Membrane Breakdown Research Articles

On-Chip Single-Cell Bioelectrical Analysis for Identification of Cell Electrical Phenotyping in Response to Sequential Electric Signal Modulation.

In recent years, an interesting biomarker called membrane breakdown voltage has been examined using artificial planar lipid bilayers. Even though they have great potential to identify cell electrical phenotyping for distinguishing similar cell lines or cells under different physiological conditions, the biomarker has not been evaluated in the context of living cell electrical phenotyping. Herein, we present a single-cell analysis platform to continuously measure the electric response in a large number of cells in parallel using electric frequency and voltage variables. Using this platform, we measured the direction of cell displacement and transparent cell image alteration as electric polarization of the cell responds to signal modulation, extracting the dielectrophoretic crossover frequency and membrane breakdown voltage for each cell, and utilizing the measurement results in the same spatiotemporal environment. We developed paired parameters using the dielectrophoretic crossover frequency and membrane breakdown voltage for each cell and evaluated the paired parameter efficiency concerning the identification of two different breast cancer cells and cell drug response. Moreover, we showed that the platform was able to identify cell electrical phenotyping, which was generated by subtle changes in cholesterol depletion-induced cell membrane integrity disruption when the paired parameter was used. Our platform introduced in this paper is extremely useful for facilitating more accurate and efficient evaluation of cell electrical phenotyping in a variety of applications, such as cell biology and drug discovery.

Preprint Highlight: Mitotic clustering of pulverized chromosomes from micronuclei.

Chromosomes in micronuclei are fragmented into small pieces, sometimes catastrophically. Such severely damaged chromosomes can lead to genomic rearrangements, but how the chromosome fragments segregate during mitosis is unknown. Using live-cell imaging to follow micronuclei during mitosis, this study shows that chromosome fragments in micronuclei cluster together during mitosis and segregate into one daughter cell. The authors found that the DNA damage response complex CIP2A-TOPBP1 is responsible for clustering chromosome fragments by binding to the damaged DNA after micronuclei membrane breakdown. This work suggests that biased segregation of chromosome fragments following micronucleation could explain the lack of DNA copy number errors in various cancers.

Changes in α-Farnesene and Phenolic Metabolism and the Expression of Associated Genes during the Development of Superficial Scald in Two Distinct Pear Cultivars.

Superficial scald is a postharvest physiological disorder that occurs in pear during and after cold storage. In this study, the superficial scald index; α-farnesene and its oxidation products, conjugated trienols (CTols); phenolic content; and the expression of its related genes were investigated in two different pear cultivars, ‘Wujiuxiang’ (Pyrus communis L.) and ‘Yali’ (Pyrus bretschneideri R.), following 115 days of cold storage at 0 °C followed by 7 days of shelf life at 20 °C. The results indicated that the superficial scald occurred after 115 days of cold storage and became more severe during the shelf life of the ‘Wujiuxiang’ pear, whereas no scald was observed in ‘Yali’. The α-farnesene levels increased rapidly at first and then decreased, while the CTols contents increased significantly in ‘Wujiuxiang’ as compared to ‘Yali’, and the expression levels of the genes involved in α-farnesene and CTols metabolism (HMGR1, HMGR2, GSTU7, GPX5, and GPX6), as well as the phenolic synthesis (PAL1, PAL2, C4H1, 4CL2, C3H, and ANR) of the peel, were significantly up-regulated at the onset of the superficial scald. In addition, the relative conductivity and contents of catechin and epicatechin were higher, and the expression level of the laccase gene (LAC7) significantly increased with the development of superficial scald, while lower contents of chlorogenic acid, arbutin, and isorhamnetin-3-3-glucoside, as well as the lower expression levels of a phenolic-synthesis-related gene (C4H3) and polyphenol oxidase genes (PPO1 and PPO5), were noticed in ‘Wujiuxiang’ as compared to ‘Yali’. The results indicated that the onset and progression of superficial scald were associated with the accumulation of CTols, cell membrane breakdown, and higher catechin, epicatechin, and rutin contents, as well as the expression of associated genes of the peels of pear fruit.

