SYTOX Green Research Articles

461 Colchicine Inhibits Neutrophil Extracellular Trap Formation in Acute Coronary Syndrome Patients Post Percutaneous Coronary Intervention

Release of neutrophil extracellular traps (NETs) after PCI in ACS patients is associated with peri-procedural myocardial infarction, as a result of microvascular obstruction via pro-inflammatory and pro-thrombotic pathways. Colchicine is a potent, well-established anti-inflammatory agent with growing evidence to support its use in patients with coronary disease. However, its effects on post-PCI NET formation in ACS patients has not been explored. 60 patients (40 ACS; 20 stable angina pectoris[SAP]) were prospectively recruited and allocated to colchicine (1.5 mg) or no treatment. Within 24 h of treatment, serial coronary sinus blood samples were collected during PCI. Isolated neutrophils from 10 ACS patients post-PCI and 4 healthy controls were treated with colchicine (25 nM) and stimulated with either ionomycin (5 μM) or phorbol 12-myristate 13-acetate (PMA, 50 nM) in vitro. Extracellular DNA was quantified using Sytox Green and fixed cells were stained with hoechst and anti-alpha tubulin. Baseline characteristics were similar across both treatment and control arms. ACS patients had higher NET release versus SAP patients (p<0.001), which was reduced with colchicine treatment (AUC: 0.58 vs 4.29; p<0.001). In vitro, colchicine suppressed spontaneous (p=0.004), PMA-induced (p=0.03) and ionomycin-induced (p=0.02) NET formation in neutrophils isolated from ACS patients post-PCI, but not healthy controls. Tubulin organisation was impaired in neutrophils from patients with ACS but was restored by colchicine treatment. Colchicine suppresses NET formation in ACS patients post-PCI by restoring cytoskeletal dynamics. These findings warrant further investigation in randomised trials powered for clinical endpoints.

Identification of Peptides in the Leaves of &amp;lt;i&amp;gt;Bauhinia rufescens&amp;lt;/i&amp;gt; Lam (Fabaceae) and Evaluation of Their Antimicrobial Activities Against Pathogens for Aquaculture

Bauhinia rufescens Lam is a Cameroonian medicinal plant belonging to the Fabaceae family. It is used by indigenous people in the treatment of gout, diarrhea, dysentery, diabetes, malaria. It has antibacterial, antiparasitic, antifungal and antioxidant potential. The aim of this study is to identify peptides from the leaves of Bauhinia rufescens and assess their antimicrobial activities against certain pathogens in aquaculture. Previous studies have showed the antimicrobial properties of liquid extract on water against human pathogenic bacteria and the influenza virus. These properties have been mainly attributed to phenolic compounds. Phenolic compounds play an important role in human health due to their various pharmacological activities as anti-inflammatory, anti-allergic, antimicrobial, antiviral, and vasodilatory. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. In this work, we studied peptide extracts from the leaves of Bauhinia rufescens Lam. Mass spectrometry and analysis of peptides gave 3 to 3.6 kDa, among them, peptides rich in cysteine were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Cameroonian aquaculture. In addition, membrane bleeding on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and the SYTOX Green permeabilization test showed the ability to disturpt the bacterial membrane. The results obtained show that the peptides exert their action by destroying the bacterial membrane. Based on the results obtained, the medicinal value of this plant could be attributed to the presence of secondary metabolites.

An innovative sequence of hypoxia-reoxygenation on adult mouse cardiomyocytes in suspension to perform multilabeling analysis by flow cytometry.

Cardiovascular diseases remain the leading cause of death worldwide. Although major therapeutic progress has been made during the past decades, a better understanding of the underlying mechanisms will certainly help to improve patient's prognosis. In vitro models, particularly adult mouse cardiomyocytes, have been largely used; however, their fragility and large size are major obstacles to the use of flow cytometry. Conventional techniques, such as cell imaging, require the use of large numbers of animals and are time consuming. Here, we described a new, simple, and rapid one-day protocol using living adult mouse cardiomyocytes in suspension exposed to hypoxia-reoxygenation that allows a multilabeling analysis by flow cytometry. Several parameters can be measured by fluorescent probes labeling to assess cell viability (propidium iodide, calcein-AM, and Sytox Green), mitochondrial membrane potential [DilC1(5) and TMRM], reactive oxygen species production (MitoSOX Red), and mitochondrial mass (MitoTracker Deep Red). We address the robustness and sensitivity of our model using a cardioprotective agent, cyclosporine A. Overall, our new experimental set-up offers a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes exposed to hypoxia-reoxygenation. Our model might be interesting to investigate other cellular stresses (oxidative and inflammation) or to perform pharmacological screening.

