicides in agriculture may result in substantial accumulation of active residues in soil, which affect crop health and yield. We investigated the response of Raphanus sativus (white radish) to fungicides in soil and potential beneficial interactions of radish plants with fungicide-tolerant plant growth-promoting rhizobacteria (PGPR). The PGPR were isolated from cabbage and mustard rhizospheres. Morphological and biochemical characteristics measured using standard methods, together with analysis of partial 16S rRNA gene sequences, revealed that fungicide-tolerant PGPR, isolates PS3 and AZ2, were closely related to Pseudomonas spp. These PGPR survived in the presence of high fungicide concentrations i.e., up to 2400 μg mL−1 carbendazim (CBZM) and 3200 μg mL−1 hexaconazole (HEXA). Bacterial isolates produced plant growth stimulants even under fungicide stress, though fungicides induced surface morphological distortion and alteration in membrane permeability of these bacteria, which was proved by a set of microscopic observations. Fungicides considerably affected the germination efficiency, growth, and physiological development of R. sativus, but these effects were relieved when inoculated with PGPR isolates. For instance, CBZM at 1500 mg kg−1 decreased whole dry biomass by 71%, whole plant length by 54%, total chlorophyll by 50%, protein content by 61%, and carotenoid production by 29%. After applying isolate AZ2 for white radish grown in CBZM (10 mg kg−1)-amended soil, it could improve plant growth and development with increased whole plant dry weight (10%), entire plant length (13%) and total chlorophyll content (18%). Similarly, isolate PS3 enhanced plant survival by relieving plant stress with declined biomarkers, i.e., proline (12%), malondialdehyde (3%), ascorbate peroxidase (6.5%), catalase (18%), and glutathione reductase (4%). Application of isolates AZ2 and PS3 could be effective for remediation of fungicide-contaminated soil and for improving the cultivation of radish plants while minimizing inputs of fungicides.

Pristimerin isolated from Salacia crassifolia (Mart. Ex. Schult.) G. Don. (Celastraceae) roots as a potential antibacterial agent against Staphylococcus aureus

Ethnopharmacological relevancePristimerin is a triterpenoid considered the main component of Salacia crassifolia extracts. This terpene has shown promising antitumor, anti-inflammatory, and antimicrobial effects. Likewise, S. crassifolia has been used in traditional medicine to treat cancer and as an antimicrobial and anti-inflammatory agent. Aim of the studyThis study aimed to evaluate the antibacterial activity of the hexane extract of Salacia crassifolia roots (HER) and its isolate, pristimerin, against pathogenic bacteria. Materials and methodsFirst, we evaluated the spectrum of action of HER and pristimerin by the determination of the minimum inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). Subsequently, we analyzed the time-kill curve of these plant-derived compounds against Staphylococcus aureus. Then, we examined their mode of action by three different assays: the crystal violet methodology, the release of intracellular material, and transmission electron microscopy methods (TEM). Finally, we evaluated the effect of HER and pristimerin on the pre-formed biofilm of S. aureus by the crystal violet assay, the synergistic effect by the checkerboard method, the cytotoxicity against Vero cells, and the in silico activity using the online software PASS. ResultsHER and pristimerin presented a narrow spectrum of action against Gram-positive bacteria (MIC 0.195–25 μg/mL), and their primary mode of action is the alteration of membrane permeability of S. aureus. Our results show that the compounds disrupted the pre-formed biofilm of S. aureus in a dose-dependent manner. Furthermore, HER and pristimerin presented a significant synergic effect after the combination with well-known antibiotics, which was associated with the ability of these phytomedicines to change membrane permeability. Regarding the cytotoxic effect, the selective index (SI) of HER ranged from 0.37 to 11.86, and the SI of pristimerin varied from 0.24 to 30.87, according to the bacteria tested. ConclusionsOverall, HER and pristimerin showed a promising antibacterial effect in vitro through the alteration of membrane permeability of S. aureus.

Enhanced Chemotherapy for Glioblastoma Multiforme Mediated by Functionalized Graphene Quantum Dots

