Showing papers on "Lysis published in 2018"
TL;DR: The outer membrane of Gram-negative bacteria is shown to be at least as stiff as the cell wall, and this property enables it to protect cells from mechanical pertubations.
Abstract: Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.
324 citations
TL;DR: An overview of the viral structures, key protein players and mechanisms underlying phage DNA entry to bacteria, and then escape of the newly-formed virus particles from infected hosts is presented.
Abstract: Monoderm bacteria possess a cell envelope made of a cytoplasmic membrane and a cell wall, whereas diderm bacteria have and extra lipid layer, the outer membrane, covering the cell wall Both cell types can also produce extracellular protective coats composed of polymeric substances like, for example, polysaccharidic capsules Many of these structures form a tight physical barrier impenetrable by phage virus particles Tailed phages evolved strategies/functions to overcome the different layers of the bacterial cell envelope, first to deliver the genetic material to the host cell cytoplasm for virus multiplication, and then to release the virion offspring at the end of the reproductive cycle There is however a major difference between these two crucial steps of the phage infection cycle: virus entry cannot compromise cell viability, whereas effective virion progeny release requires host cell lysis Here we present an overview of the viral structures, key protein players and mechanisms underlying phage DNA entry to bacteria, and then escape of the newly-formed virus particles from infected hosts Understanding the biological context and mode of action of the phage-derived enzymes that compromise the bacterial cell envelope may provide valuable information for their application as antimicrobials
104 citations
TL;DR: For the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h, and prominent cellular heterogeneity in protein expressive profiling was observed.
Abstract: In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells. (1) In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 μm i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 μm i.d. nano-LC packing column with 3 μm i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7-170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of our previously developed iPAD-100 system. (1) Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.
81 citations
TL;DR: The results of the present study indicate that the purified flavonoids from G. glandulosum possess antimicrobial activities, which are in some cases equal to, or higher than those of ciprofloxacin used as reference antibiotic.
Abstract: The search for new antimicrobials should take into account drug resistance phenomenon. Medicinal plants are known as sources of potent antimicrobial compounds including flavonoids. The objective of this investigation was to evaluate the antimicrobial activities of flavonoid glycosides from Graptophyllum grandulosum, as well as to determine their mechanism of antibacterial action using lysis, leakage and osmotic stress assays. The plant extracts were prepared by maceration in organic solvents. Column chromatography of the n-butanol extract followed by purification of different fractions led to the isolation of five flavonoid glycosides. The antimicrobial activities of extracts/compounds were evaluated using the broth microdilution method. The bacteriolytic activity was evaluated using the time-kill kinetic method. The effect of extracts on the red blood cells and bacterial cell membrane was determined by spectrophotometric methods. Chrysoeriol-7-O-β-D-xyloside (1), luteolin-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside (2), chrysoeriol-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside (3), chrysoeriol-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-(4"-hydrogeno sulfate) glucopyranoside (4) and isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside (5) were isolated from G. grandulosum and showed different degrees of antimicrobial activities. Their antibacterial activities against multi-drug-resistant Vibrio cholerae strains were in some cases equal to, or higher than those of ciprofloxacin used as reference antibiotic. The antibacterial activities of flavonoid glycosides and chloramphenicol increased under osmotic stress (5% NaCl) whereas that of vancomycin decreased under this condition. V. cholerae suspension treated with flavonoid glycosides, showed a significant increase in the optical density at 260 nm, suggesting that nucleic acids were lost through a damaged cytoplasmic membrane. A decrease in the optical density of V. cholerae NB2 suspension treated with the isolated compounds was observed, indicating the lysis of bacterial cells. The tested samples were non-toxic to normal cells highlighting their good selectivity index. The results of the present study indicate that the purified flavonoids from G. glandulosum possess antimicrobial activities. Their mode of antibacterial activity is due to cell lysis and disruption of the cytoplasmic membrane upon membrane permeability.