Reversibility of membrane permeabilization upon pulsed electric field treatment in Lactobacillus plantarum WCFS1

Pulsed electric field (PEF) treatment, or electroporation, can be used to load molecules into cells. The permeabilizing effect of the PEF treatment on the cellular membrane can be either reversible or irreversible depending on the severity of the PEF treatment conditions. The influence of PEF on the reversibility of membrane permeabilization in Lactobacillus plantarum WCFS1 by two different fluorescent staining methods was investigated in this study. Whereas staining with propidium iodide (PI) before and after PEF treatment indicated small reversible permeabilized fractions of maximum 14%, the use of a double staining method with PI and SYTOX Green suggested larger reversible permeabilized fractions up to 40% of the population. This difference shows that the choice for a fluorescent staining method affects the conclusions drawn regarding reversibility of membrane permeabilization. Additionally, the effect of PEF treatment conditions on membrane integrity was compared, indicating a relation between critical electric field strength, cell size and membrane permeabilization. Overall this study showed the possibilities and limitations of fluorescent membrane integrity staining methods for PEF studies.

Viability of pico- and nanophytoplankton in the Baltic Sea during spring

Phytoplankton cell death is an important process in marine food webs, but the viability of natural phytoplankton communities remains unexplored in many ecosystems. In this study, we measured the viability of natural pico- and nanophytoplankton communities in the central and southern parts of the Baltic Sea (55°21′ N, 17°06′ E–60°18′ N, 19°14′ E) during spring (4th–15th April 2016) to assess differences among phytoplankton groups and the potential relationship between cell death and temperature, and inorganic nutrient availability. Cell viability was determined by SYTOX Green cell staining and flow cytometry at a total of 27 stations representing differing hydrographic regimes. Three general groups of phytoplankton (picocyanobacteria, picoeukaryotes, and nanophytoplankton) were identified by cytometry using pigment fluorescence and light scatter characteristics. The picocyanobacteria and picoeukaryotes had significantly higher cell viability than the nanophytoplankton population at all depths throughout the study area. Viability correlated positively with the photosynthetic efficiency (Fv/Fm, maximum quantum yield of photosystem II) as measured on the total phytoplankton community. However, an anticipated correlation with dissolved organic carbon was not observed. We found that the abiotic factors suggested to affect phytoplankton viability in other marine ecosystems were not as important in the Baltic Sea, and other biotic processes, e.g. processes related to species succession could have a more pronounced role.

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure to PEF indicate a strong coupling between these parts of the cell.

Antioxidant Enzyme-Mimetic Activity and Neuroprotective Effects of Cerium Oxide Nanoparticles Stabilized with Various Ratios of Citric Acid and EDTA.

Cerium oxide (CeO2) nanoparticles (CeNPs) are potent antioxidants that are being explored as potential therapies for diseases in which oxidative stress plays an important pathological role. However, both beneficial and toxic effects of CeNPs have been reported, and the method of synthesis as well as physico-chemical, biological, and environmental factors can impact the ultimate biological effects of CeNPs. In the present study, we explored the effect of different ratios of citric acid (CA) and EDTA (CA/EDTA), which are used as stabilizers during synthesis of CeNPs, on the antioxidant enzyme-mimetic and biological activity of the CeNPs. We separated the CeNPs into supernatant and pellet fractions and used commercially available enzymatic assays to measure the catalase-, superoxide dismutase (SOD)-, and oxidase-mimetic activity of each fraction. We tested the effects of these CeNPs in a mouse hippocampal brain slice model of ischemia to induce oxidative stress where the fluorescence indicator SYTOX green was used to assess cell death. Our results demonstrate that CeNPs stabilized with various ratios of CA/EDTA display different enzyme-mimetic activities. CeNPs with intermediate CA/EDTA stabilization ratios demonstrated greater neuroprotection in ischemic mouse brain slices, and the neuroprotective activity resides in the pellet fraction of the CeNPs. The neuroprotective effects of CeNPs stabilized with equal proportions of CA/EDTA (50/50) were also demonstrated in two other models of ischemia/reperfusion in mice and rats. Thus, CeNPs merit further development as a neuroprotective therapy for use in diseases associated with oxidative stress in the nervous system.

