Complete Cell Lysis Research Articles

High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine.

The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while β-mercaptoethanol (βME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2′-dithiodipyridine (DTDP) and observed that βME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals.

Efficient delivery of oncolytic enterovirus by carrier cell line NK-92

Many members of the enterovirus family are considered as promising oncolytic agents; however, their systemic administration is largely inefficient due to the rapid neutralization of the virus in the circulation and the barrier functions of the endothelium. We aimed to evaluate natural killer cells as carriers for the delivery of oncolytic enteroviruses, which would combine the effects of cell immunotherapy with virotherapy. We tested four strains of nonpathogenic enteroviruses against the glioblastoma cell line panel and evaluated the produced infectious titers. Next, we explored whether these virus strains could be delivered to the tumor by natural killer cell line NK-92, which is being actively evaluated as a clinically acceptable therapeutic. Several strains of enteroviruses demonstrated oncolytic properties, but only coxsackievirus A7 (CVA7) could replicate in NK-92 cells efficiently. We compared the delivery efficiency of CVA7 in vivo, using NK-92 cells and direct intravenous administration, and found significant advantages of cell delivery even after a single injection. This suggests that the NK-92 cell line can be utilized as a vehicle for the delivery of the oncolytic strain of CVA7, which would improve the clinical potential of this viral oncolytic for the treatment of glioblastoma multiforme and other forms of cancer.

Features of obtaining the protein complex of vegetative cultures Bacillus anthracis for proteomic mapping of strains

Introduction. The study of the protein composition of the causative agent of anthrax — Bacillus anthracis, allows you to identify both general species and individual characteristics of strains that differ in phenotypic properties, manifested mainly in the vegetative form and which are important for virulence, immunogenicity and the ability to adapt to different vegetation conditions.Purpose of the work. In the group of anthrax microbe strains having different plasmid composition and virulence, different methods of extraction of the total proteome from vegetative bacillus cells have been tested.Results. In the course of the work, it was shown that the rate of spore formation varies significantly between individual strains of the anthrax microbe and can have a significant impact on the efficiency of extraction and the composition of the protein complex. Preliminary treatment with lysozyme, which affects the cell membrane, promotes a more complete lysis of cells, and ultramicrocentrifuge filtration provides complete specific sterility of the obtained samples.Conclusion. A culture preparation scheme was developed for B. anthracis, which allows one to obtain a culture in the vegetative phase of the life cycle and to efficiently extract proteins in combination with reliable disinfection of samples.

Daudi cell stroma: An alternative to dithiothreitol to resolve daratumumab interference in pretransfusion testing.

The Daudi B-cell line was found to express high levels of CD38 and was sonicated in a sonication buffer to achieve complete cell lysis. The resulting stroma preparation was centrifuged at 20 000g for 20 minutes and then mixed with 250 μL of DARA-plasma and incubated for 10 minutes at 37°C. The stroma-DARA-plasma mixture was centrifuged again, and the supernatant was collected and subjected to four additional rounds of adsorption with fresh stroma. DARA-depleted plasma was tested by gel indirect antiglobulin test (IAT). CD38 expression on Daudi cells was confirmed by flow cytometry. Gel IAT analysis showed that the incubation of plasma from DARA-treated patients with Daudi cells stroma resulted in a significant depletion of DARA but allowing detection of other alloantibodies of interest such as anti-K, anti-Yta , and anti-Gya . Daudi cell stroma is inexpensive, easy to prepare in large batches, and can be used as an off-the-shelf reagent. Incubation of plasma from DARA-treated patients with Daudi cell stroma can efficiently overcome DARA interference in serologic testing without affecting DTT- or trypsin-sensitive antigens.

Growth inhibition of Microcystis aeruginosa and adsorption of microcystin toxin by the yeast Aureobasidium pullulans, with no effect on microalgae.

