Changes In Membrane Integrity Research Articles

3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex.

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block-face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase-quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age-related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age-related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue-dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

An Impedance-Based Approach for Sensing Cyclodextrin-Mediated Modulation of Membrane Cholesterol.

Cyclodextrin molecules are increasingly being used in biological research and as therapeutic agents to alter membrane cholesterol content, yet there is much to learn about their interactions with cell membranes. We present a biomembrane-based organic electronic platform capable of detecting interactions of cell membrane constituents with methyl-β-cyclodextrin (MβCD). This approach enables label-free sensing and quantification of changes in membrane integrity resulting from such interactions. In this work, we employ cholesterol-containing supported lipid bilayers (SLBs) formed on conducting polymer-coated electrodes to investigate how MβCD impacts membrane resistance. By examining the outcomes of MβCD interactions with SLBs of varying cholesterol content, we demonstrate that changes in membrane permeability or resistance can be used as a functional measure for predicting cyclodextrin-mediated cholesterol extraction from cellular membranes. Furthermore, we use the SLB platforms to electronically monitor cholesterol delivery to membranes following exposure to MβCD pre-loaded with cholesterol, observing that cholesterol enrichment is commensurate with an increase in resistance. This biomembrane-based bioelectronic sensing system offers a tool to quantify the modulation of membrane cholesterol content using membrane resistance and provides information regarding MβCD-mediated changes in membrane integrity. Given the importance of membrane integrity for barrier function in cells, such knowledge is essential for our fundamental understanding of MβCD as a membrane cholesterol modulator and therapeutic delivery vehicle.

Estimating the optimal efflux inhibitor concentration of carvacrol as a function of the bacterial physiological state.

Our aim was to find the optimal efflux inhibitor concentration of a natural component, carvacrol, as a function of the physiological state of Escherichia coli. Using fluorescence-based measurements with two strains of E. coli, the effect of carvacrol was assessed at 17 sub-inhibitory concentrations, at which the bacterial efflux mechanism was compromised. The efficacy of carvacrol, as an efflux inhibitor, was compared to synthetic inhibitors and we found carvacrol the most efficient one. We considered the accumulation of Ethidium Bromide (EtBr) as a proxy for drugs spreading in the cell, thus measuring the efflux activity indirectly. The change in membrane integrity caused by the exposure to carvacrol was monitored using the LIVE/DEAD BacLight Bacterial Viability kit. To find the optimal inhibitory concentration of carvacrol, we used predictive microbiology methods. This optimum varied with the bacterial physiological state, as non-growing cultures were less susceptible to the effect of carvacrol than growing cultures were. Moreover, we point out, for the first time, that the efflux-mediated resistance of untreated cultures was also stronger in the non-growing than in the growing phase at population level.

Cell mechanical responses to subcellular perturbations generated by ultrasound and targeted microbubbles

The inherently dynamic and anisotropic microenvironment of cells imposes not only global and slow physical stimulations on cells but also acute and local perturbations. However, cell mechanical responses to transient subcellular physical signals remain unclear. In this study, acoustically activated targeted microbubbles were used to exert mechanical perturbations to single cells. The cellular contractile force was sensed by elastic micropillar arrays, while the pillar deformations were imaged using brightfield high-speed video microscopy at a frame rate of 1k frames per second for the first 10s and then confocal fluorescence microscopy. Cell mechanical responses are accompanied by cell membrane integrity changes. Both processes are determined by the perturbation strength generated by microbubble volumetric oscillations. The instantaneous cellular traction force relaxation exhibits two distinct patterns, correlated with two cell fates (survival or permanent damage). The mathematical modeling unveils that force-induced actomyosin disassembly leads to gradual traction force relaxation in the first few seconds. The perturbation may also influence the far end subcellular regions from the microbubbles and may propagate into connected cells with attenuations and delays. This study carefully characterizes the cell mechanical responses to local perturbations induced by ultrasound and microbubbles, advancing our understanding of the fundamentals of cell mechano-sensing, -responsiveness, and -transduction. Statement of significanceSubcellular physical perturbations commonly exist but haven't been fully explored yet. The subcellular perturbation generated by ultrasound and targeted microbubbles covers a wide range of strength, from mild, intermediate to intense, providing a broad biomedical relevance. With µm2 spatial sensing ability and up to 1ms temporal resolution, we present spatiotemporal details of the instantaneous cellular contractile force changes followed by attenuated and delayed global responses. The correlation between the cell mechanical responses and cell fates highlights the important role of the instantaneous mechanical responses in the entire cellular reactive processes. Supported by mathematical modeling, our work provides new insights into the dynamics and mechanisms of cell mechanics.