Abstract P1109: Cholesterol Regulation of Hydrogen Sulfide-Mediated Vasodilation

Hydrogen sulfide (H 2 S) is produced within the vascular wall by cystathionine γ-lyase (CSE). CSE is expressed throughout the vascular tree however, the sensitivity to H 2 S-induced dilation in small resistance size arteries (&lt;100 μm) is greater than the dilation sensitivity in large arteries (&gt;300 μm). Although H 2 S can induce vasodilation in different size arteries, it is not clear how H 2 S signaling mechanisms differ in large and small arteries, garnering H 2 S-induced dilation prominent and important in small resistance arteries. We previously demonstrated that within endothelial cells, H 2 S-mediated vasodilation involves activation of transient receptor potential cation channel subfamily V member 4 (TRPV4)-dependent Ca 2+ influx and large conductance Ca 2+ -activated potassium (BK) channel. Based on previous reports that membrane cholesterol negatively regulates TRPV4 mobility and BK activity, we hypothesized that H 2 S-mediated vasodilation is diminished by membrane cholesterol and that lower sensitivity in large arteries might be due to higher levels of membrane cholesterol in endothelial cells. Isolated pressurized mesenteric arteries (300-380 μm) were treated luminally with vehicle (HEPES) or the cholesterol-depleting agent, methyl β cyclodextran (MBCD, 100 μM) and exposed to H 2 S donor, NaHS (10 μM). Analysis of H 2 S-induced dilation (expressed as % Ca 2+ free diameter) showed that MBCD treatment significantly enhanced H 2 S-mediated vasodilation (vehicle: 1.2% ± 1.2, MBCD: 8.9% ± 2.3, p = 0.012, n = 5 animals/group). Additionally, mesenteric arteries of different orders were luminally loaded with the fluorescent cholesterol-binding agent, filipin III (20 μg/ml, ex/em: 405/461 nm) the endothelial glycocalyx marker DyLight 594-tomato lectin (20 μg/ml, ex/em: 561/613 nm) and the nuclei stain SYTOX green (ex/em: 488/518 nm). Analysis of endothelial membrane cholesterol content (filipin III /tomato lectin fluorescence) was significantly greater in larger arteries (2 nd order: 1.019 ± 0.158, 5 th order: 0.344 ± 0.179, p = 0.0054, n = 3-5 animals/group). These studies suggest that higher membrane cholesterol decreases H 2 S-mediated vasodilation.

Effects of the peptide H-OOWW-NH2 and its derived lipopeptide C12-OOWW-NH2 on controlling of citrus postharvest green mold

The present study evaluated the antifungal properties of tetrapeptide H-OOWW-NH2 (O3TR) and its derivative lipopeptide C12-OOWW-NH2 (C12O3TR) against Penicillium digitatum, one of the main postharvest pathogens in citrus, and the possible mechanisms of their antifungal action. The results showed that the peptides O3TR and C12O3TR could inhibit conidial germination, induce conidia death and reduce the survival of mycelia of P. digitatum in vitro. The antifungal properties of O3TR and C12O3TR against P. digitatum were thermostable (40 °C–80 °C), insensitive to the change of pH (3–10) and varying sensitive to the presence of cations (Na+, Ca2+). In addition, the two peptides could effectively control green mold on citrus in vivo study. In terms of safety evaluation, the hemolytic activity of O3TR was neglectable, and significantly lower than that of C12O3TR, both of which were much lower than that of commercial prochloraz. The signals and intensity of fluorescent dye SYTOX Green (SG) and Propidium Iodide (PI) showed that O3TR and C12O3TR could enhance the mycelial and conidial membrane permeabilization. The antifungal action of O3TR and C12O3TR was further demonstrated by the release of cellular constituents and extracellular conductivity. In conclusion, the two peptides have a promising prospect to be applied as antifungal agents for the control of the green mold of citrus postharvest diseases.