This study evaluates the inhibitory effect of a yeast strain, Aureobasidium pullulans KKUY0701, isolated from decayed cyanobacterial bloom against harmful cyanobacterium Microcystis aeruginosa and determines the ability of this strain to remove microcystin (MC) toxin from the water. The antialgal activity of this yeast strain was assayed by co-cultivation with M. aeruginosa, diatom, and green algal species. The MC adsorption experiment was conducted in the presence of living and heat-inactivated yeast cells. Both yeast cells and filtrates caused a rapid reduction in the growth of M. aeruginosa, with complete death and cell lysis occurring after 3days. The yeast strain did not exhibit any inhibitory effect on either green algae or diatoms. Both living and heat-inactivated yeast cells were capable of adsorption of MC on their surfaces. Inactivated yeast exhibited higher adsorption capacity and lower intensity than living yeast for the adsorption of MC toxin. The results of this study suggest that this yeast strain could be employed to selectively reduce cyanobacterial blooms in freshwaters. Moreover, the application of heat-inactivated yeast's biomass for toxin adsorption gives new possibilities in drinking water treatment plants.

Effect of Viral Infection on the Functioning and Lysis of Black Sea Microalgae Tetraselmis viridis (Chlorophyta) and Phaeodactylum tricornutum (Bacillariophyta)

Experimental studies have been performed on Tetraselmis viridis (Rouchijajnen) R.E. Norris, Hori & Chihara (Chlorophyta) and Phaeodactylum tricornutum Bohlin (Bacillariophyta) cultures of Black Sea microalgae using TsV-S1 and PtV-S18 algal virus strains isolated from the Black Sea ecosystem. The study assesses the effects of light intensity and the initial abundance of microalgae cells on the onset of their infection by viruses and the decline in their abundance. As early as on the second or third day, the cells changed their shape and increased in volume in the cultures infected by viruses. At this time, a decrease was observed in chlorophyll a red autofluorescence, variable fluorescence per cell, and photochemical efficiency of photosystem 2. In the studied cultures, a reduction in cell abundance due to viral lysis was noted 1 to 2 days later. The threshold cell abundance at which this process was observed was independent of light conditions and amounted to 3 × 105 cells/mL for T. viridis and 18 × 105 cells/mL for Ph. tricornutum. The complete lysis of algae cells was detected by the end of the fourth to sixth day. In the dark, the effect of viruses on microalgae was insignificant.

Local Effects of Two Intravenous Formulations of Pulmonary Vasodilators on Airway Epithelium.

Intravenous formulations of epoprostenol are frequently delivered via nebulizer to treat pulmonary hypertension in acutely ill patients. Although their efficacy as pulmonary vasodilators has been shown to be comparable to inhaled nitric oxide, the local effects of these formulations within the airways have not been determined. We hypothesized that the alkaline diluents of these compounds would lead to increased airway epithelial cell death and ciliary cessation. Human bronchial epithelial cells were exposed to epoprostenol in glycine and arginine diluents or control fluid. Ciliary beat frequency, lactate dehydrogenase, and total RNA levels were measured before and after exposure. Results were compared between exposure and control groups. Ciliary beat frequency ceased immediately after exposure to epoprostenol with both diluents. Lactate dehydrogenase levels increased by 200% after exposure to epoprostenol and glycine diluent (P = .002). Total RNA levels were undetectable after exposure to epoprostenol and arginine, indicating complete cell death and lysis (P = .015). Ciliary beat frequency ceased after 30 s of exposure to epoprostenol and glycine (P = .008). There was no difference between cells exposed to epoprostenol and those exposed only to diluent. Exposure to intravenous formulations of epoprostenol in glycine and arginine caused increased cell death and ciliary cessation in bronchial epithelial cells. These findings suggest that undesired local effects may occur when these compounds are delivered as inhaled aerosols to patients.