Effect of Thioketal Antioxidants on Islet Cell Transplantation

AbstractPancreatic islet transplantation is an effective strategy for restoring glucose regulation for highly selected patients with type 1 diabetes. However, an undesirable level of islet cell death is caused by oxidative stress that occurs during islet isolation, culture, and transplantation. The loss of islets throughout the transplantation procedure often necessitates multiple human donors per recipient to achieve insulin independence. Administration of exogenous antioxidants has shown promise in both preserving islet cell viability and functionality post‐transplantation. Herein, thioketal (TK) antioxidant is evaluated using neonatal porcine islets and Beta‐TC‐6 cells exposed to H2O2 for changes in membrane integrity, oxygen consumption rates, and lipid peroxidation. Diabetic BALB/c mice are transplanted with a marginal dose of syngeneic islets, ±48‐h TK pre‐treatment, under the kidney capsule. Graft function is measured by nonfasting blood glucose and glucose tolerance testing. It is found that 200 µM of TK was not cytotoxic, reduced reactive oxygen species‐mediated oxidative islet damage, preserved in vitro islet cell functionality, and facilitated superior murine syngeneic marginal islet mass engraftment. These results demonstrate that the antioxidant attributes of TK reduce the deleterious effects of reactive oxygen species experienced in vitro and enhance marginal islet mass transplant outcomes.

Single-Cell Level Measurements of Photocatalytic Damage to Escherichia coli Cells

The increase in water demand due to the increasing population and the addition of emerging contaminants like drug-resistant bacteria, pharmaceuticals, and treatment-resistant chemicals such as PFAS are straining the current water treatment infrastructure. This straining infrastructure could be complemented by photocatalysis and other Advanced oxidation processes (AOP) [1] [2]. Photocatalytic water treatment methods, specifically, have generated a lot of interest due to their ability in eliminating pollutants like dyes and other toxic organic pollutants as well as eliminating pathogens such as bacteria, fungi, and viruses while minimizing the use of material resources. The elimination of bacteria by photocatalysis has been reported widely, with many reports focusing on the kinetics of photocatalytic disinfection, as well as some aspects of process scale-up. However, the mechanism for photocatalytic damage to the bacterial cells has thus far proven difficult to study [3] [4].It is critical to determine the mechanisms of photocatalytic disinfection for the development of better methods of catalyst discovery and reactor design. The mechanisms can be determined if quantitative data on how single bacterial cells interact with catalysts is available. However, experimental measurements at a single-cell level are relatively few and assays for evaluating photocatalyst performance in real-time have not been developed to our knowledge. In this work, we addressed these shortcomings with a combination of light microscopy and particle-tracking algorithms. Using nanowire-shaped titanium dioxide (TiO2) catalyst particles, which maintain a high surface area while remaining visible using the established techniques for light microscopy, we investigated the interactions between catalyst particles and bacteria. We quantified cell motility during the course of photocatalytic disinfection as a loss in motility is expected to faithfully track damage to the membrane [5]. We further correlated the kinetics of antibacterial action of the photocatalytic treatment in E. coli with real-time changes in membrane integrity. Our investigations are anticipated to reveal the mechanisms by which photocatalytic treatments cause cell damage. Such single-cell approaches are expected to help build libraries of photocatalysts and optimize the use of antibacterial agents against superbugs, with a range of future applications in water-disinfection reactors to hospitals.REFERENCES[1] X. Qu, J. Brame, Q. Li, P.J. Alvarez, Nanotechnology for a safe and sustainable water supply: enabling integrated water treatment and reuse, Accounts of chemical research, 46 (2013) 834-843.[2] A. Boretti, L. Rosa, Reassessing the projections of the world water development report, NPJ Clean Water, 2 (2019) 1-6.[3] O.K. Dalrymple, E. Stefanakos, M.A. Trotz, D.Y. Goswami, A review of the mechanisms asnd modeling of photocatalytic disinfection, Applied Catalysis B: Environmental, 98 (2010) 27-38.[4] H.A. Foster, I.B. Ditta, S. Varghese, A. Steele, Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity, Applied microbiology and biotechnology, 90 (2011) 1847-1868.[5] R. Gupta, K. Y. Rhee, S. D. Beagle, R. Chawla, N. Perdomo, S. W. Lockless and P. P. Lele, Indole modulates cooperative protein-protein interactions in the flagellar motor, PNAS Nexus (2022)

