Release Of Intracellular Proteins Research Articles

Baicalin enhances antioxidant, inflammatory defense, and microbial diversity of yellow catfish (Pelteobagrus fulvidraco) infected with Aeromonas hydrophila.

The aim of this research was to clarify the mechanism through which baicalin exerts its inhibitory effects on Aeromonas hydrophila infection. The antibacterial efficacy of baicalin was assessed by determining its minimum inhibitory concentration (MIC) against A. hydrophila. Various parameters, including the growth curve, cell wall integrity, biofilm formation, AKP content, and morphological alterations of A. hydrophila, were analyzed. In vivo experiments involved the administration of A. hydrophila 4 h postintraperitoneal injection of varying doses of baicalin to induce infection, with subsequent monitoring of mortality rates. After a 3 d period, liver, spleen, and intestinal tissues were harvested to evaluate organ indices, antioxidant and immune parameters, as well as intestinal microbial composition. The findings indicated that baicalin treatment resulted in the disruption of the cell wall of A. hydrophila, leading to the loss of its normal structural integrity. Furthermore, baicalin significantly inhibited biofilm formation and facilitated the release of intracellular proteins (P < 0.05). In vivo, baicalin enhanced the survival rates of yellow catfish infected with A. hydrophila. Compared to the control group, the liver index of yellow catfish was elevated, while the spleen and intestinal indices were reduced in the baicalin-treated group (P < 0.05). Additionally, baicalin at an appropriate dosage was found to increase levels of SOD, GSH, CAT, ACP, and AKP in yellow catfish (P < 0.05), while simultaneously decreasing MDA accumulation and the mRNA expression of inflammatory markers such as Keap1, IL1, IFN-γ, and TNF-α, (P < 0.05). Moreover, baicalin significantly enhanced the operational taxonomic unit (OTU) count in A. hydrophila-infected yellow catfish (P < 0.05), restoring the abundance of Barnesiellaceae, Enterobacteriaceae, Plesiomonas, and UBA1819 (P < 0.05). In summary, baicalin demonstrates the potential to improve the survival rate of yellow catfish subjected to A. hydrophila infection, augment antioxidant and immune responses, mitigate inflammation, and enhance intestinal microbial diversity.

Dellaglioa Algida Cell-Free Supernatant Inhibits Pseudomonas Fluorescence and Pseudomonas Fragi by Destroying Cell Membranes.

The aim of this study was to examine the components of the cell-free supernatant (CFS) derived from a novel strain of psychrophilic Lactobacillus, Dellaglioa algida, and to further elucidate the impact of this CFS on various cellular processes. Specifically, we sought to understand its effects on the cell membrane, protein and DNA release, protease activity, and metabolites of Pseudomonas fluorescens and Pseudomonas fragi, thereby clarifying the antibacterial mechanism involved. The CFS components were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS), the Coomassie Brilliant Blue method, and the phenol-sulfuric acid method. The inhibitory effect of the CFS on Pseudomonas fluorescens and Pseudomonas fragi was assessed using the ethidium bromide (EB) assay, Oxford cup assay, and ultramicroassay. Additionally, we analyzed the metabolites produced by Pseudomonas fluorescens and Pseudomonas fragi when treated with the CFS. The findings reveal that the CFS of Dellaglioa algida contains 94 volatile components, with protein and sugar concentrations of 32.857 ± 0.9705 mg/mL and 98.250 ± 4.210 mg/L, respectively. The CFS induces varying degrees of damage to the cell membranes of both Pseudomonas fluorescens and Pseudomonas fragi, leading to the release of intracellular proteins and DNA. Furthermore, the CFS reduced the protease activity and metabolic capacity of Pseudomonas fluorescens and Pseudomonas fragi. These results enhance our understanding of the mechanism by which psychrophilic Dellaglioa algida inhibits Pseudomonas fluorescens and Pseudomonas fragi, confirming that its inhibitory effect predominantly occurs through damage to the biological cell membranes of Pseudomonas. Dellaglioa algida is a newly identified cold-adapted inhibitor of Pseudomonas, indicating that its CFS is an effective microbial inhibitor in cold environments. This discovery suggests potential applications in inhibiting the growth and reproduction of Pseudomonas fluorescens and Pseudomonas fragi in food, pharmaceuticals, perfumes, and other chemicals, providing a valuable new reference for industrial preservation.