Isolation and Genomic Characterization of phiVibrioH1 a Myoviridae Phage for Controlling Pathogenic Vibrio parahaemolyticus from Seafood and Human

Vibrio parahaemolyticus is a zoonotic pathogen causing vibriosis in marine fish and associated with food poisoning outbreaks in humans. This study aimed to isolate and characterize bacteriophage against multidrug resistant (MDR) Vibrio parahaemolyticus from seafood and humans, and also to assess the lytic efficacy of phage on growth. Vibrio Parahaemolyticus was isolated from flesh of 80 white shrimp, 70 blue crabs and 50 mullets, and from 50 hand swabs of humans (fish handlers) on Thiosulphate Citrate bile salts sucrose agar media. The suspected colonies were biochemically identified. Pathogenic V. parahaemolyticus carrying thermostable direct hemolysin (tdh+) gene was molecularly detected . The pathogenic tdh+V. parahaemolyticus (n=25) isolates were explored for antimicrobial vulnerability resistant to 12 antimicrobials employing the disc diffusion method. Fifteen isolates of tdh+V. parahaemolyticus were 100% resistant to five antimicrobials. Bacteriophage was isolated from sewage water using spot test and double over layer agar assay. The phiVibrioH1 was belonged to family Myoviridae according to transmission electron microscopy. This study revealed a polyvalent phage infecting wide host range of MDR V. parahaemolyticus, V. vulnificus, V. fluvialis, V. alginolyticus, Pseudomonas aeruginosa, E. coli O26 and Proteus vulgaris. The genome of phage was sensitive to digestion with BamH1 and ECoR1 restriction enzymes indicating double stranded DNA. From 24 to 42 hours post-treatment of pathogenic V. parahaemolyticus with the PhiVibrioH1 phage showed complete lysis of bacterial cells. This study confirmed that the phiVibrioH1 is a lytic phage and has a high potential to control pathogenic V. parahaemolyticus strains recovered from seafood and humans.

Comparison of the efficiency of different cell lysis methods and different commercial methods for RNA extraction from Candida albicans stored in RNAlater

BackgroundObtaining sufficient RNA yield and quality for comprehensive transcriptomic studies is cumbersome for clinical samples in which RNA from the pathogen is present in low numbers relative to the nucleic acids from the host, especially for pathogens, such as yeasts, with a solid cell wall. Therefore, yeast cell lysis including cell wall disruption constitutes an essential first step to maximize RNA yield. Moreover, during the last years, different methods for RNA extraction from yeasts have been developed, ranging from classic hot phenol methods to commercially available specific kits. They offer different RNA yield and quality, also depending on the original storage medium, such as RNAlater.ResultsWe observed that, for C. albicans cells stored in Tryptic Soy Broth with 15% glycerol, 10 min of bead beating in a horizontal position in RiboPure Lysis Buffer provided complete cell lysis. Cell lysis efficiency was decreased to 73.5% when cells were stored in RNAlater. In addition, the RiboPure Yeast Kit (Ambion) offered the highest RNA yield in comparison with the automated platform NucliSENS easyMAG total nucleic extraction (bioMérieux) and the RNeasy Mini Kit (Qiagen) according to NanoDrop and Fragment Analyzer. Moreover, we showed that, in spite of the decrease of cell lysis efficiency after RNAlater storage, as compared to storage in TSB + 15% glycerol, RNAlater increased RNA yield during RNA extraction with both RiboPure Yeast Kit and easyMAG, as confirmed by Fragment Analyzer analysis and by RT-qPCR of the RNA from the Internal Transcribed Spacer 2.ConclusionsIn our hands, the most efficient cell lysis and highest RNA yield from C. albicans cells stored in RNAlater was obtained by horizontal bead beating in RiboPure Lysis Buffer followed by RNA extraction with the RiboPure Yeast Kit.