The design of a cell-selective fowlicidin-1-derived peptide with both antimicrobial and anti-inflammatory activities

Fowlicidin-1 (Fowl-1), a cathelicidin expressed in chicken intestine, is known to have both antimicrobial and anti-inflammatory properties. However, its pharmaceutical development has been ultimately compromised by its high host cytotoxicity. In this study, a series of N- and C-terminal-truncated 19-meric Fowl-1 peptides were synthesized. Among these truncated peptides, Fowl-1 (8–26) exhibited broad-spectrum antimicrobial activity without human erythrocyte cytotoxicity while reducing anti-inflammatory activity. Further, Fowl-1 (8–26)-WRK was designed via Thr5→Trp, Ile7→Arg, and Asn11→Lys substitutions in Fowl-1 (8–26) to exhibit more amphipathicity. The results revealed that it exhibited both antimicrobial and anti-inflammatory properties. This study also demonstrated that the inhibitory activity of Fowl-1 (8–26)-WRK against LPS-induced inflammation was mainly due to the binding of LPS to the peptide. Interestingly, compared with human cathelicidin LL-37 and melittin, Fowl-1 (8–26)-WRK showed more potent activity against drug-resistant bacteria. It was also resistant to physiological salts and human serum and acted synergistically in combination with conventional antibiotics, such as chloramphenicol, ciprofloxacin, and oxacillin, suggesting that combined with conventional antibiotics, it is a promising adjuvant. Furthermore, membrane depolarization, SYTOX Green uptake, and flow cytometry revealed that it kills bacteria by damaging their membrane integrity. Therefore, this study suggests that Fowl-1 (8–26)-WRK has considerable potential for future development as an antimicrobial and anti-inflammatory agent for treating antibiotic-resistant infections.

Evaluating microalgal cell disruption upon ultra high pressure homogenization

The impact of (ultra) high pressure homogenization on the degree of cell disruption was investigated for Nannochloropsis sp. suspensions. The degree of cell disruption was studied by combining four evaluation methods: turbidity measurement, scanning electron microscopy, hexane:isopropanol extraction efficiency, and fluorescence microscopy using the viability stain SYTOX green. Applying an ultra high pressure of 250 MPa obviously reduced the number of homogenization passes required to obtain a specific degree of cell disruption compared to 100 MPa. However, heating of the sample occurred at 250 MPa, resulting in extensive aggregate formation of the released intracellular material after multiple homogenization passes. Furthermore, cell wall integrity was not necessarily linked to membrane integrity, implying that moderate (U)HPH conditions are possibly sufficient for certain applications by damaging the cell membrane, without achieving full rupture of the cell wall. Once again it was proven that different methods for evaluation of cell disruption should be combined to get comprehensive insight into the disruption of microalgae.

Abstract 4739: Induction of cell death by HDAC, Aromatase and MDM2 inhibitors in MCF-7 cells

Abstract The histone deacetylase (HDAC) enzymes are evolving as key enzymes in the regulation of many physiological processes, including chromatin remodeling, genome stability, DNA repair, regulation of transcription, protein secretion, cellular metabolism, cell cycle progression, differentiation, migration, and cell death. Among different regulators of HDACs, suberoylanilide hydroxamic acid (SAHA) is a potent inhibitor and it is known to cause strong growth arrest, differentiation and apoptosis of many tumor types in both in vitro and in vivo experimental models. Interestingly, treatment of MCF-7 breast cancer cells with SAHA was found to trigger necroptosis, which is a caspase independent form of cell death. Typically, stimulation of death domain receptors (DDR) by suitable ligands lead to the activation of RIP1, RIP3 and MLKL proteins which are the critical regulators of necroptosis. It appears that, during SAHA induced necroptosis elevation of p21 may be the main signal that triggers recruitment of RIP1, Phospho-RIP3 and MLKL intracellularly in MCF-7 cells. Also, our results confirmed that the apoptosis marker such as BAX and caspases are not elevated by SAHA in MCF-7 cells after 24 hrs of treatment. In addition, the fluorescence assay conducted with SYTOX Green after 10 and 50 µM necrostatin pre-treatments, confirmed that SAHA treatment activates RIP1 mediated necroptosis pathway in MCF-7 cells. The induction of p21 expression was also down-regulated significantly by 10 and 50 µM necrostatin pre-treatments, which further suggests that p21 is up-stream of the necroptosis executioners in MCF-7 cells. On the other hand, when the MCF-7 cells were treated with Letrazole and RG-7388, BAX appears to be involved in mediating cell death. Interestingly, both Letrazole and RG-7388 were not able to elevate p21 in MCF-7 cells. So far, our results suggest that SAHA can induce BAX independent necroptosis while Letrazole and RG-7388 induce cell death through elevation of BAX and BAK in MCF-7 cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Umamaheswari Natarajan, Vijayaraghavan Radhakrishnan, Shila Samuel, Appu Rathinavelu. Induction of cell death by HDAC, Aromatase and MDM2 inhibitors in MCF-7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4739.

