Hypotonic Lysis Research Articles

Decontamination of Escherichia coli O157:H7 on fresh Romaine lettuce using a novel bacteriophage lysin

Raw vegetables are a key food for a healthy diet, but their increased consumption brings a higher risk for foodborne disease. Contamination of salad greens with Shiga toxin-producing Escherichia coli (STEC) O157:H7 has caused severe disease and important economic losses almost yearly in the United States over the last 10 years. To curb the risk of infections from contaminated produce, approaches based on bacterial virus – commonly known as bacteriophage or phage – have recently started to draw interest among other antimicrobial strategies. Phages enter bacterial cells to reproduce and cause cellular lysis to release their phage progeny at the end of their infection cycle. This lytic effect is caused by lysins, phage-encoded enzymes that have evolved to degrade the bacterial cell wall resulting in hypotonic lysis. When applied externally in their purified form, such enzymes are able to kill sensitive bacteria on contact in a similar way. Their unique bactericidal properties have made lysins effective antimicrobial agents in a variety of applications, from treating multidrug-resistant infections in humans to controlling bacterial contamination in several areas, including microbiological food safety. Here we describe a novel lysin, namely PlyEc2, with potent bactericidal activity against key gram-negative pathogens including E. coli, Salmonella, Shigella, Acinetobacter and Pseudomonas. PlyEc2 displayed high bactericidal activity against STEC to a concentration of 12.5 μg/ml under different pH conditions. This lysin was also able to reduce the bacterial titer of several pathogenic strains in vitro by more than 5 logarithmic units, resulting in complete sterilization. Importantly, PlyEc2 proved to be a powerful produce decontamination agent in its ability to clear 99.7% of contaminating STEC O157:H7 in our Romaine lettuce leaf model. PlyEc2 was also able to eradicate 99.8% of the bacteria contaminating the washing solution, drastically reducing the risk of cross-contamination during the washing process. A sensory evaluation panel found that treatment with PlyEc2 did not alter the visual and tactile quality of lettuce leaves compared to the untreated leaves. Our study is the first to describe a highly effective lysin treatment to control gram-negative pathogenic contamination on fresh lettuce without the addition of membrane destabilizing agents.

Improved pharmacokinetic and biodistribution of 5-fluorouracil loaded biomimetic nanoerythrocytes decorated nanocarriers for liver cancer treatment

Nanoerythrocytes membrane (NEs) has recently been used to improve pharmacokinetics and biodistribution for successful drug therapy. NEs intended to enhance the drug targeting due to immune evasion and long circulation. In this work, NEs could serve as efficient 5- fluorouracil (5-FU) carriers to target liver cells. NEs decorated 5-FU-loaded chitosan coated-poly (lactide-co-glycolic acid) nanoparticles (5-FU-C-NPs-NEs), chitosomes (5-FU-C-LPs-NEs) and 5-FU-NEs were prepared by hypotonic lysis and extrusion procedures. Moreover, 5-FU loaded-chitosan coated 5-FU-NPs (5-FU-C-NPs) and chitosomes (5-FU-C-LPs) for the compared issues were prepared. They were characterized in terms of particle size, encapsulation efficiency (EE), membrane protein content, phosphatidylserine exposure, surface morphology, and in vitro release profiles. Also, their cytotoxic efficacy was determined. Furthermore, pharmacokinetics and biodistribution studies were investigated for optimized formulation. The results revealed that 5-FU-C-NPs-NEs have narrow particle size distribution, desirable EE%, and retained the erythrocyte membrane properties as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, it displayed a sustained release profile up to 72 h of 5-FU-C-NPs-NEs compared to other formulations. In comparison to 5-FU solution and 5-FU-C-NPs, 5-FU-C-NPs-NEs extended the drug release time in vivo with highly uptake by the liver. These results suggest that the 5-FU-C-NPs-NEs could be used to deliver 5-FU and enhance its targetability to liver cancer.

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells.

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

Paclitaxel-Loaded Macrophage Membrane Camouflaged Albumin Nanoparticles for Targeted Cancer Therapy.