Insights into the antibacterial mechanism of ozone water combined with tea polyphenols against Shewanella putrefaciens: membrane disruption and oxidative stress

SummaryShewanella putrefaciens (S. putrefaciens) is the main specific spoilage organism (SSO) of marine fish during cold logistic process. To explore the antimicrobial mechanism of ozone water combined with tea polyphenol (TP) against S. putrefaciens, the growth condition, endogenous oxidative stress, changes of membrane integrity and energy metabolism enzyme were evaluated, respectively. As evidenced by the growth curve and confocal laser scanning microscopy (CLSM) analysis, bacterial growth was inhibited, and death threat occurred. The increase of reactive oxygen species (ROS) suggested intracellular oxidative stress was activated. Additionally, membrane integrity and metabolism functions were destroyed according to the results of alkaline phosphatase (AKPase), β‐galactosidase, transmembrane electrical potential, scanning electron microscope (SEM), adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDHase). In summary, the inactivation of S. putrefaciens could be induced by the combination of ozone water and TP, which provided a theoretical basis for the sterilisation in aquatic products.

Functional enhancement of hydroxypropyl cellulose‐based bionanocomposite films incorporating chitosan nanoparticles

SummaryThe aims of the present study were to investigate the effect of the incorporation of chitosan nanoparticles (ChiNPs) on the physicochemical properties and antifungal activity of hydroxypropyl cellulose (HPC) bionanocomposite films. The films of 4% HPC and ChiNP at various dosages (0%, 0.1%, 0.5%, 1%, 1.5%, 3% w/w of HPC) were prepared. Atomic force microscopy (AFM) verified the dispersion of spherical‐shaped ChiNP in a nanoscale size range (14.75–56.74 nm). Incorporation of nanofiller affected the functional properties by increasing the film–water contact angle to >53°, improving the optical properties, and reducing water vapour permeability by 5.73–12.54%. These results were influenced by surface roughness and microstructure characteristics of a bionanocomposite film, which were confirmed using AFM and scanning electron microscopy (SEM). Tensile assays of the films showed slight increases in tensile strength from 8.87 to 11.08 MPa and Young's modulus from 0.10 to 0.15 GPa with increasing proportions of ChiNP (1.5%). An effective concentration of ChiNP of 1.5% (w/w of HPC) was able to improve the performance of HPC and offered antifungal activity against Botrytis cinerea, as confirmed through mycelial growth inhibition assays and by membrane integrity changes. The HPC/ChiNP bionanocomposite films may have potential applications for active packaging, particularly for fresh fruit commodities.

Biopolymer gels as components of protective medium for cryopreservation of spermatogonial stem cells.