Functional Ultrastructure of Hymenopteran Stingers: Devastating Spear or Delicate Syringe.

In this study, we tested the hypothesis that a micro-serrated edge on the honey bee Apis mellifera stinger tip serves as a tool for more intensive crushing of cell membranes in the victim's tissues. This could have mechanical consequences as well as initiate metabolic pathways linked to cell membrane breakdown (e.g., production of biogenic amines). Accordingly, we found that hymenopteran species that use their stingers as an offensive or defensive weapon to do as much damage to the victim's body as possible had this cuticular microstructure. In parasitic hymenopterans, on the other hand, this structure was missing, as stingers are solely used to delicately transport venom to the victim's body in order to do little mechanical harm. We also demonstrated that the stinger lancets of the honey bee A. mellifera are living organs with sensilla innervated by sensory neurons and containing other essential tissues, rather than mere cuticular structures.

In Vitro anti-metastasis of Perilla frutescens leaf water extract on aggressive human breast cancer cells

Background: Perilla frutescens is a long-established plant that is often used in foods and traditional medicines in Asian countries. The perilla leaf contains a considerable number of bioactive substances, such as phenolics and flavonoids, which have been demonstrated to possess anticancer activity in vitro and in vivo. Objectives: We aimed to study anti-metastatic activity, anti-invasion activity, and anti-migration activity of perilla leaf water extract (PLW) at 90°C for 1-5 min in MDA-MB-231 aggressive human breast cancer cells. Materials and methods:Dry perilla leaves were extracted using hot water for 1-5 min to obtain crude extract and then lyophilized for PLW powder. PLW was evaluated for total phenolic, total flavonoid, and rosmarinic acid (RA) contents by Folin-Ciocalteau reagent, aluminum chloride colorimetric assay, and ultra-high-pressure liquid chromatography, respectively. Antioxidant activity of PLW was determined by DPPH and ABTS assays. MTT assay was performed to evaluate the cytotoxicity of PLW on MDA-MB-231 cells. Effective PLW was further determined its inhibitory effect on human breast cancer cell metastasis by a Boyden chamber-based transmembrane assay, the MMP-9 activity, and the proteolytic type IV collagenase activity. Results: PLW by 5-min infusion showed the highest amount of total phenolic and flavonoid contents, as well as RA. Moreover, by the 5-min infusion, PLW had the highest antioxidant capacity when compared to PLW by infusions for 1-4 min. Following that, cytotoxicity testing revealed that the PLW is not toxic to MDA-MB-231 cells after a 24-hr exposure. The PLW at non-toxic doses (12.5-100 µg/mL) intensely presents an inhibitory effect on cell invasion and migration. The gelatinolytic activity showed that the PLW at concentrations of 12.5-100 µg/mL decreases MMP-9 activity in a dose-related manner. Furthermore, after treatment with the PLW, the proteolytic type IV collagenase activity was reduced considerably in a dose-related manner. Conclusion: Our findings further showed that the PLW samples inhibit proteolytic enzymes involved in basement membrane breakdown, which might explain the anti-invasion and anti-migration properties of breast cancer cells. From the result, the application of perilla leaf might be developed as an herbal tea and used as an anti-metastatic agent for breast cancer prevention and treatment.

Spatiotemporal analysis of axonal autophagosome-lysosome dynamics reveals limited fusion events and slow maturation.

Macroautophagy is a homeostatic process required to clear cellular waste. Neuronal autophagosomes form constitutively in the distal tip of the axon and are actively transported toward the soma, with cargo degradation initiated en route. Cargo turnover requires autophagosomes to fuse with lysosomes to acquire degradative enzymes; however, directly imaging these fusion events in the axon is impractical. Here we use a quantitative model, parameterized and validated using data from primary hippocampal neurons, to explore the autophagosome maturation process. We demonstrate that retrograde autophagosome motility is independent of fusion and that most autophagosomes fuse with only a few lysosomes during axonal transport. Our results indicate that breakdown of the inner autophagosomal membrane is much slower in neurons than in nonneuronal cell types, highlighting the importance of this late maturation step. Together, rigorous quantitative measurements and mathematical modeling elucidate the dynamics of autophagosome-lysosome interaction and autophagosomal maturation in the axon.