BackgroundMelanoma is the most common symptom of aggressive skin cancer, and it has become a serious health concern worldwide in recent years. The metastasis rate of malignant melanoma remains high, and it is highly difficult to cure with the currently available treatment options. Effective yet safe therapeutic options are still lacking. Alternative treatment options are in great demand to improve the therapeutic outcome against advanced melanoma. This study aimed to develop albumin nanoparticles (ANPs) coated with macrophage plasma membranes (RANPs) loaded with paclitaxel (PTX) to achieve targeted therapy against malignant melanoma.MethodsMembrane derivations were achieved by using a combination of hypotonic lysis, mechanical membrane fragmentation, and differential centrifugation to empty the harvested cells of their intracellular contents. The collected membrane was then physically extruded through a 400 nm porous polycarbonate membrane to form macrophage cell membrane vesicles. Albumin nanoparticles were prepared through a well-studied nanoprecipitation process. At last, the two components were then coextruded through a 200 nm porous polycarbonate membrane.ResultsUsing paclitaxel as the model drug, PTX-loaded RANPs displayed significantly enhanced cytotoxicity and apoptosis rates compared to albumin nanoparticles without membrane coating in the murine melanoma cell line B16F10. RANPs also exhibited significantly higher internalization efficiency in B16F10 cells than albumin nanoparticles without a membrane coating. Next, a B16F10 tumor xenograft mouse model was established to explore the biodistribution profiles of RANPs, which showed prolonged blood circulation and selective accumulation at the tumor site. PTX-loaded RANPs also demonstrated greatly improved antitumor efficacy in B16F10 tumor-bearing mouse xenografts.ConclusionAlbumin-based nanoscale delivery systems coated with macrophage plasma membranes offer a highly promising approach to achieve tumor-targeted therapy following systemic administration.

Tumor Cell Membrane-Based Peptide Delivery System Targeting to Tumor Microenvironment for Cancer Immunotherapy and Diagnosis

\The development of effective delivery system for peptides targeting to the tumor microenvironment has always been a hot topic in the field of cancer diagnosis and therapy. A multifunctional delivery system by encapsulating superparamagnetic iron oxide nanoparticles (SPIO NPs) with tumor cell membrane obtained by hypotonic lysis followed by mechanical fragmentation was constructed to effectively deliver therapeutic peptides. SPIO nanoparticles were encapsulated with H460 lung cancer cell membranes (SPIO NP@M) and peptides consisted of PD-L1 inhibitory peptide (TPP-1) and MMP2 substrate peptide (PLGLLG) was conjugated to H460 membrane (SPIO NP@M-P). The abilities of homologous targeting, cytotoxicity, pharmacokinetics, and tumor targeting ability of SPIO NP@M-P were evaluated. TPP-1 peptide was delivered and released to the tumor microenvironment through the homotypic effect of tumor cell membrane and specific digestion by the tumor specific enzyme, MMP2. The newly developed delivery system (SPIO NP@M-P) for PD-L1 inhibitory peptide could effectively extend the half-life of the peptides (60 times longer) and meanwhile maintain the ability to re-activate T cell and inhibit the tumor growth in vitro and in vivo. Furthermore, SPIO NPs in the system could be used as a tumor imaging agent and thus indicate the effect of peptide treatment. The SPIO NP@M might provide a promising theranostic platform for therapeutic peptide application in cancer therapy.

N-Glycanase 1 Transcriptionally Regulates Aquaporins Independent of Its Enzymatic Activity

Patients with pathogenic mutations in NGLY1 cannot make tears and have global developmental delay and liver dysfunction. Traditionally, NGLY1 cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins before proteasomal degradation. We demonstrate that Ngly1-null mouse embryonic fibroblasts, NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient mouse embryonic fibroblasts swell slower and have reduced aquaporin1 mRNA and protein expression. Ngly1 knockdown and overexpression confirms that Ngly1 regulates aquaporin1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 knockout cellsshow reduced aquaporin11 mRNA, supporting NGLY1 as regulating expression of multiple aquaporins across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restores normal hypotonic lysis and aquaporin1 protein. We show that transcription factors Atf1/Creb1 regulate aquaporin1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient mouse embryonic fibroblasts. These results identify a non-enzymatic, regulatory function of NGLY1 in aquaporin transcription, possibly related to alacrima and neurological symptoms.