Biopolymer gels attract a lot of attention in a field of biothechnology due to their excellent compatibility and degradation. Their application is also promising for cryopreservation of spermatogonial stem cells (SSCs) which is so necessary to preserve the fertility of young patients. The aim of the study was to determine the effectiveness of biopolymer gels as a component of cryopreservation medium for SSCs of immature rats at the stage of exposure to cryoprotectants. It was found that 30-min exposure to cryopreservation media based on collagen or fibrin gel with an addition of 5% Me2SO or 6% glycerol did not lead to significant changes in membrane integrity, cytochrome C content, metabolic, mitochondrial and antioxidant activities in SSCs compared to the control (Leibovitz-based cryomedium). But fibrin gel more than collagen reduced the toxic effects of Me2SO and glycerol on SSCs increasing exposure time up to 45 min without significant changes in cell viability. The same cryoprotectants in Leibovitz-based media showed significant toxicity starting from the 15th minute of exposure. Necrosis was the main cause of cell death at this stage of cryopreservation in all experimental groups. The obtained results can be used to optimize SSC cryopreservation protocols for further cell autotransplantation for spermatogenesis initiation in boys who undergo gonadotoxic therapy in prepubertal age.

The Physiological and Biochemical Response of Field Bean (Vicia faba L. (partim)) to Electromagnetic Field Exposure Is Influenced by Seed Age, Light Conditions, and Growth Media

Research interest into the exposure of plants to magnetic fields (MF), including electromagnetic fields (EMF), has increased recently but results often vary depending on factors such as plant species and treatment dose. In this study, we exposed young (one year) and old (four years) field bean (Vicia faba L. (partim)) seeds to EMF (50 Hz, 7 mT) and observed seed germination and seedling growth under different conditions (growth media and light). The results indicated a stimulation by EMF of germination and early root growth of Petri dish-sown old seeds in continuous darkness and inhibition of germination of the pot-sown young seeds under long-day conditions. Root growth of two-week-old seedlings from pot-sown young seeds was stimulated by EMF treatment while their stem growth was inhibited. Some selected biochemical traits were examined, showing specific changes in membrane integrity, amylase activity, H2O2 levels, photosynthetic pigments, and content of the main groups of phytohormones, depending on seed age. The results indicate that priming of field bean seeds with EMF (50 Hz, 7 mT) could be a eustress factor that influences germination, early growth, and cellular activities and could positively influence the ability of field bean plants to grow and develop in more stressful conditions at later stages.

In vitro antibacterial activities of silver nanoparticles synthesised using the seed extracts of three varieties of Phoenix dactylifera.

Green synthesis of ‏silver nanoparticles (AgNPs) is an ecofriendly, cost-effective and promising approach for discovery of novel therapeutics. The aim of the current work was to biogenic synthesize, characterize AgNPs using seed extracts of three economically important varieties of date palm (Iklas, Irziz and Shishi), and assess their anti-pathogenic bacterial activities. AgNPs were synthesised then characterised using electron microscopy and Fourier transform infrared analyses. The bactericidal activities of AgNPs against five different bacterial pathogens, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae, were determined in vitro. In particular, changes in membrane integrity of virulent bacterial strains in response to AgNPs were investigated. Results of lactate dehydrogenase, alkaline phosphatase activity assays, and measurement of membrane potential revealed that the cytotoxic effects of the AgNPs were mainly centred on the plasma membrane of bacterial cells, leading to loss of its integrity and eventually cell death. In conclusion, green synthesis of AgNPs is an efficient, cost-effective and promising strategy to combat virulent antibiotic-resistant strains.

Antifungal features and properties of chitosan/sandalwood oil Pickering emulsion coating stabilized by appropriate cellulose nanofiber dosage for fresh fruit application

A novel composite edible coating film was developed from 0.8% chitosan (CS) and 0.5% sandalwood oil (SEO). Cellulose nanofibers (CNFs) were used as a stabilizer agent of oil-in-water Pickering emulsion. We found four typical groups of CNF level-dependent emulsion stabilization, including (1) unstable emulsion in the absence of CNFs; (2) unstable emulsion (0.006–0.21% CNFs); (3) stable emulsion (0.24–0.31% CNFs); and (4) regular emulsion with the addition of surfactant. Confocal laser scanning microscopy was performed to reveal the characteristics of droplet diameter and morphology. Antifungal tests against Botrytis cinerea and Penicillium digitatum, between emulsion coating stabilized with CNFs (CS-SEOpick) and CS or CS-SEO was tested. The effective concentration of CNFs (0.24%) may improve the performance of CS coating and maintain CS-SEO antifungal activity synergistically confirmed with a series of assays (in vitro, in vivo, and membrane integrity changes). The incorporation of CNFs contributed to improve the functional properties of CS and SEO-loaded CS including light transmission at UV and visible light wavelengths and tensile strength. Atomic force microscopy and scanning electron microscopy were employed to characterize the biocompatibility of each coating film formulation. Emulsion-CNF stabilized coating may have potential applications for active coating for fresh fruit commodities.