Effect of pulsed electric field treatment on cell-membrane permeabilization of potato tissue and the quality of French fries

The effect of pulsed electric field (PEF) treatment on the cell membrane permeabilization of potato tissue and the quality of French fries was investigated. Pulses with an electric field strength of 0.5, 1.5, and 2.5 kV/cm and a width of 20 μs were applied to the potato. PEF treatment permeabilized the membrane of potato cells. The magnitude of cell-membrane permeabilization was estimated by ion leaching and biological impedance tests and verified by microscopic observation. As the PEF field strength increased, the accumulation of neutral red dye decreased due to increased cell rupture. The index Z-values (relative cell membrane breakdown values) for 0.5-, 1.5-, and 2.5-kV/cm PEF-treated samples were 0.01, 0.28, and 0.52, respectively. PEF treatment at 2.5 kV/cm reduced the cutting force of potatoes by 33%; it also increased the degree of the crispness of French fries by 64% and decreased crude fat content by 28%. The total reducing sugar content was decreased by PEF treatment, which could be attributed to increased lightness and yellowness after frying. Therefore, PEF treatment improved the quality of French fries by increasing crispness, improving color, and reducing crude fat content.

Alleviation of pulp breakdown in harvested longan fruit by acidic electrolyzed water in relation to membrane lipid metabolism

This study investigated the effect of acidic electrolyzed water (AEW) on the inhibition of pulp breakdown of postharvest longan fruit. The harvested longan fruit were immersed in AEW (pH 2.5 and available chlorine concentration 80 mg L−1) for 10 min, then air-dried and stored at 25 °C. It was revealed that the AEW treatment reduced the cell membrane permeability and breakdown incidence of pulp by modulating membrane lipid metabolism. Compared to the untreated fruit, AEW treatment significantly suppressed the activities of lipoxygenase (LOX), lipase, and phospholipase D (PLD), down-regulated the expression of DlPLD1, DlPLD2, Dllipase1, Dllipase2, DlLOX1 and DlLOX2, and reduced the hydrolysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) and the accumulation of phosphatidic acid (PA). In addition, a lower saturated fatty acids (SFAs) proportion, but a higher unsaturated fatty acids (USFAs) proportion, the index of unsaturated fatty (IUFA), and unsaturated to saturated fatty acids ratio (U/S) were found in AEW-treated longan fruit. These findings indicated that AEW treatment inhibited the activities of membrane lipid metabolism-related enzymes (LOX, lipase, and PLD) and their expressions of associated genes, and altered the cell membrane lipid composition, which was conductive to maintaining the integrity of cell membrane, ultimately alleviating the progress of pulp breakdown in harvested longan fruit. Consequently, AEW treatment may be a reliable method to reduce the occurrence of pulp breakdown and improve the storability of harvested longan fruit.

(Digital Presentation) Fabrication and Electrochemical Characterization of Inkjet Printed IrO2 Electrodes for Water Electrolysis