Antibacterial activity of 2-hydroxy-4-methoxybenzaldehyde and its possible mechanism against Staphylococcus aureus.

Staphylococcus aureus causes several complicated infections. Despite decades of research on developing new antimicrobials, methicillin-resistant S. aureus (MRSA) remains a global health problem. Hence, there is a dire need to identify potent natural antibacterial compounds as an alternative to antimicrobials. In this light, the present work divulges the antibacterial efficacy and the action mechanism of 2-hydroxy-4-methoxybenzaldehyde (HMB) isolated from Hemidesmus indicus against S. aureus. Antimicrobial activity of HMB was assessed. HMB exhibited 1024µg ml-1 as the minimum inhibitory concentration (MIC) and 2×MIC as the minimum bactericidal concentration against S. aureus. The results were validated by spot assay, time kill, and growth curve analysis. In addition, HMB treatment increased the release of intracellular proteins and nucleic acid contents from MRSA. Additional experiments assessing the structural morphology of bacterial cells using SEM analysis, β-galactosidase enzyme activity, and the fluorescence intensities of propidium iodide and rhodamine123 dye divulged that the cell membrane as one of the targets of HMB to hinder S. aureus growth. Moreover, the mature biofilm eradication assay revealed that HMB dislodged nearly 80% of the preformed biofilms of MRSA at the tested concentrations. Further, HMB treatment was found to sensitize MRSA cells upon combining tetracycline treatment. The present study suggests that HMB is a promising compound with antibacterial and antibiofilm activities and could act as a lead structure for developing new antibacterial drugs against MRSA.

Electrochemically driven Fe0-activated sulfite oxidation for enhancing sludge dewaterability

Sulfate radical (SO4·-)-based advanced oxidation processes (SR-AOPs) are promising for sludge dewatering. In this work, an electrochemically driven Fe0-activated sulfite system (E/Fe/sulfite) was developed and demonstrated for sustainably releasing Fe2+ to activate sulfite for dewatering waste activated sludge. Under the optimal conditions (sulfite dosage = 40 mg/g dry solids, current density = 30 mA/cm2, pH = 6, and time = 60 min), the reduction of capillary suction time and water content of sludge cake were 87.9 ± 2.8 % and 73.1 ± 0.8 %, respectively. The improved dewatering performance was attributed to the degradation of tightly-bound extracellular polymeric substances, along with the decreased live/dead cell ratio and the increased permeability of sludge flocs. The thermal effect (∼21–59 °C) in the E/Fe/sulfite system accelerated the release of intracellular proteins, which could be effectively degraded by the strong oxidizing SO4·-. The results also suggested that electrochemical oxidation and thermal effect in the E/Fe/sulfite system had a synergistic impact on improving sludge dewaterability. According to the density functional theory calculation, the hydrophobic amino acids produced from protein degradation were favorable for forming the drainage channels and enhancing sludge dewaterability. This study provides new insights for understanding the SR-AOPs and their applications to sludge treatment.

Proteomic characterization of pilot scale hot-water extracts from the industrial carrageenan red seaweed Eucheuma denticulatum