Tethered Lipid Membranes as a Nanoscale Arrangement towards Non-Invasive Analysis of Acute Pancreatitis.

Extracellular heat shock proteins (HSPs) mediate immunological functions and are involved in pathologies such as infection, stress, and cancer. Here, we demonstrated the dependence of an amount of HSP70 and HSP90 in serum vs. severity of acute pancreatitis (AP) on a cohort of 49 patients. Tethered bilayer lipid membranes (tBLMs) have been developed to investigate HSPs’ interactions with tBLMs that can be probed by electrochemical impedance spectroscopy (EIS). The results revealed that HSP70 and HSP90 interact via different mechanisms. HSP70 shows the damage of the membrane, while HSP90 increases the insulation properties of tBLM. These findings provide evidence that EIS offers a novel approach for the study of the changes in membrane integrity induced by HSPs proteins. Herein, we present an alternative electrochemical technique, without any immunoprobes, that allows for the monitoring of HSPs on nanoscaled tBLM arrangement in biologics samples such us human urine. This study demonstrates the great potential of tBLM to be used as a membrane based biosensor for novel, simple, and non-invasive label-free analytical system for the prediction of AP severity.

Carvacrol exhibits rapid bactericidal activity against Streptococcus pyogenes through cell membrane damage

Streptococcus pyogenes is an important human pathogen worldwide. The identification of natural antibacterial phytochemicals has renewed interest due to the current scarcity of antibiotic development. Carvacrol is a monoterpenoid found in herbs. We evaluated carvacrol alone and combined with selected antibiotics against four strains of S. pyogenes in vitro. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvacrol against S. pyogenes were 125 µg/mL (0.53 mM) and 250 µg/mL (1.05 mM), respectively. Kill curve results showed that carvacrol exhibits instantaneous bactericidal activity against S. pyogenes. We also demonstrated the potential mechanism of action of carvacrol through compromising the cell membrane integrity. Carvacrol induced membrane integrity changes leading to leakage of cytoplasmic content such as lactate dehydrogenase enzymes and nucleic acids. We further confirmed dose-dependent rupturing of cells and cell deaths using transmission electron microscopy. The chequerboard assay results showed that carvacrol possesses an additive-synergistic effect with clindamycin or penicillin. Carvacrol alone, combined with clindamycin or penicillin, can be used as a safe and efficacious natural health product for managing streptococcal pharyngitis.

Inhibition of Botrytis cinerea by alginate/cajuput essential oil and the composite’s surface properties as potential antifungal coating

Inhibition of B. cinerea by alginate (Alg) / cajuput essential oil (Caj) at different concentration (0%, 0.25%, 0.5%, 0.75%) and the composite’s surface properties were investigated. Among the samples, the treated spore with Caj 0.75% showed the lowest germination percentage and germ tube length. The regarded antifungal action was confirmed by observing the changes of membrane integrity stained with propidium iodide under fluorescence microscope. Moreover, the film composite roughness was characterized which Caj affected significantly (P < 0.05) on arithmetical mean high of Alg based film. Hence, the results of the present study suggest that Alg - Caj blended coating could be an alternative of natural coating agents applied on fruit or vegetable.