79 citations
TL;DR: The potential for L-form switching in the host environment is demonstrated and the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity are highlighted, which can continue to proliferate in the presence of antibiotic.
75 citations
TL;DR: It is shown that increased phage production resulted directly from a lysis delay caused by the relative shortage of holin in filamented bacterial hosts in the presence of sublethal concentrations of stress-inducing substances, such as antibiotics and reactive oxygen species (ROS).
Abstract: When phages infect bacteria cultured in the presence of sublethal doses of antibiotics, the sizes of the phage plaques are significantly increased. This phenomenon is known as phage-antibiotic synergy (PAS). In this study, the observation of PAS was extended to a wide variety of bacterium-phage pairs using different classes of antibiotics. PAS was shown in both Gram-positive and Gram-negative bacteria. Cells stressed with β-lactam antibiotics filamented or swelled extensively, resulting in an increase in phage production. PAS was also sometimes observed in the presence of other classes of antibiotics with or without bacterial filamentation. The addition of antibiotics induced recA expression in various bacteria, but a recA deletion mutant strain of Escherichia coli also showed filamentation and PAS in the presence of quinolone antibiotics. The phage adsorption efficiency did not change in the presence of the antibiotics when the cell surfaces were enlarged as they filamented. Increases in the production of phage DNA and mRNAs encoding phage proteins were observed in these cells, with only a limited increase in protein production. The data suggest that PAS is the product of a prolonged period of particle assembly due to delayed lysis. The increase in the cell surface area far exceeded the increase in phage holin production in the filamented host cells, leading to a relatively limited availability of intracellular holins for aggregating and forming holes in the host membrane. Reactive oxygen species (ROS) stress also led to an increased production of phages, while heat stress resulted in only a limited increase in phage production.IMPORTANCE Phage-antibiotic synergy (PAS) has been reported for a decade, but the underlying mechanism has never been vigorously investigated. This study shows the presence of PAS from a variety of phage-bacterium-antibiotic pairings. We show that increased phage production resulted directly from a lysis delay caused by the relative shortage of holin in filamented bacterial hosts in the presence of sublethal concentrations of stress-inducing substances, such as antibiotics and reactive oxygen species (ROS).
65 citations
TL;DR: This study uses molecular dynamics simulations combined with experiments to resolve how and to what extent models of the IM, OM, and cell wall respond to changes in surface tension, and calculated the area compressibility modulus of all three components in simulations from tension-area isotherms.
62 citations
TL;DR: A plasmid encoded novel bacteriocin BMP11 produced by Lactobacillus crustorum MN047 was innovatively identified and found to have rich α-helix conformation after prediction and had promising potential as antimicrobial to control foodborne pathogens in dairy products.
57 citations
TL;DR: Findings indicated that that FAC6 has promising potential to be developed as a multifunctional food additive.
Abstract: This work aims to prepare ferulic acid alkyl esters (FAEs) through the lipase-catalyzed reaction between methyl ferulate and various fatty alcohols in deep eutectic solvents and ascertain their antibacterial activities and mechanisms. Screens of antibacterial effects of FAEs against Escherichia coli ATCC 25922 ( E. coli) and Listeria monocytogenes ATCC 19115 ( L. monocytogenes) revealed that hexyl ferulate (FAC6) exerted excellent bacteriostatic and bactericidal effects on E. coli and L. monocytogenes (minimum inhibitory concentration (MIC): 1.6 and 0.1 mM, minimum bactericidal concentration (MBC): 25.6 and 0.2 mM, respectively). The antibacterial mechanism of FAC6 against E. coli was systematically studied to facilitate its practical use as a food additive with multifunctionalities. The growth and time-kill curves implied the partial cell lysis and inhibition of the growth of E. coli caused by FAC6. The result related to propidium iodide uptake and cell constituents' leakage (K+, proteins, nucleotides, and β-galactosidase) implied that bacterial cytomembranes were substantially compromised by FAC6. Variations on morphology and cardiolipin microdomains and membrane hyperpolarization of cells visually verified that FAC6 induced cell elongation and destructed the cell membrane with cell wall perforation. SDS-PAGE analysis and alterations of fluorescence spectra of bacterial membrane proteins manifested that FAC6 caused significant changes in constitutions and conformation of membrane proteins. Furthermore, it also could bind to minor grooves of E. coli DNA to form complexes. Meanwhile, FAC6 exhibited antibiofilm formation activity. These findings indicated that that FAC6 has promising potential to be developed as a multifunctional food additive.