Water electrolysis is believed to be one of the most promising technologies to produce green hydrogen. However, the current challenge in commercialising this technology is the high relative cost. According to a recent report on cost breakdown of Polymer Electrolyte Membrane (PEM) electrolyser [1], the cell stack contributes to 35% - 45% of the total cost. Catalyst materials on the electrodes are believed to be the primary areas for innovation and cost reduction in PEM electrolysers. Several approaches have been applied to reduce the stack cost, with a focus on reducing material usage while enhancing the performance of PEM electrolysers. This project aims to reduce catalyst loading on the anode of PEM water electrolyser while maintaining high performance, through developing an inkjet printing technique to create a thin, porous and active catalyst layer. Conventional catalyst fabrication techniques such as spray coating produce large amount of material waste and non-homogenous distribution on the surface [2]. Inkjet printing will provide controlled catalyst loading, ensuring well catalyst distribution, creating porous structure, and having the ability to create patterned electrodes [3].Due to its high activity, Iridium Oxide (IrO2) is selected as the catalyst. The ink was formulated to be compatible with the printing requirement, with 7.5 wt% of IrO2 powder, 7.9 wt% of Nafion, and 84.5 wt% of solvents mixture of Isopropanol and Propylene Glycol. Full coverage of the catalyst on the Titanium substrate was proven by Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX). The effect on varying the number of printed layers was analysed in terms of catalyst loading and activity to show a linear increase in activity with the number of layers. Moreover, electrochemical performance was inspected using cyclic voltammetry in a 3-electrodes cell. The printed electrodes showed comparable stability to conventional electrodes. Electrochemical surface area (ECSA) was higher for the 10-layered electrodes as compared to the 2-layered electrodes indicating higher catalyst utilisation. Printed electrodes showed activity of 300 mA/mg IrO2 with ECSA of 225 cm2 at reduced catalyst loading of 0.8 mg IrO2/cm2. These results outperform conventionally fabricated electrodes by spray and decal transfer coatings [4].

Physodic acid sensitizes LNCaP prostate cancer cells to TRAIL-induced apoptosis

In spite of the extensive research for developing new therapies, prostate cancer is still one of the major human diseases with poor prognosis and high mortality. Therefore, with the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of lichen secondary metabolite physodic acid on cell growth in human prostate cancer cells. In addition, we tested the apoptotic activity of physodic acid on TRAIL-resistant LNCaP cells in combination with TRAIL. The cell viability was measured using MTT assay. LDH release, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation and caspase-3 activity assay were employed. The expression of proteins was detected by Western blot analysis. It was observed that physodic acid showed a dose–response relationship in the range of 12.5–50 μM concentrations in LNCaP and DU-145 cells, activating an apoptotic process. In addition, physodic acid sensitizes LNCaP cells to TRAIL-induced apoptosis. The combination of physodic acid with other anti-prostate cancer therapies could be considered a promising strategy that warrants further investigations.

Magnetic field therapy enhances muscle mitochondrial bioenergetics and attenuates systemic ceramide levels following ACL reconstruction: Southeast Asian randomized-controlled pilot trial.

BackgroundMetabolic disruption commonly follows Anterior Cruciate Ligament Reconstruction (ACLR) surgery. Brief exposure to low amplitude and frequency pulsed electromagnetic fields (PEMFs) has been shown to promote in vitro and in vivo murine myogeneses via the activation of a calcium–mitochondrial axis conferring systemic metabolic adaptations. This randomized-controlled pilot trial sought to detect local changes in muscle structure and function using MRI, and systemic changes in metabolism using plasma biomarker analyses resulting from ACLR, with or without accompanying PEMF therapy.Methods20 patients requiring ACLR were randomized into two groups either undergoing PEMF or sham exposure for 16 weeks following surgery. The operated thighs of 10 patients were exposed weekly to PEMFs (1 ​mT for 10 ​min) for 4 months following surgery. Another 10 patients were subjected to sham exposure and served as controls to allow assessment of the metabolic repercussions of ACLR and PEMF therapy. Blood samples were collected prior to surgery and at 16 weeks for plasma analyses. Magnetic resonance data were acquired at 1 and 16 weeks post-surgery using a Siemens 3T Tim Trio system. Phosphorus (31P) Magnetic Resonance Spectroscopy (MRS) was utilized to monitor changes in high-energy phosphate metabolism (inorganic phosphate (Pi), adenosine triphosphate (ATP) and phosphocreatine (PCr)) as well as markers of membrane synthesis and breakdown (phosphomonoesters (PME) and phosphodiester (PDE)). Quantitative Magnetization Transfer (qMT) imaging was used to elucidate changes in the underlying tissue structure, with T1-weighted and 2-point Dixon imaging used to calculate muscle volumes and muscle fat content.ResultsImprovements in markers of high-energy phosphate metabolism including reductions in ΔPi/ATP, Pi/PCr and (Pi ​+ ​PCr)/ATP, and membrane kinetics, including reductions in PDE/ATP were detected in the PEMF-treated cohort relative to the control cohort at study termination. These were associated with reductions in the plasma levels of certain ceramides and lysophosphatidylcholine species. The plasma levels of biomarkers predictive of muscle regeneration and degeneration, including osteopontin and TNNT1, respectively, were improved, whilst changes in follistatin failed to achieve statistical significance. Liquid chromatography with tandem mass spectrometry revealed reductions in small molecule biomarkers of metabolic disruption, including cysteine, homocysteine, and methionine in the PEMF-treated cohort relative to the control cohort at study termination. Differences in measurements of force, muscle and fat volumes did not achieve statistical significance between the cohorts after 16 weeks post-ACLR.ConclusionThe detected changes suggest improvements in systemic metabolism in the post-surgical PEMF-treated cohort that accords with previous preclinical murine studies. PEMF-based therapies may potentially serve as a manner to ameliorate post-surgery metabolic disruptions and warrant future examination in more adequately powered clinical trials.The Translational Potential of this ArticleSome degree of physical immobilisation must inevitably follow orthopaedic surgical intervention. The clinical paradox of such a scenario is that the regenerative potential of the muscle mitochondrial pool is silenced. The unmet need was hence a manner to maintain mitochondrial activation when movement is restricted and without producing potentially damaging mechanical stress. PEMF-based therapies may satisfy the requirement of non-invasively activating the requisite mitochondrial respiration when mobility is restricted for improved metabolic and regenerative recovery.