Lyse-Reseal Erythrocytes for Transfection of Plasmodium falciparum

Simple and efficient transfection methods for genetic manipulation of Plasmodium falciparum are desirable to identify, characterize and validate the genes with therapeutic potential and better understand parasite biology. Among the available transfection techniques for P. falciparum, electroporation-based methods, particularly electroporation of ring-infected RBCs is routinely used. Nonetheless, transfection of P. falciparum remains a resource-intensive procedure. Here, we report a simple and economic transfection method for P. falciparum, which is termed as the lyse-reseal erythrocytes for transfection (LyRET). It involved lysis of erythrocytes with a hypotonic RBC lysis buffer containing the desired plasmid DNA, followed by resealing by adding a high salt buffer. These DNA-encapsulated lyse-reseal erythrocytes were mixed with P. falciparum trophozoite/schizont stages and subjected to selection for the plasmid-encoded drug resistance. In parallel, transfections were also done by the methods utilizing electroporation of DNA into uninfected RBCs and parasite-infected RBCs. The LyRET method successfully transfected 3D7 and D10 strains with different plasmids in 63 of the 65 attempts, with success rate similar to transfection by electroporation of DNA into infected RBCs. The cost effectiveness and comparable efficiency of LyRET method makes it an alternative to the existing transfection methods for P. falciparum, particularly in resource-limited settings.

N-Glycanase 1 Regulates Aquaporins Independent of Its Enzymatic Activity

Patients with pathogenic mutations in NGLY1 cannot make tears (alacrima), have global developmental delay and liver dysfunction. Traditionally, N-glycanase 1 (NGLY1) cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins prior to their proteasomal degradation. We found that Ngly1-null mouse embryonic fibroblasts (MEFs), NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient MEFs swell slower and have reduced aquaporin 1 (Aqp1) mRNA and protein expression. Ngly1 knockdown and overexpression confirmed that Ngly1 regulates Aqp1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 KO cells show reduced AQP11 mRNA, supporting NGLY1 regulates expression of multiple AQPs across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restored normal hypotonic lysis and Aqp1 protein. We show that transcription factor Creb1 regulates Aqp1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient MEFs. These results identify a novel, non-enzymatic, regulatory function of NGLY1 in AQP transcription possibly related to alacrima.

Extremophiles - Platform Strains for Sustainable Production of Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) are biodegradable polyesters, which are produced by various bacteria including numerous halophiles. Employment of halophilic strain for PHA production brings numerous benefits such as robustness of the process against contamination by ubiquitous mesophiles or possibility to isolate polymer from bacterial biomass via hypotonic lysis. In this work, we screened three moderate halophiles – Halomonas halophila, Halomonas organivorans and Halomonas salina for the presence of phaC gene encoding for PHA synthase and, subsequently, we have investigated their PHA production potential on various sugars. Among tested strains, H. organivorans demonstrated the extraordinary capacity of PHA production in particular on galactose and mannose since on these saccharides PHA content in dried bacterial cells reached 83 and 90 wt. % on mannose and galactose, respectively. Therefore, H. organivoras can be considered being promising PHA producing strain in particular suitable for the valorization of lignocellulose materials rich in galactomannans such as spent coffee grounds.