The three cytokines IL-1β, IL-18, and IL-1α share related but distinct secretory routes

Interleukin (IL)-1 family cytokines potently regulate inflammation, with the majority of the IL-1 family proteins being secreted from immune cells via unconventional pathways. In many cases, secretion of IL-1 cytokines appears to be closely coupled to cell death, yet the secretory mechanisms involved remain poorly understood. Here, we studied the secretion of the three best-characterized members of the IL-1 superfamily, IL-1α, IL-1β, and IL-18, in a range of conditions and cell types, including murine bone marrow–derived and peritoneal macrophages, human monocyte–derived macrophages, HeLa cells, and mouse embryonic fibroblasts. We discovered that IL-1β and IL-18 share a common secretory pathway that depends upon membrane permeability and can operate in the absence of complete cell lysis and cell death. We also found that the pathway regulating the trafficking of IL-1α is distinct from the pathway regulating IL-1β and IL-18. Although the release of IL-1α could also be dissociated from cell death, it was independent of the effects of the membrane-stabilizing agent punicalagin, which inhibited both IL-1β and IL-18 release. These results reveal that in addition to their role as danger signals released from dead cells, IL-1 family cytokines can be secreted in the absence of cell death. We propose that models used in the study of IL-1 release should be considered context-dependently.

A Next-Generation Fc-Bearing CD3-Engaging Bispecific DART® Platform with Extended Pharmacokinetic and Expanded Pharmacologic Window: Characterization As CD123 x CD3 and CD19 x CD3 DART Molecules

Background: CD3-engaging DART molecules direct T cells to tumor-expressed antigens. Flotetuzumab, a CD123 x CD3 bispecific DART molecule that targets the differential expression of the IL-3 receptor alpha chain (CD123) on acute myeloid leukemia (AML) blasts and leukemic stem cells, is currently being investigated in a phase 1 study in relapsed/refractory AML and myelodysplastic syndrome (MDS), with initial evidence of clinical activity1. A common adverse event in redirected T-cell killing interventions is cytokine release syndrome (CRS), a Cmax-associated event that may limit their therapeutic window. Owing to its short circulating half-life, flotetuzumab is administered as continuous infusion, which affords prolonged exposure with limited Cmax excursions. In contrast, long-acting Fc-bearing, CD3-engaging molecules would eliminate the need for continuous dosing, but would be especially impacted by CRS, given the high Cmax levels required to maintain adequate trough concentrations over several days or weeks. We previously reported the development of a CD3-engaging DART molecule with reduced affinity for CD3 that maintained maximal target cell killing and T-cell proliferation, albeit at higher concentrations than its wild-type (WT) counterpart, but with reduced cytokine release2. We report here further optimization and validation of the approach.Methods: Anti-CD3 variants spanning a range of affinities were formatted as Fc-bearing DART molecules with CD123- or CD19-targeting arms and evaluated for cell binding as well as for antigen-dependent induction of T-cell proliferation, cytolytic activity and cytokine release. A therapeutic index (TI) was determined from the ratio of in vitro cell killing activity to that of target cell-induced cytokine release. In vivo anti-tumor activity was assessed in human immune cell-reconstituted mouse tumor models or in mice expressing the epitope recognized by the CD3 arm of the DART proteins (human CD3 k/i mice). Safety, pharmacokinetic (PK) and biological activity of selected molecules was evaluated in cynomolgus monkeys.Results: Six Fc-bearing DART molecules (from a panel of 23 CD123 x CD3 DART variants) were selected for characterization. These molecules spanned a relatively small interval of low CD3 affinities, but varied greatly in activity, from inactive at all concentrations tested to being capable of lysing tumor cells, including primary leukemic and MDS blasts, to the same maximal level of killing observed with the parental WT DART molecule. Higher concentrations of the active variants were needed, owing to a deliberate design incorporating decreased CD3 binding. At levels capable of complete tumor cell lysis, active DART variants showed greatly reduced pro-inflammatory cytokine release (e.g., IL-2, IFN-γ and TNF-α) in vitro and in human PBMC-reconstituted mice compared to the WT version. Molecules were ranked based on their TI and a version with a highly favorable index was engineered as a B-cell targeting CD19 x CD3 DART molecule to conveniently ascertain its biological activity in cynomolgus monkeys. The CD19 x CD3 DART variant demonstrated good tolerability at single doses up to 30 mg/kg. B-cell depletion in the circulation and lymphoid tissues occurred at 1 mg/kg and achieved maximal depletion at ≥10 mg/kg, equivalent to the degree of reduction observed with 0.1 mg/kg of the WT molecule. However, only minimal circulating pro-inflammatory cytokines (including IL-6) were observed at all doses of the DART variant, while a substantial increase in IL-2, IL-6 and IFN-γ (among others) was observed with 0.1 mg/kg of the WT counterpart. The PK profile of the DART variant in cynomolgus monkeys appeared linear in the 1-30 mg/kg interval, with a half-life of 5 to 10 days, consistent with possible clinical dosing at weekly or longer intervals.Conclusions: A next-generation CD3-engaging Fc-bearing DART platform was generated through CD3 arm affinity modulation. These DART variants feature extended PK and an expanded TI suitable for potential clinical development in hematological malignancies.1. Uy et al, Blood 2017, vol. 130, Suppl 1 6372. Huang et al, Keystone Symposia: Antibodies as Drugs, 2/25-3/1/2018, Whistler, BC, Canada DisclosuresBonvini:MacroGenics: Employment, Equity Ownership. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Huang:MacroGenics, Inc.: Employment, Equity Ownership. Lam:MacroGenics, Inc.: Employment, Equity Ownership. Kaufman:MacroGenics, Inc.: Employment, Equity Ownership. Liu:MacroGenics, Inc.: Employment, Equity Ownership. Alderson:MacroGenics, Inc.: Employment, Equity Ownership. Stahl:MacroGenics, Inc.: Employment, Equity Ownership. Brown:MacroGenics, Inc.: Employment, Equity Ownership. Li:MacroGenics, Inc.: Employment, Equity Ownership. Sharma:MacroGenics, Inc.: Employment, Equity Ownership. Tamura:MacroGenics, Inc.: Employment, Equity Ownership. Johnson:MacroGenics, Inc.: Consultancy, Equity Ownership. Moore:MacroGenics, Inc.: Employment, Equity Ownership.