Effect of Tetracycline Antibiotics on Anaerobic Digestion of Chicken Manure Based on Data Mining.

Although anaerobic bioengineering treatment of chicken manure has the advantages of low energy consumption, less pollution, and recyclable biomass energy, antibiotics are usually added in the process of modern intensive farming. And antibiotics still exist in feces and pose a threat to human health. Therefore, this study aimed to deeply understand the role of TC in the anaerobic digestion of chicken manure and to analyze the effect of tetracycline antibiotics on the anaerobic digestion of chicken manure based on data mining. In this study, chicken manure was used as raw material for anaerobic fermentation, and the effects of tetracycline (TC) on anaerobic and anaerobic fermentation of chicken manure were compared through batch and sequence experiments. Also, this study analyzes the fermentative transformation to elucidate the effect of TC on anaerobic manure conversion in chicken manure, which further studies the effect of TC on the anaerobic fermentation of chicken manure. The experimental results in this study show that when the TC concentration is 50–150 mg/L, the content of tryptophan and tyrosine proteins in the treatment group is also higher than that in the control group. It shows that at low concentrations (10, 25 mg/L), TC mainly promotes the degradation of LEPS by promoting the dissolution of intracellular substances in the sludge. With the increase of TC concentration (50–300 mg/L), TC mainly promotes the breakdown of the sludge cell membrane by promoting the rupture of the sludge cell membrane, thereby promoting the degradation of LEPS.

A new cost-minimizing power-allocating strategy for the hybrid electric bus with fuel cell/battery health-aware control

The two primary challenges preventing the commercialization of fuel cell hybrid electric vehicles (FCHEV) are their high cost and limited lifespan. Improper use usage can could also hasten the breakdown of proton exchange membrane fuel cell (PEMFC). This paper proposes a new cost-minimizing power-allocating technique with fuel cell/battery health-aware control to optimize the economic potential of fuel cell/battery hybrid buses. The proposed framework quantifies the fuel cell (FC) deterioration of the whole working zone in a real hybrid electric bus using a long short-term memory network (LSTM), which reduces the time required to get the key lifetime parameters. A new FC lifespan model is embedded into the control framework, together with a battery aging model, to balance hydrogen consumption and energy source durability. In addition, in the speed prediction step, an enhanced online Markov prediction approach with stochastic disturbances is presented to increase the forecast accuracy for future disturbances. Finally, comparative analysis is used to verify the efficacy of the suggested approach, which shows that when compared to the benchmark method, the proposed strategy may extend the FC lifetime and lower operating costs by 5.04% and 3.76%, respectively.