Nano-erythrocyte membrane-chaperoned 5-fluorouracil liposomes as biomimetic delivery platforms to target hepatocellular carcinoma cell lines

Nano-erythrocyte coating has been developed as an interesting biomimetic platform to provide hybrid nano-carriers with innate functions to target liver cancer. This goal was achieved by coating nano-erythrocyte membranes (NEMs) onto 5-fluorouracil (5-FU)-loaded liposomes (LPs) to produce NEM-5-FU-LPs. This framework is used to promote the escape of 5-FU-LPs from degradation during systemic circulation. NEMs were obtained by hypotonic lysis of erythrocytes to produce ghost erythrocytes (GEs) followed by extrusion through polycarbonate membranes. Chimeric NEM-5-FU-LPs were fabricated via the fusion of NEMs and artificial LPs. The resultant chaperoned LPs were characterized based on particle size, morphology, entrapment efficiency (EE %), stability, protein content and phosphatidylserine exposure and their in vitro release profiles and cytotoxic efficacy were also determined. The present results revealed that 5-FU-LPs, NEM-5-FU and NEM-5-FU-LPs exhibited nanosize, spherical shapes and unimodal size distributions <0.3. In addition, the vesicles presented a zeta potential with EE% of 24.6–30.7% and an appropriate stability for 3 weeks. NEM-5-FU-LPs retained the erythrocyte membrane proteins as confirmed by PAGE and displayed a sustained release profile up to 48 h when compared to NEM-5-FU and the 5-FU solution. Moreover, hybrid NEM-5-FU-LPs induced a late cytotoxic effect after 48 h compared to the other formulations. Thus, mantling of 5-FU-LPs by NEMs could enhance vesicle controllability and their targetability to liver cancer cells.

Multimodal analysis of Plasmodium knowlesi-infected erythrocytes reveals large invaginations, swelling of the host cell, and rheological defects.

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block‐face scanning electron microscopy to explore the topography of P. knowlesi‐infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi‐infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python‐based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi‐infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei.

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme's protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Effects of Interferon-Gamma 1-b (IFN-γ) on Neutrophil Function and Biochemistry in Patients with Chronic Granulomatous Disease

Introduction: Chronic Granulomatous Disease (CGD) is a genetic disorder of the Nox2 enzyme system in phagocytic cells. Neutrophils and monocytes from patients with CGD are deficient in Nox2 components, unable to produce superoxide anion and other reactive oxygen species (ROS), and cannot kill certain types of bacteria and fungi. Patients with CGD suffer from recurrent, life-threatening infections. Treatment includes prophylaxis with antibiotic and antifungal agents and therapy with IFN-γ. Results from clinical trials of IFN-γ in patients with CGD have demonstrated decreased incidence and severity of infections. The exact mechanism(s) by which IFN-γ achieves its clinical effects is not clear. Previous studies from our laboratory in healthy adult volunteers demonstrated that administration of IFN-γ induced dramatic changes in the neutrophil phenotype with significant functional implications. To investigate IFN-γ induced changes in neutrophils further we initiated a study in CGD patients.Clinical Trial and Methods: Patients with CGD were enrolled on a study protocol approved by the COMIRB at the University of Colorado Denver. Patients between 5 and 60 years with defined genetic variants of CGD, but without recent infections or medical complications were entered onto the study. IFN-γ was stopped for a week to allow a drug washout period; prophylactic antibiotics and antifungal medications were continued. Blood samples were obtained from patients off IFN-γ, 12 hours after the first dose, and after the fourth dose of IFN-γ (50 mcg./m2 given on a standard schedule three times a week). Neutrophils were isolated by Dextran sedimentation, Ficoll Hypaque density centrifugation, and hypotonic lysis of red blood cells. RNA was isolated by standard technique and gene expression completed by RNAseq analysis (results pending). Ingestion was measured with fluorescently labeled S. aureus, bactericidal activity against S. aureus with 1:1 ratio of neutrophils to bacteria in 10% normal human serum by standard technique, and chemotaxis to fMLF and C5a with calcein-AM labeled neutrophils across fluorescence blocking PET membrane in Corning HTS Fluroblock plates. Superoxide anion was measured as SOD inhibitable cytochrome c reduction in response to standard agonists, CD11b expression and F-actin assembly in response to PMA and fMLF by flow cytometry, MHC Class II antigens by flow cytometry, nitric oxide (NO) levels in neutrophil lystes using a colorimetric assay to measure the combined nitrate and nitrite levels, and apoptosis determined as the combined caspase 3 and caspase 7 activity in isolated lysates using the Caspase-Glo 3/7 Assay from Promega.Results: We report here preliminary results on the first three patients studied; all are gp91phox deficient X-linked CGD. As expected, superoxide anion was not detected from patient neutrophils off INF-γ or during treatment. Ingestion and cell motility were not affected by IFN-γ administration. In all three subjects, caspase activity was decreased during IFN-γ treatment compared to before. In all three patients bactericidal activity against S. aureus was deficient; in 2/3 patients, administration of IFN was associated with a small but specific increase in killing, without change in ROS production. Strikingly, in two patients who showed improved bactericidal activity, NO production in lysates was increased after IFN-γ treatment compared to before and showed a further increase when cells were stimulated with fMLF. CD11b expression and F-actin assembly did not appear to be altered by IFN-γ treatment. Expression of HLA Class II markers (HLADRA and CD 274) appeared on neutrophils and increased in expression after initiation of IFN treatment. This change also was seen with CD 40.Summary: IFN-γ appeared to have dramatic effects on neutrophils from patients with CGD including possible improved bactericidal activity, the generation of NO which could be bactericidal and compensate for the deficiency of generation of ROS, and expression of MHC Class II antigens with possible association to antigen processing and enhanced interaction with adaptive immune function.Conclusion: Changes in the neutrophil phenotype induced by IFN-γ may provide alternative strategies compensating for the genetic deficiency of CGD. Defining how IFN-γ enhances neutrophil function may expand the use of this drug to other medical disorders in which innate immune response is involved. DisclosuresAmbruso:Horizon Pharma Ireland Ltd.: Consultancy.