49 citations
TL;DR: H2O2 at moderate dosages could be a promising method for the biomass control, in a fast and efficient way, on M. aeruginosa blooms, and induced apoptosis-like death in terms of membrane potential dissipation, caspase-3 activation, chromatin condensation, and lysis induction.
47 citations
TL;DR: The effect of combined treatment with ethylenediaminetetraacetic acid and microwave was studied and the polymer recovered through this method was 97.21% pure, showed 2.9-fold improvement in molecular weight and better PDI, and is more eco-friendly than the sodium hypochlorite lysis method.
Abstract: Poly(3-hydroxybutyrate) (PHB) is a bacterial polymer of great commercial importance due to its properties similar to polypropylene. With an aim to develop a recombinant system for economical polymer production, PHB biosynthesis genes from Bacillus aryabhattai PHB10 were cloned in E. coli. The recombinant cells accumulated a maximum level of 6.22 g/L biopolymer utilizing glycerol in shake flasks. The extracted polymer was confirmed as PHB by GC–MS and NMR analyses. The polymer showed melting point at 171 °C, thermal stability in a temperature range of 0–140 °C and no weight loss up to 200 °C. PHB extracted from sodium hypochlorite lysed cells had average molecular weight of 143.108 kDa, polydispersity index (PDI) 1.81, tensile strength of 14.2 MPa and an elongation at break of 7.65%. This is the first report on high level polymer accumulation in recombinant E. coli solely expressing PHB biosynthesis genes from a Bacillus sp. As an alternative to sodium hypochlorite cell lysis mediated polymer extraction, the effect of combined treatment with ethylenediaminetetraacetic acid and microwave was studied which attained 93.75% yield. The polymer recovered through this method was 97.21% pure, showed 2.9-fold improvement in molecular weight and better PDI. The procedure is simple, with minimum polymer damage and more eco-friendly than the sodium hypochlorite lysis method.
TL;DR: This work demonstrated an efficient method to nondestructively probe intracellular protein levels and monitor the dynamic protein expression, with great potential to help understanding cell behaviors and functions.
Abstract: Techniques used to understand the dynamic expression of intracellular proteins are critical in both fundamental biological research and biomedical engineering. Various methods for analyzing proteins have been developed, but these methods require the extraction of intracellular proteins from the cells resulting in cell lysis and subsequent protein purifications from the lysate, which limits the potential of repetitive extraction from the same set of viable cells to track dynamic intracellular protein expression. Therefore, it is crucial to develop novel methods that enable nondestructive and repeated extraction of intracellular proteins. This work reports a hollow nanoneedle-electroporation system for the repeated extraction of intracellular proteins from living cells. Hollow nanoneedles with ∼450 nm diameter were fabricated by a material deposition and etching process, followed by integration with a microfluidic device. Long-lasting electrical pulses were coupled with the nanoneedles to permeate the cell ...
TL;DR: The comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota.