Effect of phytochemical-filled microcapsules with antifungal activity on material properties and dimensional accuracy of denture base resin for three-dimensional printing

BackgroundStudies on the material properties and dimensional accuracy of three-dimensionally (3D) printed denture base containing microcapsules with antifungal phytochemicals are lacking.MethodsTwo types of phytochemicals (phytoncide A and B) with antifungal activity were microencapsulated. The 3D-printed denture base specimens with minimum and maximum effective concentrations of microcapsules (6 and 8 wt% for phytoncide A; 15 and 25 wt% for phytoncide B) were prepared. The morphological changes of C. albicans on 3D-printed denture base with microcapsules was microscopically observed. The degree of conversion of 3D-printed denture base with microcapsules investigated. The microhardness and flexural strength values were also measured to evaluate the mechanical properties of 3D-printed denture bases. The dimensional accuracy (trueness) of the specimens with microcapsules was measured as root-mean-square values (RMS) for the whole, upper, and side surfaces of the specimens as well as their total height. For the degree of conversion, microhardness, and flexural strength values, the Kruskal–Wallis analysis and a post-hoc comparison using Mann–Whitney U test was performed. For the analysis of trueness (RMS), the one-way analysis of variance and a post-hoc comparison using Tukey’s method was conducted (α = 0.05).ResultsAt both maximum and minimum effective concentrations of microcapsules, cell surface disruption or membrane breakdown of fungal cells were observed in the specimens. The groups with microcapsules (both phytoncide A- and B-filled) showed significantly lower microhardness and elastic modulus values than the control group (all, P = 0.001). For the trueness, all the RMS values of the whole, upper, and side surfaces of the specimens with microcapsules were less than 100 µm, although significantly higher than those without (all, P = 0.001). The mean flexural strength values of the groups with phytoncide A-filled microcapsule were higher than 65 MPa, not statistically different from that of the control group (all, P > 0.05). However, the groups with phytoncide B-filled microcapsules showed significantly lower values than the control (all, P = 0.001).ConclusionsWithin the limitations of this in-vitro study, the 3D-printed denture base containing 6 wt% of phytoncide A-filled microcapsules was clinically acceptable in terms of antifungal activity, dimensional accuracy, and flexural strength.

Key Facilitator Proteins that Mediate Sarcolemma Membrane Repair have Potential as Therapeutics for Muscle Disease and Injury

A common aspect of many diseases and traumatic injuries affecting skeletal muscle is the necrotic death of muscle fibers. Necrotic death of muscle fibers involves the breakdown of the sarcolemmal membrane that can be exacerbated by increased fragility of the membrane or by compromised endogenous sarcolemmal membrane repair processes. Increasing the efficacy of these repair mechanisms could act as a therapeutic approach for several muscular diseases and injuries. Limb‐girdle muscular dystrophy 2B (LGMD2B), a subtype of LGMD, is an autosomal recessive neuromuscular disorder caused by mutations in the DYSF gene that encodes dysferlin, a protein that is vital in membrane repair. The dysferlin protein has been previously shown to facilitate membrane repair in skeletal muscle and knockout mice for dysferlin develop progressive muscular dystrophy. When dysferlin is absent or inactivated through mutation, membrane damage that cannot be repaired accumulates until muscle fibers undergo necrosis that eventually overwhelms the regenerative capacity of the muscle. This causes skeletal muscle deterioration in LGMD2B patients, particularly in the pelvic and shoulder girdle muscles. There is currently no cure for LGMD2B and treatments that stimulate the membrane repair process in muscle fibers have been greatly overlooked and underutilized as a potential therapeutic approach. This project investigates a novel membrane repair signaling cascade in skeletal cells by utilizing SC79, an Akt activator, to increase membrane repair capacity in skeletal muscle. We hypothesize that activation of the phosphoinositide‐3 kinase (PI3K)/Akt1 signaling axis regulates membrane repair in cultured muscle cells and mouse tissue. Based on our data, we find that SC79 injection leads to decreased sarcolemmal membrane injury during treadmill exercises where injection of SC79 decreases the entry of Evans blue dye into muscle fibers. We determined that various doses of SC79 can increase membrane repair in cultured muscle cells and Bla/J dysferlin deficient mice. In these studies, multi‐photon infrared laser microscopy was used to damage the cell membrane of myoblasts, transdifferentiated from skin fibroblasts isolated from LGMD2B patients, in the presence of FM4‐64 dye, a lipophilic dye that fluoresces when it enters the cells and binds to the phospholipids of the cell membrane and intracellular organelles. The extent of localized FM4‐64 dye fluorescence provides a measurement of how well the membrane is repaired. We found SC79 could increase membrane repair in these human patient myoblasts in a dose dependent fashion. This assay was also used on isolated complete muscles from the Bla/J dysferlin deficient mice, where SC79 also increased membrane repair responses. We conclude that activation of the PI3K/Akt1 signaling axis increases membrane repair in dysferlin deficient skeletal muscle. This project explores a novel signaling cascade controlling membrane repair that could be leveraged to develop new therapies for muscle disease and injury.