PHA granules help bacterial cells to preserve cell integrity when exposed to sudden osmotic imbalances

Polyhydroxyalkanoates (PHA) are microbial polyesters which accumulate as intracellular granules in numerous prokaryotes and mainly serve as storage materials; beyond this primary function, PHA also enhance the robustness of bacteria against various stress factors. We have observed that the presence of PHA in bacterial cells substantially enhances their ability to maintain cell integrity when suddenly exposed to osmotic imbalances. In the case of the non-halophilic bacterium Cupriavidus necator, the presence of PHA decreased plasmolysis-induced cytoplasmic membrane damage during osmotic up-shock, which subsequently enabled the cells to withstand subsequent osmotic downshock. In contrast, sudden induction of osmotic up- and subsequent down-shock resulted in massive hypotonic lysis of non-PHA containing cells as determined by Transmission Electron Microscopy and Thermogravimetrical Analysis. Furthermore, a protective effect of PHA against hypotonic lysis was also observed in the case of the halophilic bacterium Halomonas halophila; here, challenged PHA-rich cells were capable of retaining cell integrity more effectively than their PHA-poor counterparts. Hence, it appears that the fact that PHA granules, as an added value to their primary storage function, protect halophiles from the harmful effect of osmotic down-shock might explain why PHA accumulation is such a common feature among halophilic prokaryotes. The results of this study, apart from their fundamental importance, are also of practical biotechnological significance: because PHA-rich bacterial cells are resistant to osmotic imbalances, they could be utilized in in-situ bioremediation technologies or during enrichment of mixed microbial consortia in PHA producers under conditions of fluctuating salinity.