P3.01-83 Use of Blood Outgrowth Endothelial Cells as a Cellular Carrier for Oncolytic Vesicular Stomatitis Virus in Preclinical Models of NSCLC

Oncolytic virus therapy has demonstrated efficacy in numerous tumor models including non-small cell lung cancer. One of the limitations of viral therapy for metastatic lung cancer is that systemic administration can be hindered by complement and antiviral immunity. Thus, we investigated the possibility of using ex-vivo infected blood outgrowth endothelial cells with tumor-homing properties to deliver oncolytic VSV-IFNβ in preclinical models of NSCLC. BOECs were obtained from either human donors or C57/Bl6 mice. Cells were confirmed to maintain the BOEC phenotype and growth characteristics. Indiana strains of VSV were engineered to produce GFP or IFNβ and were titered on Vero cells using either plaque assay or limiting dilution assay. Human NSCLC cell lines, H2009 and H2030 were used for in vitro assays. A549 expressing firefly luciferase cells were used to induce lung metastasis in NOD/SCID mice and treated with BOECs, VSV-IFNβ, or BOECs-infected with VSV-IFNβ. Additionally, syngeneic murine adenocarcinoma cell line, LM2 was used in vivo in A/J mice (n=5). BOEC cells were able to home to metastatic LM2 lung tumors and were retained there for up to 72 hours post-infusion. BOEC cells were retained within lung tumors of mice bearing tumors, but there were none detected in lungs of mice without lung tumors. Both human and murine BOECs could be infected and lysed by VSV-GFP and VSV-IFNβ, however, VSV-IFNβ was attenuated compared to VSV-GFP. Maximal viral titer was obtained at 24 or 48 hours for VSV-GFP and VSV-IFNβ, respectively. Co-culture experiments showed near complete lysis of H2009 cells using infected BOECs. Both H2009 and H2030 cells were lysed efficiently by infected BOECs while naked VSV was completely inhibited in the presence anti-VSV neutralizing antibodies. Using Firefly luciferase-expressing A549 cells, metastatic lung tumors were induced in NOD/SCID mice. Compared to BOEC alone and PBS-treated mice, VSV-IFNβ-infected BOECs resulted in superior antitumor efficacy as measured by luciferase activity (p<0.02). Infected BOECs resulted in superior survival of mice compared to VSV-IFNβ alone (n=10, p<0.05). Using immune competent A/J mice, infected BOECs trended toward improved antitumor efficacy to BOEC alone and intravenous VSV-IFNβ treatment (n=5, p=0.09). Replicating virus was recovered only from lungs of infected BOEC treated mice. BOECs can be used as cellular carrier for systemic delivery of oncolytic VSV-IFNβ. For clinical translation, the use of cellular carriers might be an effective method of virotherapy for metastatic NSCLC.