Urban PM2.5 Induces Cellular Toxicity, Hormone Dysregulation, Oxidative Damage, Inflammation, and Mitochondrial Interference

Epidemiological studies have found air pollution to be a driver of adverse pregnancy outcomes but biological mechanisms are not clear. A first trimester trophoblast cell line (HTR-8/SVneo) was exposed to various concentrations of PM2.5 in order to elucidate the effect of urban particulate matter (PM) of size &lt;2.5 μm on placental function. PM2.5 were collected at a site representative of urban traffic and dispersed in cell media by indirect and direct sonication. The HTR-8 cells were grown under standard conditions. Cellular uptake was studied after 24 and 48 h of exposure by transmission electron microscopy (TEM). The secretion of human chorionic gonadotropin (hCG), progesterone, and Interleukin-6 (IL-6) was measured by ELISA. Changes in membrane integrity and H2O2 production were analyzed using the CellToxTM Green Cytotoxicity and ROSGloTM assays. Protease activity was evaluated by MitoToxTM assay. Mitochondrial function was assessed through high resolution respirometry in an Oroboros O2k-FluoRespirometer, and mitochondrial content was quantified by citrate synthase activity. Results: TEM analysis depicted PM2.5 cellular uptake and localization of the PM2.5 to the mitochondria after 24 h. The cells showed aggregated cytoskeleton and generalized necrotic appearance, such as chromatin condensation, organelle swelling and signs of lost membrane integrity. The mitochondria displayed vacuolization and disruption of cristae morphology. At 48 h exposure, a significant drop in hCG secretion and a significant increase in progesterone secretion and IL-6 production occurred. At 48 h exposure, a five-fold increase in protease activity and a significant alteration of H2O2 production was observed. The HTR-8 cells exhibited evidence of increased cytotoxicity with increasing exposure time and dose of PM2.5. Conclusion: PM may contribute to the placental dysfunction associated with pregnancy outcomes, such as preeclampsia and intrauterine growth restriction, through their direct and indirect effects on trophoblast protein secretion, hormone regulation, inflammatory response, and mitochondrial interference.

One-pot synthesis of natural amine-modified biocompatible carbon quantum dots with antibacterial activity

In the present study, the thermal decomposition of citric acid in the presence of biogenic amine was used to synthesize four different functionalized carbon quantum dots (CQDs), namely, histamine-(HCQDs), putrescine-(PCQDs), cadaverine-(CCQDs) and spermine-(SCQDs). The thermal decomposition of the precursors resulted in a decrease in stability and the formation of surface amides via a cross-linking process between the carboxyl and amine groups. The deposition of biogenic amines was confirmed by a structural characterization of the synthesized CQDs. The resulting CQDs, with a net zero charge, exhibited excellent stability in environments with different pH values. Through a set of different cytotoxicity tests, the absence of gene mutations, apoptosis, necrosis or disruption in cell membranes revealed the high biocompatibility of the CQDs. The antimicrobial activity of the synthesized CQDs was investigated against different bacterial species (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia). We determined the growth kinetics, production of reactive oxygen species (ROS), cell viability and changes in membrane integrity by scanning electron microscopy (SEM). The minimal inhibitory concentrations (MICs) for S. aureus ranged from 3.4 to 6.9 µg/mL. Regarding E.coli and K. pneumonia, all CQD formulations reduced growth, and the MICs were determined for CCQDs and HCQDs (6.9–19.4 µg/mL). The antibacterial activity mechanism was attributed to the oxidative stress generated after CQD treatment, which resulted in the destabilization of the bacterial membrane. The bacterial permeability to propidium iodide indicated a change in membrane integrity, and the effect of CQDs on the morphology of the bacterial cells was evidenced by SEM.

Water availability effects on germination, membrane stability and initial root growth of Agave lechuguilla and A. salmiana

Water availability during seed germination and seedling development is critical to plant development and establishment. The aim of this study was to determine the effects of four water potentials (ΨW) (−0.03, −0.48, −1.19 and −2.04 MPa) on seed germination, seedling root growth and root membrane stability of Agave lechuguilla Torr. and A. salmiana Otto ex Salm-Dyck. to explain the paradoxical situation of vegetatively propagated species producing large quantities of seeds in a dry climate. The results showed that seed water uptake and germination percentage were significantly lower at −1.19 and −2.04 MPa than at −0.03 MPa. The time to reach maximum seed germination increased at low ΨW. The final root length and root growth rate were higher in A. salmiana than in A. lechuguilla and decreased at ΨW of −1.19 and −2.04 MPa. Both Agave species increased root electrolyte leakage at ΨW of −1.19 MPa, which suggests changes in membrane integrity and stability in response to the water deficit. These germination and initial root growth responses could ensure the establishment of both Agave species during the rare periods with sufficient moisture availability and allow them to tolerate initial periods of low water availability, although with low establishment probability.