Comparative methodological studies of L-asparaginase encapsulation into erythrocytes

Background. L-asparaginase is an enzyme, widely used in the therapy of acute lymphoblastic leukemia in children and adults, but its use is limited due to a wide range of side effects and anaphylactic reactions. L-asparaginase loaded into erythrocytes can solve these problems. This enzyme is protected from the immune system and plasma proteases due to erythrocyte membrane, but continues to work inside the cell because its membrane is permeable to L-asparagine. Thus, the half-life of the drug increases and anaphylactic reactions reduce. The encapsulation of L-asparaginase into erythrocytes can be performed by various osmotic methods. Each of them is characterized by the amount of encapsulated enzyme, the cell yield, as well as by the quality indices of the survived erythrocytes. An important parameter of each method is the possibility to provide sterility of this dosage form for the clinical use.The aim of the study was the comparing of three osmotic methods of L-asparaginase encapsulation into erythrocytes (hypo-osmotic lysis, dialysis and flow dialysis) to select the most promising method for clinical use.Materials and methods. A suspension of erythrocytes of healthy donors (hematocrit 60–70%) was mixed with L-asparaginase from E. сoli. The procedures of hypotonic reversible lysis, dialysis in dialysis bags, or flow dialysis using pediatric dialyzers were performed. The physiological osmolality was restored in suspensions after the procedure by the addition of a hypertonic solution, and they were incubated for 30 min at 37 °C. Then the cells were washed in isotonic phosphate-buffered saline with pH 7.4. Activity of L-asparaginase, volume, hematocrit, hematological indices and osmotic cell fragility of erythrocytes were measured in the suspensions of erythrocytes before and after the enzyme encapsulation procedure.Results. An optimal osmolality of the hypotonic buffer for each method was selected and was equal to 90–110 mOsm/kg. The yields of encapsulation were 4.2 ± 2.0, 6.0 ± 2.3 and 16.2 ± 2.2 % for hypotonic lysis, dialysis and flow dialysis, respectively. The hematological indices of the obtained erythrocyte-carriers differed from the corresponding parameters of the initial erythrocytes, but did not differ significantly for different methods.Conclusion. Comparative investigation of mentioned above parameters allowed choosing the method of flow dialysis as the most promising for clinical use.

Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines

Rapid and high quality preparation of peripheral blood leucocytes (PBL) is important in fish immunology research and in particular for fish vaccine development, where multiple immune parameters can be monitored on the same fish over time. Fish PBL are currently prepared by density separation using Percoll or Hispaque-1.077, which is time consuming, costly and prone to erythrocyte contamination. We present here a modified PBL preparation method that includes a 20 s hypotonic lysis of erythrocytes and a subsequent separation of PBL from cell debris by a cell strainer. This method is simple, rapid and cost effective. The PBL obtained are similar in cellular composition to those prepared by density separation but have less erythrocyte contamination as demonstrated by FACS analysis and the expression of cell marker genes. Marker gene analysis also suggested that PBL prepared by hypotonic lysis are superior to those obtained by the gradient method in that some high-density cells (certain B cell types and neutrophils) might be lost using the latter. The PBL prepared in this way can proliferate in response to the T cell mitogen PHA, and both lymphoid and myeloid cells can phagocytose fluorescent beads and bacteria, with the latter enhanced by treatment with pro-inflammatory cytokines (IL-1β and IL-6). Furthermore, the PBL can respond to stimulation with PAMPs (LPS, poly I:C) and cytokines (IL-1β and IFNγ) in terms of upregulation of proinflammatory cytokine gene expression. Such data demonstrate the utility of this approach (hypotonic lysis of erythrocytes) for PBL isolation and will enable more studies of their role in disease protection in future immunological and vaccine development research in fish.

Comparison Between K3EDTA and Lithium Heparin as Anticoagulant to Isolate Bovine Granulocytes From Blood.