Seaweeds have a long history as a resource for polysaccharides/hydrocolloids extraction for use in the food industry due to their functionality as stabilizing agents. In addition to the carbohydrate content, seaweeds also contains a significant amount of protein, which may find application in food and feed. Here, we present a novel combination of transcriptomics, proteomics, and bioinformatics to determine the protein composition in two pilot-scale extracts from Eucheuma denticilatum (Spinosum) obtained via hot-water extraction. Although the quality of extracted protein appeared quite poor based on SDS-PAGE analysis, extracts were characterized by qualitative and quantitative proteomics using LC-MS/MS and a de-novo transcriptome assembly for construction of a suitable protein database. A novel concept of length-normalization for relative quantification of sub-optimal protein extracts with partial, non-specific digestion is introduced and validated against conventional methods for relative quantification of proteins. Despite a limited number of protein identifications due to poor protein quality, our data suggest that the majority of quantified protein in the extracts (>75%) is constituted by merely three previously uncharacterized proteins. Putative subcellular localization for the quantified proteins was determined by bioinformatic prediction using several predictors, and by correlating with the expected copy number from the transcriptome analysis, we find that the extracts appear highly enriched in extracellular proteins. This implies that the extraction method used predominantly extracts extracellular proteins, and thus appear ineffective for cellular disruption and subsequent release of intracellular proteins. Nevertheless, the highly abundant proteins may be potential substrates for targeted hydrolysis and release of bioactive peptides. Ultimately, this study highlight the potential of quantitative proteomics for characterization of alternative protein sources intended for use in foods and evaluating protein extraction process efficiency through novel combinations with bioinformatic analysis.

The effects of Naja sumatrana venom cytotoxin, sumaCTX on alteration of the secretome in MCF-7 breast cancer cells following membrane permeabilization

Naja sumatrana venom cytotoxin (sumaCTX) is a basic protein which belongs to three-finger toxin family. It has been shown to induce caspase-dependent, mitochondrial-mediated apoptosis in MCF-7 cells at lower concentrations. This study aimed to investigate the alteration of secretome in MCF-7 cells following membrane permeabilization by high concentrations of sumaCTX, using label-free quantitative (LFQ) approach. The degree of membrane permeabilization of sumaCTX was determined by lactate dehydrogenase (LDH) assay and calcein-propidium iodide (PI) assays. LDH and calcein-PI assays revealed time-dependent membrane permeabilization within a narrow concentration range. However, as toxin concentrations increased, prolonged exposure of MCF-7 cells to sumaCTX did not promote the progression of membrane permeabilization. The secretome analyses showed that membrane permeabilization was an event preceding the release of intracellular proteins. Bioinformatics analyses of the LFQ secretome revealed the presence of 105 significantly distinguished proteins involved in metabolism, structural supports, inflammatory responses, and necroptosis in MCF-7 cells treated with 29.8 μg/mL of sumaCTX. Necroptosis was presumably an initial stress response in MCF-7 cells when exposed to high sumaCTX concentration. Collectively, sumaCTX-induced the loss of membrane integrity in a concentration-dependent manner, whereby the cell death pattern of MCF-7 cells transformed from apoptosis to necroptosis with increasing toxin concentrations.

Inflammation disrupts epithelial barrier function and induces the release of different populations of exosomes

Abstract Introduction Age-related macular degeneration (AMD) is a degenerative retinal disease that affects central vision. Most of their phenotypical features are believed to be associated with the dysfunction of retinal pigment epithelium (RPE). The accumulation of damaged proteins in aged RPE is associated with disruption of proteolytic pathways and exocytic activity, with release of intracellular proteins via exosomes (Exo), that are important players in intercellular communication and can contribute to disease progression. However, the impact of their secretion by polarized RPE on outer blood retinal barrier (oBRB) breakdown remains largely elusive Objectives Our aim was to explore the role of inflammation on the loss of RPE integrity and to understand the relative role of directional secretion of Exo by RPE in the loss of polarity and barrier disruption Methodology We used a human RPE cell line (ARPE-19), highly polarized RPE primary cultures (pRPE) and porcine eyecups. To mimic the inflammatory conditions present in AMD, cells were treated with two inflammatory stimuli, TNF (10 ng/mL) or LPS (100 ng/mL) Results TNF and LPS do not affect the viability of the RPE cells. RPE cells developed a confluent monolayer and reached a relatively constant TER of about 40 Ω/cm2 (ARPE-19) or higher than 150 Ω/cm2 (pRPE). Treatment with TNF significantly reduces the TER, decreased immunoreactivity and co-localization of the TJ proteins ZO-1 and occludin and increases MMP-2/-9 activity in the medium. Apical Exo isolated from the RPE cells are enriched in CD63 compared to the basolateral Exo, that are enriched in CD81. The Exo isolated from porcine eyecups, especially with the LPS stimulus, are enriched in CD81 and MMP-2 but have similar levels of CD63 Conclusion Overall, our results show that inflammation induces loss of RPE integrity and release of different populations of Exo. The unravelling of novel drug targets paves the way for development of new therapeutic strategies for AMD.