Glioblastoma is the most aggressive and lethal brain cancer. Current treatments involve surgical resection, radiotherapy and chemotherapy. However, the life expectancy of patients with this disease remains short and chemotherapy leads to severe adverse effects. Furthermore, the presence of the blood–brain barrier (BBB) makes it difficult for drugs to effectively reach the brain. A promising strategy lies in the use of graphene quantum dots (GQDs), which are light-responsive graphene nanoparticles that have shown the capability of crossing the BBB. Here we investigate the effect of GQDs on U87 human glioblastoma cells and primary cortical neurons. Non-functionalized GQDs (NF-GQDs) demonstrated high biocompatibility, while dimethylformamide-functionalized GQDs (DMF-GQDs) showed a toxic effect on both cell lines. The combination of GQDs and the chemotherapeutic agent doxorubicin (Dox) was tested. GQDs exerted a synergistic increase in the efficacy of chemotherapy treatment, specifically on U87 cells. The mechanism underlying this synergy was investigated, and it was found that GQDs can alter membrane permeability in a manner dependent on the surface chemistry, facilitating the uptake of Dox inside U87 cells, but not on cortical neurons. Therefore, experimental evidence indicates that GQDs could be used in a combined therapy against brain cancer, strongly increasing the efficacy of chemotherapy and, at the same time, reducing its dose requirement along with its side effects, thereby improving the life quality of patients.

Altered paracellular permeability in intestinal cell monolayer challenged with lipopolysaccharide: Modulatory effects of pterostilbene metabolites

Epithelial barrier alteration is a central event in the pathogenesis of inflammatory bowel diseases. Lipopolysaccharide, correlated to the pathogenesis of such pathologies, has been demonstrated to cause altered membrane permeability, through the disruption and/or relocation of tight junction proteins, following redox-sensitive mitogen-activated protein kinases (MAPKs) modulation. Pterostilbene and its metabolite pinostilbene are natural stilbenoids which may reach relevant concentrations at intestinal level, together with their glucuronide and sulfate metabolites. The aim of our study was to evaluate the ability of these compounds to inhibit lipopolysaccharide-induced toxic effects on intestinal cell monolayer integrity and to explore the mechanism of action. Caco-2 cells, differentiated as enterocytes, were treated with lipopolysaccharide following pretreatment with the phenolic compounds at 1 μM physiological concentration. Caco-2 monolayer's permeability was monitored with time, measuring the transepithelial electrical resistance. Tight junction proteins were assessed by western blotting and immunofluorescence in lipopolysaccharide-treated cells, in relation to MAPK p38 and ERK1/2 activation. Pretreatment with all the phenolic compounds significantly slowed lipopolysaccharide-induced transepithelial electrical resistance decrease, preserved tight junction proteins levels and reduced MAPKs phosphorylation. The reported findings indicate that pterostilbene and its metabolites may counteract lipopolysaccharide-induced alteration of epithelial permeability, one of the initial events in the intestinal inflammatory process.

Synergistic effect on anti-methicillin-resistant Staphylococcus aureus among combinations of α-mangostin-rich extract, lawsone methyl ether and ampicillin.

α-Mangostin-rich extract (AME) exhibited satisfactory inhibitory activities against all tested MRSA strains, with minimum inhibitory concentrations (MICs) of 7·8-31·25µgml-1 , whereas lawsone methyl ether (LME) and ampicillin revealed weak antibacterial activity with MICs of 62·5-125µgml-1 . However, the combination of AME and LME showed synergistic effects against all tested MRSA strains with fractional inhibitory concentration index (FICI) values of 0·008-0·009, while the combination of AME and ampicillin, as well as LME and ampicillin produced synergistic effects with FICIs of 0·016-0·257. A time-kill assay against MRSA (DMST 20654 strain) revealed a 6-log reduction in CFU per ml, which completely inhibited bacterial growth for the combinations of AME and LME, AME and ampicillin, and LME and ampicillin at a 8-h incubation, while those against MRSA (2468 strain) were at 10-h incubation. The combination of α-mangostin and LME as well as the combinations of each compound with ampicillin synergized the alteration of membrane permeability. In addition, α-mangostin, LME and ampicillin inhibited the biofilm formation of MRSA. These findings indicated that the combinations of AME and LME or each of them in combination with ampicillin had enhanced antibacterial activity against MRSA. Therefore, these compounds might be used as the antibacterial cocktails for treatment of MRSA.