Granulocytes play a key role in the defense against invading pathogens. To study granulocyte functions, the isolation of a pure and active cell population from fresh blood is required. Anticoagulants and red blood cells (RBCs) lysis used in the isolation procedure may influence cell harvest, cell marker expression, and pre-activation of cells. In this study, the influence of the anticoagulants K3EDTA or lithium heparin and the effect of different RBCs lysis methods on bovine granulocyte population from fresh blood of healthy cows after density gradient centrifugation were investigated. Venous blood from healthy cows was collected in K3EDTA and lithium heparin tubes. Density gradient centrifugation to separate granulocytes from other cells was conducted using Biocoll. Then, RBCs were lysed with hypotonic water or 0.2% sodium chloride (NaCl). Immediately after isolation, harvest, viability, size, granularity, purity, and CD11b expression as a marker for granulocytes was analyzed by flow cytometry. In addition, as a marker for activation and reactivity of the granulocytes, we stimulated cells with phorbol-myristate-acetate to evaluate the release of reactive oxygen species. Furthermore, extracellular trap (ET) formation was investigated by confocal immunofluorescence microscopy in untreated control cells and cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin. We did not find a significant difference in percentage of dead cells when comparing the two anticoagulants or the different RBCs lysis methods. However, the percentage of granulocytes in the harvested population was significantly less using lithium heparin blood as anticoagulant compared to K3EDTA. The granulocytes harvested from lithium heparin blood and water lysis exhibited higher clumping and pre-activation of unstimulated control cells as indicated by isolation of doublet cells, increased CD11b expression, and increased oxidative burst and higher amount of ET-releasing cells. Furthermore, the combination of K3EDTA as anticoagulant and NaCl as RBCs lysis method revealed the lowest variability and highest difference between untreated and methyl-β-cyclodextrin-treated cells when quantifying ET formation. In conclusion, density gradient centrifugation of K3EDTA blood resulted in higher purity of bovine granulocytes compared to lithium heparin blood. In contrast to water lysis, NaCl lysis method is recommended to avoid pre-activation of cells which may occur during hypotonic water lysis.

Volume expansion of erythrocytes is not the only mechanism responsible for the protection by arginine-based surfactants against hypotonic hemolysis.

A novel arginine-based cationic surfactant Nα-benzoyl-arginine dodecylamide (Bz-Arg-NHC12) was synthesized in our laboratory. In this paper we study the interaction of Bz-Arg-NHC12 with sheep and human red blood cells (SRBC and HRBC respectively) due to their different membrane physicochemical/biophysical properties. SRBC demonstrated to be slightly more resistant than HRBC to the hemolytic effect of the surfactant, being the micellar structure responsible for the hemolytic effect in both cases. Moreover, besides the hemolytic effect, a dual behavior was observed for the surfactant studied: Bz-Arg-NHC12 was also able to protect red blood cells against hypotonic lysis for HRBC in a wide range of surfactant concentrations. However, the degree of protection showed for SRBC was about 50% lower than for HBRC. In this regard, a remarkable volume expansion was evidenced only for SRBC treated with Bz-Arg-NHC12, although no correlation with the antihemolytic potency (pAH) was found. On the contrary, our surfactant showed a greater pAH when human erythrocytes were submitted to hypotonic stress, with a low volume expansion, showing a higher amount of solubilized phospholipids in the supernatant when compared with SRBC behavior. Surface plasmon resonance measurements show the molecular interaction of the surfactant with lipid bilayers from HRBC and SRBC lipids, demonstrating that in the latter neither microvesicle release or lipid extraction occurred. Our results demonstrate that the volume expansion of erythrocytes is not the only mechanism responsible for the protection by surfactants against hypotonic hemolysis: volume expansion could be compensated via microvesicle release or by the extraction of membrane components upon collisions between red blood cells and surfactant aggregates depending on the membrane composition.

PO-449 Optimisation of an AlphaLISA assay for the characterisation of PROTAC-induced ternary complexes within cell lysates