Membrane-permeabilized sonodynamic therapy enhances drug delivery into macrophages.

Macrophages play a pivotal role in the formation and development of atherosclerosis as a predominant inflammatory cell type present within atherosclerotic plaque. Promoting anti-atherosclerotic drug delivery into macrophages may provide a therapeutic potential on atherosclerotic plaque. In this study, we investigated whether membrane-permeabilized sonodynamic therapy (MP-SDT) enhances drug delivery into THP-1 macrophages. Images of confocal microscopy confirmed that the optimal plasma distribution of the sonosensitizer protoporphyrin IX (PpIX) was at 1 hour incubation. The non-lethal parameter of MP-SDT was determined by cell viability as measured by a CCK-8 assay. Bright field microscopy demonstrated plasma membrane deformation in response to MP-SDT. Using SYTOX Green, a model drug for cellular uptake, we found that MP-SDT significantly induced membrane permeabilization dependent on ultrasound intensity and exposure time. Using Fluo-3 AM, intracellular calcium elevation during MP-SDT was confirmed as a result of membrane permeabilization. Membrane perforation of MP-SDT-treated cells was observed by scanning electron microscopy and transmission electron microscopy. Moreover, MP-SDT-induced membrane permeabilization and perforation were remarkably prevented by scavenging reactive oxygen species (ROS) during MP-SDT. Furthermore, we assessed the therapeutic effect of MP-SDT in combination with anti-atherosclerotic drug atorvastatin. Our results showed that MP-SDT increased the therapeutic effect of atorvastatin on lipid-laden THP-1-derived foam cells, including decreasing lipid droplets, increasing the cholesterol efflux and the expression of PPARγ and ABCG1. In conclusion, MP-SDT might become a promising approach to facilitating the delivery of anti-atherosclerotic drugs into macrophages via membrane permeabilization.

Survival of membrane-damaged Escherichia coli in a cytosol-mimicking solution

Selective permeability of cell membrane is critically important for cell survival. The damage caused to cell membrane by pore-forming antimicrobial peptides may result in the loss of selective permeability and leakage of intracellular molecules, eventually leading to cell death. Here, we examined whether the membrane-damaged Escherichia coli cells survive in a cytosol-mimicking solution (CMS), which compensates for the lethal leakage of intracellular molecules. We prepared a CMS comprising 34 low molecular weight compounds from the cytosol and found that the cells were able to grow in CMS even in the presence of a pore-forming peptide, melittin. We confirmed that the melittin-treated cells lost selective membrane permeability by staining with membrane-impermeable dyes, propidium iodide and SYTOX green. Some stained cells maintained the colony formation ability in CMS. These results provide an evidence that E.coli cells can at least partially survive in the CMS even after the temporary impairment of membrane selective permeability. This study demonstrates a technique that allows temporal loss of the selective permeability of the cell membrane while maintaining the viability of cells that may be useful for the introduction of membrane-impermeable molecules into E.coli cells.