Cytosolic LPS sensing triggers the release of a lectin DAMP that promotes inflammation

Abstract Activation of inflammatory caspases, such as caspase-11 and caspase-4, by lipopolysaccharide (LPS) from Gram-negative bacteria, is a key mechanism of innate immune defense against infections. This cytosolic LPS sensing pathway results in activation of a pore-forming protein, gasdermin D, which mediates an inflammatory form of cell death called pyroptosis. In addition, caspase-11-mediated cytosolic LPS sensing leads to the activation of caspase-1 and the maturation of IL-1b and IL-18. Importantly, cytosolic LPS-induced pyroptosis is associated with the release of intracellular proteins that act as alarmins or damage-associated molecular patterns (DAMPs) to modulate the inflammatory response and play an important role during infectious diseases and sepsis. However, the identity and function of DAMPs released downstream of caspase-11 activation are poorly characterized. Using a proteomic approach involving ProteomeLab’s PF2D fractionation system followed by mass spectrometry, we have discovered that galectin-1, a b-galactoside-binding lectin, is released as a consequence of cytosolic LPS sensing. By utilizing various mutant cell lines and primary cells we show that galectin-1 secretion is dependent on caspase-11 and gasdermin D, but not NLRP3 or caspase-1. Importantly, in vivo studies using galectin-1-deficient mice showed that galectin-1 acts as a DAMP to promote systemic inflammation and plays a detrimental role during septic shock. Additionally, human septic patients were found to have increased levels of galectin-1 in their serum. Taken together, we have characterized the secretion and function of a novel lectin DAMP released upon lytic cell death and provide new insights into outcomes of inflammasome activation.

Cell-type specific role of non-canonical inflammasome during LPS septic shock

Abstract Cytosolic sensing of LPS through caspase-11 is the key step of non-canonical inflammasome activation and innate immune defense against Gram-negative bacterial infections. Upon LPS sensing, caspase-11 induces GSDMD-mediated pore formation in the plasma membrane, followed by maturation and secretion of caspase-1 and pro-inflammatory cytokines (IL1band IL18). Excessive pore formation on the membrane leads to pyroptotic death of cell and release of intracellular proteins; many of which exacerbate the immune responses to infection, leading to cytokine storm, tissue damage, organ failure, and death. Caspase-11 sensing of LPS occurs in various cell-types, however the contribution of these cell-types to non-canonical inflammasome mediated pyroptosis, cytokine release, and lethality during LPS septic shock is poorly understood. To understand the cell type-specific role of caspase-11 during LPS septic shock, caspase-11 was conditionally deleted in multiple cell-types including macrophages by utilizing caspase-11 floxed mice and several appropriate Cre strains. The conditional KOs of caspase-11 were subjected to LPS challenge and various responses were assessed. Our in vivostudies showed cell-type specific contribution of caspase-11 to intracellular LPS-induced responses such as gasdermin D activation and lethality. Particularly, myeloid cell-intrinsic expression of caspase-11 is required for pathological manifestations of intracellular LPS sensing. Overall, our findings provide new insights into the cell type-specific role of cytosolic LPS sensing.

Rhabdomyolysis observed at forensic autopsy: a series of 52 cases.