Abstract: DNA based methods have been widely used to study the complexity of the rumen microbiota, and it is well known that the method of DNA extraction is a critical step in enabling accurate assessment of this complexity. Rumen fluid (RF) and fibrous content (FC) fractions differ substantially in terms of their physical nature and associated microorganisms. The aim of this study was therefore to assess the effect of four DNA extraction methods (RBB, PBB, FDSS, PQIAmini) differing in cell lysis and/or DNA recovery methods on the observed microbial diversity in RF and FC fractions using samples from four rumen cannulated dairy cows fed 100% grass silage (GS100), 67% GS and 33% maize silage (GS67MS33), 33% GS and 67% MS (GS33MS67), or 100% MS (MS100). An ANOVA statistical test was applied on DNA quality and yield measurements, and it was found that the DNA yield was significantly affected by extraction method (p < 0.001) and fraction (p < 0.001). The 260/280 ratio was not affected by extraction (p = 0.08) but was affected by fraction (p = 0.03). On the other hand, the 260/230 ratio was affected by extraction method (p < 0.001) but not affected by fraction (p = 0.8). However, all four extraction procedures yielded DNA suitable for further analysis of bacterial, archaeal and anaerobic fungal communities using quantitative PCR and pyrosequencing of relevant taxonomic markers. Redundancy analysis (RDA) of bacterial 16S rRNA gene sequence data at the family level showed that there was a significant effect of rumen fraction (p = 0.012), and that PBB (p = 0.012) and FDSS (p = 0.024) also significantly contributed to explaining the observed variation in bacterial community composition. Whilst the DNA extraction method affected the apparent bacterial community composition, no single extraction method could be concluded to be ineffective. No obvious effect of DNA extraction method on the anaerobic fungi or archaea was observed, although fraction effects were evident for both. In summary, the comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota.
TL;DR: Investigation of the impacts of three widely used oxidants on Pseudanabaena sp.
TL;DR: Chlorine dioxide was found to increase the permeability of outer and cytoplasmic cell membranes and consequently resulting in the release of vital nuclear materials which strongly correlated with loss of cell activity or death, however, from TEM micrographs significant morphological damages or cells lysis was not observed.
Abstract: This study evaluated the inactivation kinetics and the bactericidal mechanism of chlorine dioxide towards Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 29213) on a laboratory scale with the view of determining the optimal operational conditions of its application as an alternative disinfectant in water treatment. Bacteria inactivation was conducted in batch reactors at varied disinfectant concentrations, pH, temperature and initial bacteria densities in buffered disinfectant demand free water. The bactericidal mechanism in terms of the effect on the permeability of the outer and cytoplasmic cell membranes and the morphology of the cells were explored. At the highest studied concentration (5.0 mg/L), at least 5-log reductions in bacterial population were observed for each strain of bacteria. Chlorine dioxide inactivation showed a stronger sensitivity to changes in water pH conditions with the inactivation rate at 8.5 being at least 4-fold of what pertained at 6.5 but efficiency was less impacted by changes in the initial bacteria density. A rise in temperature from 4 °C to 15 °C resulted in approximately 56% increase in the inactivation rate of S. aureus. Chlorine dioxide was found to increase the permeability of outer and cytoplasmic cell membranes and consequently resulting in the release of vital nuclear materials which strongly correlated with loss of cell activity or death. However, from TEM micrographs significant morphological damages or cells lysis was not observed. These results provide vital data on operational strategies to enhance efficient disinfection of water with chlorine dioxide whilst monitoring regulatory requirements on disinfection by-products.
TL;DR: The results showed that a water matrix can significantly affect the effectiveness of K2FeO4 on cyanobacteria inactivation, and could serve as a post-oxidant to inactivate cyanobacterial cells and degrade MCs effectively, depending on the specific water matrix.
TL;DR: The findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues.
Abstract: Many bacteria are resistant to killing (tolerant) by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A penicillin binding proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. Transposon insertion sequencing (TnSeq) analyses revealed that lipopolysaccharide (LPS) and cell wall biogenesis genes, as well as the sigma E cell envelope stress response, were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for postantibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.
TL;DR: It is concluded that the LplT/Aas system is important for maintaining the integrity of the membrane envelope in Gram-negative bacteria, and may help inform new therapeutic strategies to enhance host sPLA2 antimicrobial activity.
TL;DR: Urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates, which led to a reduction in the negative effects often observed for such modifications.