How myristyltrimethylammonium bromide enhances biomass harvesting and pigments extraction from Synechocystis sp. PCC 6803

Myristyltrimethylammonium bromide (MTAB) is a cationic surfactant used to improve biomass harvesting and pigment extraction form microalgae, but the mechanisms underlying its effectiveness are poorly defined. We document the mechanisms for enhanced harvesting and pigment extraction for the cyanobacterium Synechocystis sp. PCC 6803 using measurements from flow cytometer, zeta potential, release of soluble components, and microscopy. Harvesting efficiency increased as the MTAB/Biomass dose increased from 0 to 40%. A low MTAB dose (≤ 8%) mainly brought about coagulation and flocculation, which led to aggregation that improved harvesting, but 40% MTAB had the highest harvesting efficiency, 62%. Adding MTAB above a MTAB/Biomass dose of 8% also increased cell-membrane permeability, which allowed the solvent (ethyl acetate) to pass into the cells and resulted in a large increase in extraction efficiency of pigments: An MTAB/Biomass ratio of 60% for 180 min achieved the highest extraction efficiencies of chlorophyll and carotenoids, 95% and 91%, respectively. Combining harvesting and extraction performances with results from flow cytometry, zeta potential, release of soluble components, and microscopy lead to the following mechanistic understandings. MTAB dose from 8% to 40% solubilized EPS, which lowered the biomass's negative charge, but caused breakup of the large aggregates. An increase of cell permeability also in this stage allowed ethyl acetate to pass into the cells and achieve better pigment extraction. MTAB >40% led to cell lysis and a large increase in soluble organics, but complete cell lysis was not required to achieve the maximum extraction efficiency. The MTAB/Biomass % ratio for optimizing harvest efficiency and pigment extraction lay in the range of 40%–60%.

Listeria Inactivation by the Combination of High Hydrostatic Pressure and Lactocin AL705 on Cured-Cooked Pork Loin Slices

The effect of the bacteriocin lactocin AL705 in combination with high hydrostatic pressure (HHP) on the inactivation of Listeria innocua 7, a nonpathogenic indicator for Listeria monocytogenes, deliberately inoculated (ca. 6.4 log CFU/g) onto the surface of ready-to-eat (RTE) sliced cured-cooked pork loin, was evaluated. Nontreated pork slices (control) and treatments subjected to lactocin AL705 (105 AU/ml) and/or HHP (400 or 600 MPa) were prepared. L. innocua 7 was monitored at days 1, 20, and 40 of storage at 4 °C. The results showed a complete inhibition of L. innocua 7 after the combined treatment with lactocin AL705 and 600 MPa and no regrowing of cells up to 40-day storage. The treatment at 600 MPa alone was not enough to avoid regrowth of L. innocua. Ultrastructural cell damage was observed at the cytoplasm and cell membrane/wall levels with all treatments; however, complete cell lysis was observed only with the combined treatment. HHP in combination with lactocin AL705 provided a wider margin of safety as post-processing listericidal treatment of RTE cured-cooked meat products.

Antagonistic Activity and Mode of Action of Phenazine-1-Carboxylic Acid, Produced by Marine Bacterium Pseudomonas aeruginosa PA31x, Against Vibrio anguillarum In vitro and in a Zebrafish In vivo Model.