Rhabdomyolysis is characterized by skeletal muscle injury resulting in the release of intracellular proteins (such as myoglobin) and electrolytes into the blood circulation, which cause acute kidney injury, myoglobinuria and electrolyte imbalances. Clinical diagnosis of rhabdomyolysis is made on the basis of biochemical analysis; however, for forensic autopsies, biochemical data are often not available, and it is necessary to diagnose rhabdomyolysis via histopathological examinations. This study analyzed 52 cases with rhabdomyolysis and applied myoglobin immunohistochemistry to kidney, urine and blood samples. We found that blunt force injuries were the most common cause of rhabdomyolysis across all age groups, and drugs were the second most common cause. The drugs included ketamines, amphetamines, synthetic cathinones, entheogens, benzodiazepines, opioid analgesics, and anesthesia. Less than 60% of our cases had biochemical data, including myoglobin (92.5~416,978ng/mL), creatine kinase (220~774,015U/L), potassium (1.6~10.3meq/L), calcium (2.7~29.2mg/dL), and phosphorus (2.6~14.2mg/dL). In the kidney tissue sections, we found that 95% of the rhabdomyolysis cases were positive for myoglobin immunohistochemistry and that 96% were associated with acute tubular necrosis. Our findings describe the features of fatal rhabdomyolysis in a large series and suggest that myoglobin immunohistochemistry can be used in post-mortem blood and urine cell blocks to detect myoglobin.

Microencapsulation extends mycelial viability of Streptomyces lividans 66 and increases enzyme production

BackgroundFilamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics.ResultsHere, we applied immobilization of Streptomyces lividans 66 using alginate as semi-solid matrix. This alginate-mediated micro-encapsulation increased the production of the extracellular enzyme tyrosinase more than three-fold. The increased production was accompanied by extended viability of the mycelium and a dramatic reduction in the release of intracellular proteins into the culture broth.ConclusionsOur data demonstrate the utility of micro-encapsulation as a powerful technique to achieve higher yields and lower downstream-processing costs of streptomycetes.

Permeability enhancement of Escherichia coli by single-walled carbon nanotube treatment.

This research investigated the use of single-walled carbon nanotubes (SWNTs) as an additive to increase the permeability of a bacterial cell wall. Recombinant Escherichia coli BL21 (DE3) that expressed β-lactamase were exposed to SWNTs under various levels of concentration and agitation. Activity of β-lactamase in the culture fluid and transmission electron microscopy (TEM) were used to determine the amount of released protein, and visually examine the permeability enhancement of the cells. It was found that β-lactamase release in the culture fluid occurred in a dose-dependent manner with treatment by SWNTs and was also dependent on agitation rate. Based on TEM, this treatment successfully caused an increase in permeability without significant damage to the cell wall. Consequently, SWNTs can be used as an enhancement agent to cause the release of intracellular proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:654-657, 2017.

Biological Effect and Inactivation Mechanism of Bacillus subtilis Exposed to Pulsed Magnetic Field: Morphology, Membrane Permeability and Intracellular Contents

Biological effect and inactivation mechanism of Bacillus subtilis exposed to pulsed magnetic field (PMF) were investigated. Cell morphology, membrane permeability, release of intracellular proteins and damage of deoxyribonucleic acid (DNA) were monitored. The inactivation effect of PMF treatment on B. subtilis was enhanced with the increase in intensity and pulse number. Scanning electron microscopy (SEM) analysis showed that the PMF treated bacterial cells had rough surfaces and damaged cellular membranes. Flow cytometry measurements showed that the cell membrane permeability increased after the PMF treatment. Leakage of intracellular contents, measured as OD, significantly increased with the increase in PMF intensity and pulsed number. A maximum leakage of cytoplasmic contents was detected at intensity of 3.30 T and 30 pulses. Enterobacterial repetitive intergenic consensus - polymerase chain reaction (ERIC-PCR) fingerprint signaled that the DNA was fragmented after the PMF treatment at the intensity of 3.0 T with 30 pulses and 3.3 T with 10, 20 and 30 pulses. These findings may give clues to the mechanism of bacterial cell death due to the PMF treatment.