Introduction Proteolysis Targeting Chimaeras (PROTACs) offer a powerful strategy to degrade cancer-associated proteins. PROTACs recruit an E3 ubiquitin ligase, such as von Hippel-Lindau (VHL), to a protein of interest (POI) to form a ternary complex. Measuring the formation of this ternary species is important for understanding PROTAC mode of action. Proximity AlphaLISA assays can be used for this purpose but have, to date, focused on using purified recombinant protein in vitro . Here, we report the development and optimisation of an AlphaLISA assay for monitoring ternary complex formation using full-length POI within cell lysates. Material and methods AlphaLISA assays were used to profile PROTAC-induced complex formation of FLAG-tagged full-length POI in cell lysate to purified biotinylated VCB. Signal was measured from close proximity of FLAG-tagged acceptor beads to streptavidin donor beads and benchmarked against a well-characterised PROTAC, MZ1, purified His-tagged Brd4 bromodomain 2 (BD2) and biotinylated VCB. A FLAG-tagged full-length POI containing an insert of Brd4 BD2 (POI-fusion) was utilised, such that MZ1 could be employed as a tool compound for assay development. Results and discussions Signal intensity was dependent on total lysate protein and VCB concentration, allowing for optimisation of component concentrations for maximal signal. A mild, detergent-free hypotonic cell lysis buffer gave enhanced signal over harsher buffers containing detergents. Additionally, the cell lysate cytosolic fraction provided superior signal compared to nuclear fractions and whole cell lysates, further highlighting the reliance of transient ternary complex formation on assay conditions. By utilising these optimal assay conditions determined with the POI-fusion, it is now possible to detect ternary complex formation between POI-targeting PROTACs and full-length POI. Conclusion To our knowledge, this optimised cell lysate AlphaLISA assay provides the first report and proof of concept for the interrogation of PROTAC-induced complex formation of full-length proteins with E3 ligases, which will be a valuable tool to the burgeoning PROTAC field. Consideration of assay parameters that permit optimal conditions for ternary complex formation in a POI-dependent manner allows the application of the AlphaLISA method as a screening platform. This application could profile PROTAC efficiency for a wide range of full-length, cell-derived proteins that are more relevant to the endogenous context encountered by the PROTAC intracellularly.

Beyond ERAD: N‐glycanase will bring you to tears

Patients with mutations in NGLY1 cannot make tears (alacrima), have global developmental delay, movement disorder and liver dysfunction. N‐glycanase 1 (NGLY1) de‐glycosylates misfolded N‐glycosylated proteins in the cytoplasm as part of the ERAD pathway prior to their proteasomal degradation. Surprisingly, NGLY1‐deficient patient cells do not accumulate cytoplasmic misfolded N‐glycoproteins, suggesting a more complex function. Interestingly, new studies show NGLY1 is needed for transcription factor NFE2L1/NRF1 activation and normal mitochondrial function.Using WT and NGLY1‐deficient mouse embryonic fibroblasts (MEFs) we found that NGLY1‐deficient cells were resistant to hypotonic lysis compared to WT. The same was seen in NGLY1‐patient fibroblasts. Additionally, MEFs deficient in both NGLY1 and ENGase (another deglycosylation enzyme) were even more resistant to hypotonic lysis compared to NGLY1‐deficient cells. Water influx and cell swelling precedes cell lysis. We found that NGLY1‐deficient MEFs swell slower than WT MEFs. Since aquaporins (AQP) transport water, we hypothesized that AQP levels might be disrupted in NGLY1‐deficient cells. We found both AQP1 mRNA and protein were reduced in NGLY1‐deficient MEFs. shRNA knockdown of AQP1 in WT MEFs decreased hypotonic lysis, suggesting AQP1 is associated with hypotonic lysis. NGLY1 shRNA and CRISPR studies confirmed that NGLY1 regulates AQP1 levels and hypotonic cell lysis. Preliminary studies show that complementing NGLY1‐deficient MEFs with NGLY1 increases AQP1 and restores hypotonic lysis. Current efforts are directed towards understanding whether NGLY1 enzyme activity is necessary for AQP regulation. 13 types of AQP are expressed in different cell types. Our preliminary data suggest that AQP11 levels are disrupted in NGLY1‐deficient patient cells and CRISPR cells, suggesting that NGLY1 might regulate different types of AQP.We have identified a novel function of NGLY1, i.e. to directly/indirectly regulate AQPs. This finding may relate to NGLY1‐deficient patients' inability to make tears. Future efforts will try to identify NGLY1‐dependent transcription factors responsible for regulating AQPs. This work is supported by the Bertrand Might Research Fund and NGLY1.org.Support or Funding InformationThis work is supported by the Bertrand Might Research Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.