Phenazine and its derivatives are very important secondary metabolites produced from Pseudomonas spp. and have exhibited broad-spectrum antifungal and antibacterial activities. However, till date, there are few reports about marine derived Pseudomonas and its production of phenazine metabolites. In this study, we isolated a marine Pseudomonas aeruginosa strain PA31x which produced natural product inhibiting the growth of Vibrio anguillarum C312, one of the most serious bacterial pathogens in marine aquaculture. Combining high-resolution electro-spray-ionization mass spectroscopy and nuclear magnetic resonance spectroscopy analyses, the functional compound against V. anguillarum was demonstrated to be phenazine-1-carboxylic acid (PCA), an important phenazine derivative. Molecular studies indicated that the production of PCA by P. aeruginosa PA31x was determined by gene clusters phz1 and phz2 in its genome. Electron microscopic results showed that treatment of V. anguillarum with PCA developed complete lysis of bacterial cells with fragmented cytoplasm being released to the surrounding environment. Additional evidence indicated that reactive oxygen species generation preceded PCA-induced microbe and cancer cell death. Notably, treatment with PCA gave highly significant protective activities against the development of V. anguillarum C312 on zebrafish. Additionally, the marine derived PCA was further found to effectively inhibit the growth of agricultural pathogens, Acidovorax citrulli NP1 and Phytophthora nicotianae JM1. Taken together, this study reveals that marine Pseudomonas derived PCA carries antagonistic activities against both aquacultural and agricultural pathogens, which broadens the application fields of PCA.

How to trigger periplasmic release in recombinant Escherichia coli: A comparative analysis.

Recombinant protein production in Escherichia coli usually leads to accumulation of the product inside the cells. To capture the product, cells are harvested, resuspended, and lysed. However, in cases where the product is transported to the periplasm, selective disruption of the outer membrane leads to much purer crude extracts compared to complete cell lysis, as only 4-8% of the native E. coli host cell proteins are located in the periplasmic space. A variety of different strategies to enable selective release of the product from the periplasm is available. However, in most of these studies cells are harvested before they are resuspended in permeabilization agent and no differentiation between leakiness and lysis is made. Here, we tested and compared different strategies to trigger leakiness. In contrast to other studies, we performed these experiments during cultivation and quantified both leakiness and lysis. In summary, we recommend incubation with 350 mM TRIS at constant pH for several hours followed by a mild heat treatment up to 38°C to trigger leakiness with only minimal lysis. This study represents a comparative summary of different strategies to trigger E. coli leakiness and describes a solid basis for further experiments in this field.

Microbial chitosan as a biopreservative for fish sausages

Processed fish products are worthy sources to supply man with his main nutritional needs, but they are extremely susceptible to quality loss during storage. Microbial (fungal) chitosan is a bioactive polymer that has numerous applications in health promoting fields. Fungal chitosan was extracted from the grown mycelia of Aspergillus brasiliensis (niger) to investigate its potential role as antimicrobial, preservative and quality improvement agent, in processed fish sausages from Nile tilapia (Oreochromis niloticus). The produced chitosan had a molecular weight of 29kDa, deacetylation degree of 91% and solubility of 99% in acetic acid solution. Fish sausages supplementation with 1.5% chitosan resulted in sharp reductions of microbial load (Total aerobic microorganisms, coliforms, yeast & molds, E. coli, Enterobacteriaceae and Staphylococcus aureus) during cold storage, at 4°C for 28days. Sensory attributes were notably enhanced in stored chitosan-supplemented sausages, especially the odor and taste characteristics. Captured micrographs, of exposed S. aureus to chitosan, exhibited vigorous morphological alterations after 4h, and complete cell lysis after 8h of exposure period. A. brasiliensis chitosan, however, could be strongly recommended, as a supplement for Nile tilapia fish sausages, to maintain microbiological quality and enhance sensory attributes of the product during storage.