Abstract: Urea-containing buffer solutions are generally used in proteomic studies to aid protein denaturation and solubilization during cell and tissue lysis. It is well-known, however, that urea can lead to carbamylation of peptides and proteins and, subsequently, incomplete digestion of proteins. By the use of cells and tissues that had been lysed with urea, different solution digestion strategies were quantitatively assessed. In comparison with traditional proteolysis at 37 °C, urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates. Furthermore, the signal intensities and the number of carbamylated and pyroglutamic acid-modified peptides decreased. Overall, this led to a reduction in the negative effects often observed for such modifications. Data are available via ProteomeXchange with identifier PXD009426.
TL;DR: A phenomenological model traces the differential viability and ΔY of wild-type and HR cells to the difference in the effective activation energies needed to permeabilize the cells, implying that HR cells are characterized by stronger lateral interactions between molecules, such as lipids, in their cell envelope.
TL;DR: Of the seven techniques investigated, acid treatment led to the highest lipid recovery yields, and exploration of acid treatment and integration with an economic model revealed that treatment at 170 °C for 60 min at 1 wT % H2SO4 and 8 wt % yeast solids re...
Abstract: Bioderived lipids offer a potentially promising intermediate to displace petroleum-derived diesel. One of the key challenges for the production of lipids via microbial cell mass is that these products are stored intracellularly and must be extracted and recovered efficiently and economically. Thus, improved methods of cell lysis and lipid extraction are needed. In this study, we examine lipid extraction from wet oleaginous yeast in combination with seven different cell lysis approaches encompassing both physical and chemical techniques (high-pressure homogenization, microwave and conventional thermal treatments, bead beating, acid, base, and enzymatic treatments) to facilitate lipid extraction from a model oleaginous yeast strain, Lipomyces starkeyi. Of the seven techniques investigated, acid treatment led to the highest lipid recovery yields. Further exploration of acid treatment and integration with an economic model revealed that treatment at 170 °C for 60 min at 1 wt % H2SO4 and 8 wt % yeast solids re...
TL;DR: Mechanism of bacterial inactivation caused by SML was revealed through comprehensive factors including cell morphology, cellular lysis, membrane permeability, K+ leakage, zeta potential, intracellular enzyme, and DNA assay, which demonstrated that bacterial in activation against Gram-positive bacteria was primarily induced by the pronounced damage to the cell membrane integrity.
Abstract: The aim of this work was to evaluate the antibacterial activities and mode of action of sucrose monolaurate (SML) with a desirable purity, synthesized by Lipozyme TL IM-mediated transesterification in the novel ionic liquid, against four pathogenic bacteria including L. monocytogenes, B. subtilis, S. aureus, and E. coli. The antibacterial activity was determined by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and the time–kill assay. SML showed varying antibacterial activity against tested bacteria with MICs and MBCs of 2.5 and 20 mM for L. monocytogenes, 2.5 and 20 mM for B. subtilis, 10 and 40 mM for S. aureus, respectively. No dramatic inhibition was observed for E. coli at 80 mM SML. Mechanism of bacterial inactivation caused by SML was revealed through comprehensive factors including cell morphology, cellular lysis, membrane permeability, K+ leakage, zeta potential, intracellular enzyme, and DNA assay. Results demonstrated that bacterial inactivation against Gram-positive bacteria was primarily induced by the pronounced damage to the cell membrane integrity. SML may interact with cytoplasmic membrane to disturb the regulation system of peptidoglycan hydrolase activities to degrade the peptidoglycan layer and form a hole in the layer. Then, the inside cytoplasmic membrane was blown out due to turgor pressure and the cytoplasmic materials inside leaked out. Leakage of intracellular enzyme to the supernatants implied that the cell membrane permeability was compromised. Consequently, the release of K+ from the cytosol lead to the alterations of the zeta potential of cells, which would disturb the subcellular localization of some proteins, and thereby causing bacterial inactivation. Moreover, remarkable interaction with DNA was also observed. SML at sub-MIC inhibited biofilm formation by these bacteria.