Predicting emulsion breakdown in the emulsion liquid membrane process: Optimization through response surface methodology and a particle swarm artificial neural network

To anticipate emulsion breakdown in the ELM process, the Box–Behnken design was used with an artificial neural network (ANN) and a metaheuristic approach, namely particle swarm optimization (PSO) and response surface methodology (RSM). Membrane stability testing began with an experimental component to collect data. The following parameters were used to estimate membrane breakdown: emulsification time (3–7 min), surfactant loadings (2–6% v/v), internal phase concentration ([Na2CO3]: 0.01–1 mg L−1), external phase to w/o emulsion volume ratio (1–11), and internal aqueous phase to membrane volume ratio (0.5 to 1.5). The PSO algorithm was used to determine the optimal ANN parameter values. The hybrid ANN-PSO model outperformed the RSM in identifying optimal ANN parameters (weights and thresholds) and accurately forecasting emulsion breaking percentages throughout the ELM process. The hybrid ANN-PSO method may be a valuable optimization tool for predicting critical data for ELM stability under various operating conditions.

Proteomic characterization of post-mortem human brain tissue following ultracentrifugation-based subcellular fractionation.

Proteomic characterization of human brain tissue is increasingly utilized to identify potential novel biomarkers and drug targets for a variety of neurological diseases. In whole-tissue studies, results may be driven by changes in the proportion of the largest and most abundant organelles or tissue cell-type composition. Spatial proteomics approaches enhance our knowledge of disease mechanisms and changing signalling pathways at the subcellular level by taking into account the importance of cellular localization, which critically influences protein function. Density gradient-based ultracentrifugation methods allow for subcellular fractionation and have been utilized in cell lines, mouse and human brain tissue to quantify thousands of proteins in specific enriched organelles such as the pre- and post-synapse. Serial ultracentrifugation methods allow for the analysis of multiple cellular organelles from the same biological sample, and to our knowledge have not been previously applied to frozen post-mortem human brain tissue. The use of frozen human tissue for tissue fractionation faces two major challenges, the post-mortem interval, during which proteins may leach from their usual location into the cytosol, and freezing, which results in membrane breakdown. Despite these challenges, in this proof-of-concept study, we show that the majority of proteins segregate reproducibly into crude density-based centrifugation fractions, that the fractions are enriched for the appropriate organellar markers and that significant differences in protein localization can be observed between tissue from individuals with Alzheimer’s disease and control individuals.

Antioxidants and Phytohormones Act in Coordination to Regulate Sage Response to Long Term Ozone Exposure.

Antioxidants and phytohormones are hallmarks of abiotic stress responses in plants. Although it is known that they can offer cell protection or accelerate programmed cell death (PCD) depending on the level of stress, the involvement of these metabolites in stress acclimation is still not fully elucidated. Here, we showed the role of antioxidants and phytohormones in Salvia officinalis tolerance to long-term ozone (O3) exposure (120 ppb for 36 days, 5 h day−1). Salicylic acid (SA) content was increased under O3 throughout the whole experiment (+150%, as average compared with control), being required to maintain the cellular redox state and potentiate defense responses. This accumulation was induced before the production of ethylene (ET), suggesting that ET was controlled by SA during O3 exposure to modulate the magnitude of chlorosis formation and the cell redox balance (by regulating ascorbate and glutathione levels). The synthesis and/or regeneration of these antioxidants did not protect membranes from lipid peroxidation, as demonstrated by the accumulation of malondialdehyde (+23% as average). However, these processes of lipid oxidation did not include the synthesis of the membrane breakdown products, as confirmed by the unchanged values of jasmonic acid, thus indicating that this compound was not involved in the regulation of PCD strategies.