Tracking micro-optical resonances for identifying and sensing novel procaspase-3 protein marker released from cell cultures in response to toxins

The response of cells to toxins is commonly investigated by detecting intracellular markers for cell death, such as caspase proteins. This requires the introduction of labels by the permeabilization or complete lysis of cells. Here we introduce a non-invasive tool for monitoring a caspase protein in the extracellular medium. The tool is based on highly sensitive optical micro-devices, referred to as whispering-gallery mode biosensors (WGMBs). WGMBs are functionalized with antibodies for the specific and label-free detection of procaspase-3 released from human embryonic kidney HEK293 and neuroglioma H4 cells after introducing staurosporine and rotenone toxins, respectively. Additional tests show that the extracellular accumulation of procaspase-3 is concomitant with a decrease in cell viability. The hitherto unknown release of procaspase-3 from cells in response to toxins and its accumulation in the medium is further investigated by Western blot, showing that the extracellular detection of procaspase-3 is interrelated with cytotoxicity of alpha-synuclein protein (aSyn) overexpressed in H4 cells. These studies provide evidence for procaspase-3 as a novel extracellular biomarker for cell death, with applications in cytotoxicity tests. Such WGMBs could be applied to further identify as-yet unknown extracellular biomarkers using established antibodies against intracellular antigens.

Algicidal effects of aqueous leaf extracts of neem (Azadirachta indica) on Scenedesmus quadricauda (Turp.) de Brébission

The application of synthetic algaecides for the control of algae produces by-products that are sometimes toxic to the environment. There is a need for natural and cheap alternatives to synthetic algaecides. In the present study, we investigated the potential of aqueous crude extract of Azadirachta indica to inhibit the growth of Scenedesmus quadricauda. Phytochemical screening of the extract revealed the presence of groups of bioactive compounds that are capable of inhibiting microalgal growth. Chlorophyll a concentration, dry weight production and cell density of microalga decreased with increasing crude extract concentration. After three days of exposure, the 1000 mg/L extract concentration resulted in complete growth inhibition and cell lysis. Furthermore, the ability of S. quadricauda to form multi-celled coenobial structures was compromised in a concentration dependent manner. In general, catalase and peroxidase activities of the microalga were upregulated with increasing extract concentration. These results imply that aqueous neem extract may provide a cheap and ecofriendly alternative for the control of microalgae in aquatic ecosystems.

Use of lactic acid bacteria as a biological agent against the cyanobacterium Anabaena flos-aquae

In the present study, we assessed whether lactic acid bacteria (LAB) with antimicrobial activity could be used to effectively control Anabaena flos-aquae growth. In our screening of cyanobacteriacidal bacteria from 14 LAB strains belonging to 11 species, we selected six candidate strains that could lyse 90 % or more A. flos-aquae cells compared with a control. Of those, Lactobacillus paraplantarum KCTC 5045T had the strongest cyanobacteriacidal activity showing complete lysis of cyanobacterial cells at the initial densities of ≥104 cells mL−1. A host range assay revealed that L. paraplantarum strongly inhibited A. flos-aquae, Anabaena crassa, Stephanodiscus hantzschii, and Peridinium bipes but weakly inhibited or stimulated the growth of Scenedesmus actus, Pediastrum sp., Cyclotella meneghiniana, Coelastrum reticulatum, Chlamydomonas sp., and Microcystis aeruginosa. In the microcosm experiment using natural freshwater including abundant phytoplankton assemblage, moreover, the LAB under inoculation of about 105 cells mL−1 could completely terminate the co-growth of A. flos-aquae, A. crassa, Anabaena circinalis, and other Anabaena spp. (over 104 cells mL−1) without a secondary bloom by another algal and cyanobacterial species. Our observation revealed that the cyanobacteriacidal bacterium releases two or more extracellular compounds possessing cyanobacteriacidal activity without cell-to-cell contact when the host cyanobacterium necessarily exits in surrounding water. Property study of cyanobacteriacidal substance revealed the release of heat stable and hydrophobic chemical substances that do not appear to be protein and peptide compound. Taken together, our results indicate that LAB may be a potential bio-agent for future use in controlling freshwater Anabaena blooms without causing the problem of pathogenicity if there is no secondary bloom by a resistant species to this bacterium.