PS1497 NEUTROPHIL EXTRACELLULAR TRAPS (NETS), BLOOD CELL COUNTS AND THROMBOTIC RISK IN ITP PATIENTS

Background:Patients with immune thrombocytopenia (ITP) are at increased risk of vascular events (VE), but the precise mechanisms of this prothrombotic state remain largely unknown. Neutrophil extracellular traps (NETs) are DNA‐containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non‐immune disorders. However, the contribution of NETs in the prothrombotic state in ITP patients is largely unknown.Aims:To determine if ITP patients have enhanced intrinsic potential for NET formation and to evaluate their prothrombotic role in ITP, their correlation with platelet activation, and also with specific therapeutic approaches.Methods:Sixty‐three ITP patients at different stages of disease and 30 healthy controls were included. NETs were evaluated by quantifying cell free DNA (cfDNA) using SYTOX Green, and by assessing the levels of Citrullinated Histone H3 complexed to DNA (H3Cit‐DNA) via a sandwich ELISA. Platelet and white blood cell (WBC) counts were assessed by a cell counter (Sysmex), and platelet glycoproteins, size, and degranulation were evaluated by flow cytometry.Results:The levels of cfDNA and H3Cit‐DNA in the peripheral blood of ITP patients were higher than in controls (p = 0.006, and p = 0.001, respectively). The percentage of patients with detectable H3Cit‐DNA complexes (optical density &gt;0.199) was significantly different to that of controls (26.2% vs. 6.7%; p = 0.029). In patients, positivity for H3Cit‐DNA correlated with higher cfDNA (Pearson correlation: R = 0.407; p = 0.01). No significant differences in the levels of cfDNA or H3Cit‐DNA were detected between the patients in remission (n = 24) and those with active ITP (n = 39). Seven ITP patients (11.1%) had experienced previous VE and presented with increased cfDNA (0.207 vs. 0.152 ng/ml; p = 0.034), WBC count (9.6 vs. 8.0 x109/L; p = 0.037), and neutrophil count (5.4 vs. 4.3x109/L; p = 0.027) compared to patients with no history of thrombotic events. Only one of the patients with VE was under low dose prednisone. Previous thrombosis was neither associated to H3Cit‐cfDNA positivity, platelet counts, mean fluorescence intensity of platelet glycoprotein expression (GPIba, aIIb), size (FSC), nor percentage of degranulated platelets (CD62, CD63). When we evaluated whether other acquired risk factors correlated with cfDNA, neither previous splenectomy, hypertension, hypercholesterolemia, smoking, age, nor diabetes correlated with cfDNA. Notably, treatment with thrombopoietin receptor agonists, corticosteroids, or immunosuppresants at sampling were neither related to circulating NETs cfDNA or H3Cit‐DNA) nor to platelet activation.Summary/Conclusion:Our results show that plasma NET levels are elevated in ITP patients, and that this increase is especially marked in patients who have suffered from a vascular event. The fact that patients with previous thrombosis had increased WBC and neutrophil counts suggests that there may be more NETosis in these individuals, probably indicating a dysregulation between production and clearance. Other factors (platelet activation, acquired thrombotic risk factors or current therapy) did not seem to influence NET formation or platelet degradation. Similar to patients with cancer and myeloproliferative disorders, where leukocytosis has been associated with increased thromboembolic risk, we propose an association between NET generation and neutrophil counts leading to increased risk of thrombosis in ITP, which may provide new therapeutic targets to combat VE in ITP.Funding: ISCIII and FEDER [PI17/00051].

Streamlined assessment of membrane permeability and its application to membrane engineering of Escherichia coli for octanoic acid tolerance.

The economic viability of bio-production processes is often limited by damage to the microbial cell membrane and thus there is a demand for strategies to increase the robustness of the cell membrane. Damage to the microbial membrane is also a common mode of action by antibiotics. Membrane-impermeable DNA-binding dyes are often used to assess membrane integrity in conjunction with flow cytometry. We demonstrate that in situ assessment of the membrane permeability of E. coli to SYTOX Green is consistent with flow cytometry, with the benefit of lower experimental intensity, lower cost, and no need for a priori selection of sampling times. This method is demonstrated by the characterization of four membrane engineering strategies (deletion of aas, deletion of cfa, increased expression of cfa, and deletion of bhsA) for their effect on octanoic acid tolerance, with the finding that deletion of bhsA increased tolerance and substantially decreased membrane leakage.