A Noninvasive Method for Assessing Oocyte Competency

There is an increasing demand to evaluate oocyte competency and viability for oocyte cryopreservation and use for in vitro fertilization. Oocytes suffer decreased survival of cryopreservation as compared to embryos, due to physical characteristics of the oocyte. The objective of this study is to determine if a specific gravity device (SGD) can estimate oocyte viability based on oocyte descent through the buoyancy system. All experiments were performed in a research laboratory with a randomized block design with repeated measures. Three hundred-seventy-six oocytes were collected from seven mice and randomly assigned to one of four treatments: exposure to 60C for 30 min, acidified media for 1h, ethylene glycol-glycerol cryoprotectant for 1h, or standard culture to serve as control. To further analyze the relationship between oocyte descent time and viability, 98 additional oocytes were passed through SGD before and after treatment. Oocytes were stained with Coomassie Blue to determine membrane permeability and estimate viability based on treatment. Oocytes treated with 60C heat, acidified media and ethylene glycol/ glycerol cryoprotectant solution demonstrated altered descent times from control and pre-treatment oocytes (P&lt;0.05). Oocytes exposed to heat and cryoprotectants descended more rapidly through SGD than control and pre-treatment oocytes (P&lt;0.05). Oocytes treated with acidified media descended more slowly through SGD (P&lt;0.05). Permeation of stain into oocytes exposed to lethal treatments confirmed changes in membrane integrity post-treatment and further indicates SGD can detect such shifts. This suggests SGD can predict competency between live and dead oocytes. In conclusion, SGD can detect shifts in oocyte density due to altered membrane permeability, which can suggest information about oocyte competency. This information can help differentiate between high- and low-quality fresh oocytes to help select which oocytes to freeze and result in improved oocyte cryopreservation and fertilization.

Mode of action of a formulation containing hydrazones and saponins against leishmania spp. Role in mitochondria, proteases and reinfection process

Toxicity and poor adherence to treatment that favors the generation of resistance in the Leishmania parasites highlight the need to develop better alternatives. Here, we evaluated the in vitro effectiveness of hydrazone derived from chromanes 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (TC1) and 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (TC2) and the mixture of triterpene saponin hederagenin-3-O-(3,4-O-diacetyl-ß-D-xylopyranosyl-(1à3)-a-L- rhamnopyranosyl-(1à2)-a-L-arabinofuranoside, hederagenin-3-O-(3,4-O-diacetyl-a-L- arabinopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside and, hederagenin-3-O-(4-O-acetyl-ß-D-xylopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside from Sapindus saponaria (SS) on L. braziliensis and L. pifanoi. Mixtures of TC1 or TC2 with saponin were formulated for topical application and the therapeutic effectiveness was evaluated in the model for cutaneous leishmaniasis (CL) in golden hamster. The mode of action of these compounds was tested on various parasite processes and ultrastructural parasite modifications. TC1, TC2 and SS showed moderate cytotoxicity when tested independently but toxicity was improved when tested in combination. The compounds were more active against intracellular Leishmania amastigotes. In vivo studies showed that combinations of TC1 or TC2 with SS in 1:1 ratio (w/w) cured 100% of hamsters with no signs associated with toxicity. The compounds did cause changes in the mitochondrial activity of the parasite with a decrease in ATP levels and depolarization of membrane potential and overproduction of reactive oxygen species; nevertheless, these effects were not related to alterations in membrane permeability. The phagolysosome ultrastructure was also affected impacting the survival of Leishmania but the function of the lysosome nor the pH inside the phagolysosome did not change. Lastly, there was a protease inhibition which was directly related to the decrease in the ability of Leishmania to infect and multiply inside the macrophage. The results suggest that the combination of TC1 and TC2 with SS in a 1:1 ratio is capable of curing CL in hamsters. This effect may be due to the ability of these compounds to affect parasite survival and the ability to infect new cells.

Rec A disruption unveils cross talk between DNA repair and membrane damage, efflux pump activity, biofilm formation in Mycobacterium smegmatis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has emerged in recent decades as one of the leading causes of mortality worldwide. The burden of TB is alarmingly high, with one third affected global population as reported by WHO. Short-course treatment with an antibiotic is a powerful weapon to treat infection of susceptible MTB strain, however; MTB has developed resistance to anti-TB drugs, which is an escalating global health crisis. Thus there is urgent need to identify new drug targets. RecA is a 38 kilodalton protein required for the repair and maintenance of DNA and regulation of the SOS response. The objective of this study is to understand the effect of disruption of RecA gene (deletion mutant ΔdisA from previous study) in a surrogate model for MTB, Mycobacterium smegmatis. This study demonstrated that disruption of RecA causes enhanced susceptibility towards rifampicin and generation of ROS leading to lipid peroxidation and impaired membrane homeostasis as depicted by altered cell membrane permeability and efflux pump activity. Mass spectrometry based lipidomic analysis revealed decreased mycolic acid moieties, phosphatidylinositol mannosides (PIM), Phthiocerol dimycocerosate (DIM). Furthermore, biofilm formation was considerably reduced. Additionally, we have validated all the disrupted phenotypes by RT-PCR which showed a good correlation with the biochemical assays. Lastly, RecA mutant displayed reduced infectivity in Caenorhabditis elegans illustrating its vulnerability as antimycobacterial target. Together, present study establishes a link between DNA repair, drug efflux and biofilm formation and validates RecA as an effective drug target. Intricate studies are needed to further understand and exploit this therapeutic opportunity.