TL;DR: Data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type.
TL;DR: Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced.
Abstract: The alkaline comet assay, in vivo and in vitro, is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the inter-experimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H2O2, which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories.
TL;DR: The combination of scraping and freeze-thaw-cycle was optimal for harvesting and lysing adherent mammalian cells for CIL LC-MS metabolomics.
TL;DR: Spontaneously activated in aqueous environment, the RNS chemistry of Si3N4 proved much more effective in counteracting bacterial proliferation as compared to ROS formed on TiO2, which requires external energy to enhance effectiveness.
TL;DR: A 3D model is developed to quantify the impact of the changes in the cellular structure of apple (parenchyma) cells during dehydration on the tissue sorption isotherm and water permeability, and identified that the dehydrated, shrunken cellular tissue reduces the outgoing water flux compared to fresh tissue for the same water potential gradient.
TL;DR: Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity, and the process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.
Abstract: Introduction Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives. Methods In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant - Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorbate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impurities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell proteins and ds-DNA was 99.9% and 97.1%, respectively. Results & conclusion The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the developed process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.
TL;DR: The cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma, and the cell wall structure tended to shrink or distorted after high temperature.
Abstract: Fungal cells are surrounded by a tight cell wall to protect them from harmful environmental conditions and to resist lysis. The synthesis and assembly determine the shape, structure, and integrity of the cell wall during the process of mycelial growth and development. High temperature is an important abiotic stress, which affects the synthesis and assembly of cell walls. In the present study, the chitin and β-1,3-glucan concentrations in the cell wall of Pleurotus ostreatus mycelia were changed after high-temperature treatment. Significantly higher chitin and β-1,3-glucan concentrations were detected at 36 °C than those incubated at 28 °C. With the increased temperature, many aberrant chitin deposition patches occurred, and the distribution of chitin in the cell wall was uneven. Moreover, high temperature disrupts the cell wall integrity, and P. ostreatus mycelia became hypersensitive to cell wall-perturbing agents at 36 °C. The cell wall structure tended to shrink or distorted after high temperature. The cell walls were observed to be thicker and looser by using transmission electron microscopy. High temperature can decrease the mannose content in the cell wall and increase the relative cell wall porosity. According to infrared absorption spectrum, high temperature broke or decreased the glycosidic linkages. Finally, P. ostreatus mycelial cell wall was easily degraded by lysing enzymes after high-temperature treatment. In other words, the cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma.
TL;DR: This study investigates ultraviolet B (UVB) as a disruption method for the green algae Chlamydomonas reinhardtii, Dunaliella salina and Micractinium inermum to enhance solvent lipid extraction and indicates that UV disruption had a higher efficiency when used for solvent lipid extracting.
Abstract: Currently, the energy required to produce biofuel from algae is 1.38 times the energy available from the fuel. Current methods do not deliver scalable, commercially viable cell wall disruption, which creates a bottleneck on downstream processing. This is primarily due to the methods depositing energy within the water as opposed to within the algae. This study investigates ultraviolet B (UVB) as a disruption method for the green algae Chlamydomonas reinhardtii, Dunaliella salina and Micractinium inermum to enhance solvent lipid extraction. After 232 seconds of UVB exposure at 1.5 W/cm², cultures of C. reinhardtii (culture density 0.7 mg/mL) showed 90% disruption, measured using cell counting, correlating to an energy consumption of 5.6 MJ/L algae. Small-scale laboratory tests on C. reinhardtii showed bead beating achieving 45.3 mg/L fatty acid methyl esters (FAME) and UV irradiation achieving 79.9 mg/L (lipids solvent extracted and converted to FAME for measurement). The alga M. inermum required a larger dosage of UVB due to its thicker cell wall, achieving a FAME yield of 226 mg/L, compared with 208 mg/L for bead beating. This indicates that UV disruption had a higher efficiency when used for solvent lipid extraction. This study serves as a proof of concept for UV irradiation as a method for algal cell disruption.