Huddling together to survive: Population density as a survival strategy of non-spore forming bacteria under nutrient starvation and desiccation at solid-air interfaces

Acclimation and flexible response mechanisms are survival adaptations allowing prokaryotic cells to colonize diverse habitats and maintain viability in nature. Lack of water significantly impacts cellular response, which can be partially compensated for through community interactions and accessing survival means beyond the cell’s boundaries. In the present study, higher numbers of cultivable Gram-positive Arthrobacter sp. and Gram-negative Pseudomonas stutzeri cells were found on surfaces when high population density was used after prolonged periods of desiccation and nutrient starvation. Total cell counts during desiccation periods decreased slower than culturable cell counts independently from initial population density. The presence of homogenate, prepared by filtering homogenized cultures through a 0.2 µm filter, extended culturability of Arthrobacter sp. cells, while intact heat-killed cells extended the culturability of Arthrobacter sp. and P. stutzeri. Our results suggest very slow cell membrane breakdown for desiccated bacterial cells at solid-air interfaces over extended time spans, which may serve as reservoirs of nutrients, and may potentially provide trace amounts of water for surviving cells. Higher initial population density and recycling of resources from “zombie”-like cells, may support growth in a similar fashion as access to cell lysates or the contents of heat-killed cells analogous to dead-phase cultures where some cells experience cryptic growth.

The Lipoteichoic Acid-Related Proteins YqgS and LafA Contribute to the Resistance of Listeria monocytogenes to Nisin.

ABSTRACTListeria monocytogenes is a major pathogen contributing to foodborne outbreaks with high mortality. Nisin, a natural antimicrobial, has been widely used as a food preservative. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. A mariner transposon library was constructed in L. monocytogenes, leading to the identification of 99 genes associated with the innate resistance to nisin via Transposon sequencing (Tn-seq) analysis. To validate the accuracy of the Tn-seq results, we constructed five mutants (ΔyqgS, ΔlafA, ΔvirR, ΔgtcA, and Δlmo1464) in L. monocytogenes. The results revealed that yqgS and lafA, the lipoteichoic acid-related genes, were essential for resistance to nisin, while the gtcA and lmo1464 mutants showed substantially enhanced nisin resistance. Densely wrinkled, collapsed surface and membrane breakdown were shown on ΔyqgS and ΔlafA mutants under nisin treatment. Deletion of yqgS and lafA altered the surface charge, and decreased the resistance to general stress conditions and cell envelope-acting antimicrobials. Furthermore, YqgS and LafA are required for biofilm formation and cell invasion of L. monocytogenes. Collectively, these results reveal novel mechanisms of nisin resistance in L. monocytogenes and may provide unique targets for the development of food-grade inhibitors for nisin-resistant foodborne pathogens.IMPORTANCE Listeria monocytogenes is an opportunistic Gram-positive pathogen responsible for listeriosis, and is widely present in a variety of foods including ready-to-eat foods, meat, and dairy products. Nisin is the only licensed lantibiotic by the FDA for use as a food-grade inhibitor in over 50 countries. A prior study suggests that L. monocytogenes are more resistant than other Gram-positive pathogens in nisin-mediated bactericidal effects. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. Here, we used a mariner transposon library to identify nisin-resistance-related genes on a genome-wide scale via transposon sequencing. We found, for the first time, that YqgS and LafA (Lipoteichoic acid-related proteins) are required for resistance to nisin. Subsequently, we investigated the roles of YqgS and LafA in L. monocytogenes stress resistance, antimicrobial resistance, biofilm formation, and virulence in mammalian cells.