OPTIMIZATION OF CELL DISRUPTION IN RAPHIDOCELIS SUBCAPITATA AND CHLORELLA VULGARIS FOR BIOMARKER EVALUATION

Raphidocelis subcapitata and Chlorella vulgaris are bioassay microalgae with rigid cellulosic cell wall which can hinder the release of intracellular proteins often studied as toxicity biomarkers. Since cell disruption is necessary for recovering intracellular biomolecules in these organisms, this study investigated the efficiency of ultrasonication bath; ultrasonication probe; vortexer; and bead mill in disintegrating the microalgae for anti-oxidative enzyme extraction. The extent of cell disruption was evaluated and quantified using bright field microscopy. Disrupted algae appeared as ghosts. The greatest disintegration of the microalgae (83-99.6 %) was achieved using bead mill with 0.42-0.6 mm glass beads while the other methods induced little or no disruption. The degree of cell disruption using bead mill increased with exposure time, beads-solution ratio and agitation speed while larger beads caused less disruption. Findings revealed that bead milling, with specific parameters optimized, is one of the most effective methods of disintegrating the robust algal cells.

Proteomic mapping of proteins released during necrosis and apoptosis from cultured neonatal cardiac myocytes

Cardiac injury induces myocyte apoptosis and necrosis, resulting in the secretion and/or release of intracellular proteins. Currently, myocardial injury can be detected by analysis of a limited number of biomarkers in blood or coronary artery perfusate. However, the complete proteomic signature of protein release from necrotic cardiac myocytes is unknown. Therefore, we undertook a proteomic-based study of proteins released from cultured neonatal rat cardiac myocytes in response to H2O2 (necrosis) or staurosporine (apoptosis) to identify novel specific markers of cardiac myocyte cell death. Necrosis and apoptosis resulted in the identification of 147 and 79 proteins, respectively. Necrosis resulted in a relative increase in the amount of many proteins including the classical necrotic markers lactate dehydrogenase (LDH), high-mobility group B1 (HMGB1), myoglobin, enolase, and 14-3-3 proteins. Additionally, we identified several novel markers of necrosis including HSP90, α-actinin, and Trim72, many of which were elevated over control levels earlier than classical markers of necrotic injury. In contrast, the majority of identified proteins remained at low levels during apoptotic cell death, resulting in no candidate markers for apoptosis being identified. Blotting for a selection of these proteins confirmed their release during necrosis but not apoptosis. We were able to confirm the presence of classical necrotic markers in the extracellular milieu of necrotic myocytes. We also were able to identify novel markers of necrotic cell death with relatively early release profiles compared with classical protein markers of necrosis. These results have implications for the discovery of novel biomarkers of necrotic myocyte injury, especially in the context of ischemia-reperfusion injury.

Mode of Antibacterial Activity of Eclalbasaponin Isolated from Eclipta alba

The present study was undertaken to evaluate the mode of antibacterial activity of Eclalbasaponin isolated from Eclipta alba, against selected Gram-positive and Gram-negative bacteria. The probable chemical structure was determined by using various spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR) and mass spectroscopy. The antibacterial activity was evaluated by well diffusion technique, pH sensitivity, chemotaxis, and crystal violet assays. Eclalbasaponin showed clear zone of inhibition against both Bacillus subtilis and Pseudomonas aeruginosa and exhibited growth inhibition at the pH range of 5.5-9.0. The isolated saponin exhibited its positive chemoattractant property for both bacterial strains. Results of crystal violet assay and the presence of UV-sensitive materials in the cell-free supernatant confirmed the cellular damages caused by the treatment of Eclalbasaponin. The release of intracellular proteins due to the membrane damage was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Changes in the cell surface structure and membrane disruption were further revealed by FTIR and scanning electron microscopy analysis. The present study suggests that the isolated saponin from E. alba causes the disruption of the bacterial cell membrane which leads to the loss of bacterial cell viability.