Antibacterial activity and mechanism of action saponins from Chenopodium quinoa Willd. husks against foodborne pathogenic bacteria

The present study investigated the chemical composition of quinoa saponins and examined their possible antimicrobial modes of action against foodborne pathogenic bacteria.Six different compounds (Q1-Q6) were extracted from quinoa husks, separated/purified by column chromatography and identified by HPLC/MS and NMR. The anti-bactericidal effects against six types of bacteria, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Salmonella enteritidis, Pseudomonas aeruginosa, and Listeria ivanovii, were determined by the disk diffusion method and assessment of the MIC/MBC. The changes in membrane integrity were tested using a microplate reader, a flow cytometer, and a transmission electron microscopy.All the compounds exerted anti-bactericidal effects against S. aureus, S. epidermidis and B. cereus. Q4 showed the strongest activity against S. aureus and S. epidermidis, with an MIC value of 0.0625 mg/mL and an MBC value of 0.125 mg/mL. The concentration of quinoa saponin Q4 exhibited a dose-dependent relationship with its anti-bactericidal effect. In fact, a dose-effect relationship was found between the concentration of all quinoa saponins and their bacteriostatic effects. In addition, the release of nucleic acids and proteins from B. cereus increased gradually with increases in the Q4 concentration, and the membrane structure of B. cereus was destroyed by Q4.Notably, quinoa saponins caused severe damage to the tested bacteria through degradation of the cell wall followed by disruption of the cytoplasmic membrane and membrane proteins, which resulted in leakage of the cell contents.

Urban PM2.5 Induces Cellular Toxicity, Hormone Dysregulation, Oxidative Damage, Inflammation, and Mitochondrial Interference in the HRT8 Trophoblast Cell Line.

Objective: Epidemiological studies have found air pollution to be a driver of adverse pregnancy outcomes, including gestational diabetes, low term birth weight and preeclampsia. It is unknown what biological mechanisms are involved in this process. A first trimester trophoblast cell line (HTR-8/SVneo) was exposed to various concentrations of PM2.5 (PM2.5) in order to elucidate the effect of urban particulate matter (PM) of size <2.5 μm on placental function.Methods: PM2.5 were collected at a site representative of urban traffic and dispersed in cell media by indirect and direct sonication. The HTR-8 cells were grown under standard conditions. Cellular uptake was studied after 24 and 48 h of exposure by transmission electron microscopy (TEM). The secretion of human chorionic gonadotropin (hCG), progesterone, and Interleukin-6 (IL-6) was measured by ELISA. Changes in membrane integrity and H2O2 production were analyzed using the CellToxTM Green Cytotoxicity and ROSGloTM assays. Protease activity was evaluated by MitoToxTM assay. Mitochondrial function was assessed through high resolution respirometry in an Oroboros O2k-FluoRespirometer, and mitochondrial content was quantified by citrate synthase activity.Results: TEM analysis depicted PM2.5 cellular uptake and localization of the PM2.5 to the mitochondria after 24 h. The cells showed aggregated cytoskeleton and generalized necrotic appearance, such as chromatin condensation, organelle swelling and signs of lost membrane integrity. The mitochondria displayed vacuolization and disruption of cristae morphology. At 48 h exposure, a significant drop in hCG secretion and a significant increase in progesterone secretion and IL-6 production occurred. At 48 h exposure, a five-fold increase in protease activity and a significant alteration of H2O2 production was observed. The HTR-8 cells exhibited evidence of increased cytotoxicity with increasing exposure time and dose of PM2.5. No significant difference in mitochondrial respiration or mitochondrial mass could be demonstrated.Conclusion: Following exposure to air pollution, intracellular accumulation of PM may contribute to the placental dysfunction associated with pregnancy outcomes, such as preeclampsia and intrauterine growth restriction, through their direct and indirect effects on trophoblast protein secretion, hormone regulation, inflammatory response, and mitochondrial interference.