Superbright organosilica nanodots as a universal sensor for fast discrimination and accurate quantification of live/dead cells

Rapid and quantitative discrimination between live and dead cells is highly desirable yet remains a challenging task. Here, we report the use of the ultrabright green-emitting organosilica nanodots (OSiNDs, photoluminescence quantum yield: ˜100%) for fast (1 min in vitro and 5 min in vivo), wash-free, and universal labeling of the dead bacterial, fungal, and mammalian cells, which meets the demand of the green fluorescent probes that can specifically image the dead cells. The photostability of OSiNDs surpasses that of the commercial dyes, such as propidium iodide (PI) and SYTOX Green nucleic acid stain (SYTOX), making OSiNDs more suitable for long-term and stable monitoring of the cell viability. With the assistance of a commercial red fluorescent dye SYTO 60, we develop an in situ method for rapid and accurate quantification of live/dead cells. Motivated by the unique optical advantages and high sensitivity of OSiNDs to the cell viability, the OSiNDs are employed to observe the in vivo cellular changes induced by doxorubicin using zebrafish as a model, demonstrating their great potential for evaluating the toxicity/efficacy of the therapeutic agents in real time. Owing to the excellent biocompatibility and low cost, the OSiNDs can be a universal probe for efficient and accurate differentiation and quantification of the live and dead cells, which may find wide applications in the biomedical field.

Oligomerization and insertion of antimicrobial peptide TP4 on bacterial membrane and membrane-mimicking surfactant sarkosyl.

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading microorganisms. Although AMPs are known to act on bacterial membranes and increase membrane permeability, the action mechanism of most AMPs still remains unclear. In this report, we found that the TP4 peptides from Nile tilapia anchored on E. coli cells and enabled them permeable to SYTOX Green in few minutes after TP4 addition. TP4 peptides existed in small dots either on live or glutaraldehyde-fixed cells. TP4 peptides were driven into oligomers either in soluble or insoluble form by a membrane-mimicking anionic surfactant, sarkosyl, depending on the concentrations employed. The binding forces among TP4 components were mediated through hydrophobic interaction. The soluble oligomers were negatively charged on surface, while the insoluble oligomers could be fused with each other or piled on existing particles to form larger particles with diameters 0.1 to 20 μm by hydrophobic interactions. Interestingly, the morphology and solubility of TP4 particles changed with the concentration of exogenous sarkosyl or trifluoroethanol. The TP4 peptides were assembled into oligomers on or in bacterial membrane. This study provides direct evidence and a model for the oligomerization and insertion of AMPs into bacterial membrane before entering into cytosol.

719 The effect and irritation of adapalene on Cutibacterium acnes via HTS

Cutibacterium acnes is involved in acne as an opportunistic pathogen. Contrary to the previously thought, the proliferation of C. acnes is not pathogenic crucially. Instead, activation of the innate immunity induced by C. acnes might lead to this chronic inflammation. This research was to establish the screening platform of acne by nematodes and detect the anti-inflammatory activity and stimulation of adapalene. High throughput liquid screening assay was developed here to identify effective anti-infective compounds in 96-well assay plates of worms. Tetracycline was as the positive control to save infected worms in the liquid assay. Minimum inhibitory concentration was presented for visible growth of C. acnes in different conditions. The membrane permeability of C. acnes was detected by monitoring the uptake of SYTOX Green after treating with compounds. We provided results from HTS in nematodes Caenorhabditis elegans that led to the better characterization of adapalene treating acne. Compared to tetracycline, adarotene inhibited the growth of C. acnes significantly (p<0.01), while adapalene didn’t show anti-C. acnes effect, even under high concentration. Then we found that adapalene reduced the production of pro-inflammatory cytokines (IL-6, IL-8) on PMA stimulated human epidermoid carcinoma cells. But when exposure of human keratinocytes to adapalene, it resulted in the activation of the cells and the secretion of IL-6 and IL-8. The liquid C. elegans - C. acnes assay established here allows screening for anti-acne compounds that are not toxic to hosts. We confirm the utility of C. elegans as a screening platform for acne treatment, and adapalene can rescue C. acnes infected worms. Although it has anti-inflammation effects, adapalene changes the expression of IL-6 and IL-8 depending on the host inflammation conditions. Normal human keratinocytes could be triggered by the stimulation of adapalene.