Microfluidic electroporation for cellular analysis and delivery

Electroporation is a simple yet powerful technique for breaching the cell membrane barrier. The applications of electroporation can be generally divided into two categories: the release of intracellular proteins, nucleic acids and other metabolites for analysis and the delivery of exogenous reagents such as genes, drugs and nanoparticles with therapeutic purposes or for cellular manipulation. In this review, we go over the basic physics associated with cell electroporation and highlight recent technological advances on microfluidic platforms for conducting electroporation. Within the context of its working mechanism, we summarize the accumulated knowledge on how the parameters of electroporation affect its performance for various tasks. We discuss various strategies and designs for conducting electroporation at the microscale and then focus on analysis of intracellular contents and delivery of exogenous agents as two major applications of the technique. Finally, an outlook for future applications of microfluidic electroporation in increasingly diverse utilities is presented.

Release of Intracellular Proteins by Electroporation with Preserved Cell Viability

Extraction of intracellular proteins from cells is often an important first step for conducting molecular biology and proteomics studies. Although ultrasensitive detection and analytical technology at the single molecule level is becoming routine, protein extraction techniques have not followed suit and still call for complete lysis that leads to cell death. In principle, with refined extraction techniques, intracellular proteins can potentially be extracted without killing the cell. In this Letter, we demonstrate that electroporation is capable of releasing intracellular proteins from adherent Chinese hamster ovary cells while preserving the cell viability. By tuning the duration and intensity of an electric pulse, we were able to control the average amount of protein release and the percentage of viable cells after the operation. Our results indicate that a substantial fraction of the cell population was able to release proteins under electroporation and survive the procedure. Interestingly, at the single cell level, the probability for cell death does not increase with more protein release. This work paves the way to extracting and analyzing intracellular proteins while keeping cells live.

Thymus maroccanusessential oil, a membranotropic compound active on Gram-negative bacteria and resistant isolates

The effects of Thymus maroccanus essential oil (EO) on the integrity of the cell membranes and the permeability of the outer membrane (OM) and inner membrane (IM) of Escherichia coli, Enterobacter aerogenes and Salmonella enterica Typhimurium were investigated. The bacterial release of intracellular proteins, cytoplasmic β-galactosidase and periplasmic β-lactamase induced by T.maroccanus EO was compared to the membranotropic activity of polymyxin B (PB) known as an effective permeabilizer of the membrane of Gram-negative bacteria. Results showed that T.maroccanus EO increased the permeability of the OM and IM of studied bacteria and induced the release of intracellular proteins into the external medium. The effect of T.maroccanus EO on the outer membrane was comparable to that of PB, and both T.maroccanus EO and PB induce similar levels of β-lactamase release. In addition, it also promoted the release of the cytoplasmic β-galactosidase. Moreover, the lipopolysaccharide molecules and the overexpression of efflux pumps seem to play a crucial role in the level of susceptibility of studied bacteria to the permeabilizing effect of T.maroccanus EO. These results demonstrate that T.maroccanus EO can restore antibiotic activity by targeting the two bacterial membranes and would be attractive candidates for developing new adjuvants for combating resistant Gram-negative bacteria.

Action of Chitosan Against Xanthomonas Pathogenic Bacteria Isolated from Euphorbia pulcherrima

The antibacterial activity and mechanism of two kinds of chitosan were investigated against twelve Xanthomonas strains recovered from Euphorbia pulcherrima. Results indicated that both chitosans markedly inhibited bacterial growth based on OD loss. Furthermore, the release of DNA and RNA from three selected strains was increased by both chitosans. However, the release of intracellular proteins was inhibited by both chitosans at different concentration and incubation times, except chitosan A at 0.1 mg/mL for 0.5 h incubation and 0.2 mg/mL for 2.0 h incubation increased the release of proteins, indicating the complexity of the interaction and cell membranes, which was affected by incubation time, bacterial species, chitosan type and concentration. Transmission electron microscopic observations revealed that chitosan caused changes in protoplast concentration and surface morphology. In some cells, the membranes and walls were badly distorted and disrupted, while other cells were enveloped by a thick and compact ribbon-like layer. The contrary influence on cell morphology may explain the differential effect in the release of material. In addition, scanning electron microscope and biofilm formation test revealed that both chitosans removed biofilm biomass. Overall, this study showed that membrane and biofilm play an important role in the antibacterial mechanism of chitosan.