SYTOX Green Research Articles

Anti-infective Effects of a Fish-Derived Antimicrobial Peptide Against Drug-Resistant Bacteria and Its Synergistic Effects With Antibiotic.

Antimicrobial peptides (AMPs) play pivotal roles in protecting against microbial infection in fish. However, AMPs from topmouth culter (Erythroculter ilishaeformis) are rarely known. In our study, we isolated an AMP from the head kidney of topmouth culter, which belonged to liver-expressed antimicrobial peptide 2 (LEAP-2) family. Topmouth culter LEAP-2 showed inhibitory effects on aquatic bacterial growth, including antibiotic-resistant bacteria, with minimal inhibitory concentration values ranging from 18.75 to 150 μg/ml. It was lethal for Aeromonas hydrophila (resistant to ampicillin), and took less than 60 min to kill A. hydrophila at a concentration of 5 × MIC. Scanning electron microscope (SEM) and SYTOX Green uptake assay indicated that it impaired the integrity of bacterial membrane by eliciting pore formation, thereby increasing the permeabilization of bacterial membrane. In addition, it showed none inducible drug resistance to aquatic bacteria. Interestingly, it efficiently delayed ampicillin-induced drug resistance in Vibrio parahaemolyticus (sensitive to ampicillin) and sensitized ampicillin-resistant bacteria to ampicillin. The chequerboard assay indicated that topmouth culter LEAP-2 generated synergistic effects with ampicillin, indicating the combinational usage potential of topmouth culter LEAP-2 with antibiotics. As expected, topmouth culter LEAP-2 significantly alleviated ampicillin-resistant A. hydrophila infection in vivo, and enhanced the therapeutic efficacy of ampicillin against A. hydrophila in vivo. Our findings provide a fish innate immune system-derived peptide candidate for the substitute of antibiotics and highlight its potential for application in antibiotic-resistant bacterial infection in aquaculture industry.

Scorpion-Venom-Derived Antimicrobial Peptide Css54 Exerts Potent Antimicrobial Activity by Disrupting Bacterial Membrane of Zoonotic Bacteria.

Antibiotic resistance is an important issue affecting humans and livestock. Antimicrobial peptides are promising alternatives to antibiotics. In this study, the antimicrobial peptide Css54, isolated from the venom of C. suffuses, was found to exhibit antimicrobial activity against bacteria such as Listeria monocytogenes, Streptococcus suis, Campylobacter jejuni, and Salmonella typhimurium that cause zoonotic diseases. Moreover, the cytotoxicity and hemolytic activity of Css54 was lower than that of melittin isolated from bee venom. Circular dichroism assays showed that Css54 has an α-helix structure in an environment mimicking that of bacterial cell membranes. We examined the effect of Css54 on bacterial membranes using N-phenyl-1-naphthylamine, 3,3′-dipropylthiadicarbbocyanine iodides, SYTOX green, and propidium iodide. Our findings suggest that the Css54 peptide kills bacteria by disrupting the bacterial membrane. Moreover, Css54 exhibited antibiofilm activity against L. monocytogenes. Thus, Css54 may be useful as an alternative to antibiotics in humans and animal husbandry.

Bio-impedance measurement allows displaying the early stages of neutrophil extracellular traps.

Neutrophils are the most abundant immune cells in the blood. Besides common immune defense mechanisms, releasing their DNA covered with antimicrobial proteases and histones represent another strong defense mechanism: neutrophil extracellular traps. In vitro the two most common inducers of these, so called, NETs are calcium ionophores (CI) and PMA (Phorbol 12-myristate 13-acetate). Following stimulation monitoring of NET release is necessary. For now, the methods of choice are quantification of free DNA by fluorescent dyes or analysis of immunofluorescence images. As a new method we tested bio-impedance monitoring of neutrophils after stimulation with the two inducers PMA and CI in gold-electrode coated plates. Bio-impedance (cell index) was measured over time. Results were compared to the monitoring of NETs by the fluorescent DNA-binding dye Sytox Green and immunofluorescence analysis. Cell index peaked about 25 min faster following CI stimulation than following PMA stimulation. The activation in Sytox Green Assay was significantly later detectable for PMA (+ approx. 90 min) but not for CI stimulation. The earlier and faster activation by CI was also confirmed by immunofluorescence staining. Our data suggest that bio-impedance measurement allows an easy online tracking of early neutrophil activation. This offers new opportunities to monitor early phases and stimuli-dependent dynamics of NETosis.

Using flow cytometry to monitor the stress response of yeast and microalgae populations in mixed cultures developed in brewery effluents

Recently, yeast and microalgae mixed cultures have been widely used in biological effluent treatments and biofuel production because such cultures show many advantages over pure cultures. However, industrial effluents often contain toxic compounds; therefore, it is important to evaluate the cell stress response when growing in such conditions during the mixed culture development. In this work, flow cytometry (FC) was used to differentiate Rhodosporidium toruloides cells from Tetradesmus obliquus cells, based on their size, internal complexity, and chlorophyll content. FC coupled with SYTOX Green and CFDA fluorochromes was also used to characterize the cell stress response of R. toruloides and T. obliquus individual cells in a mixed culture. This work describes, for the first time, a simple and easy method to monitor individual stress response of R. toruloides and T. obliquus cells growing in mixed cultures on brewery effluents, using FC coupled with fluorescent dyes.

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

Because cardiovascular diseases still represent the leading cause of death worldwide, a better understanding of the underlying physiopathological mechanisms is therefore needed. In vitro cellular models participate to decipher the molecular mechanisms, especially adult mouse cardiomyocytes. Unfortunately, due to their high fragilitity as well as their size (150 μm), flow cytometry is usually not performed and mainly conventional techniques are used like cell imaging, which is time and animal-consuming and causes low statistical power. Here, we described a new, simple and rapid one-day protocol in living adult mouse cardiomyocytes submitted to in suspension hypoxia-reoxygenation to allow multilabeling analysis by flow cytometry. Our method enables the measurement of several physiological parameters thanks to fluorescent probes labeling, assessing notably cell viability (Propidium Iodide, Calcein-AM and Sytox Green), mitochondrial membrane potential [DilC1(5), TMRM], reactive oxygen species (ROS) production (MitoSOX Red) and mitochondrial mass (MitoTracker Deep Red). Additionally, our model can be used to perform pharmacological screening illustrated here by the protective treatment via cyclosporine A (CsA) as a test to assess robustness and sensitivity of the utilized methods. In summary, our new hypoxia-reoxygenation sequence in suspension will offer a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes, which can be extended to various cellular stress challenges (oxidative, inflammation) or pharmacological screening.

Neutrophil extracellular traps promote corneal neovascularization-induced by alkali burn

ObjectivesTo investigate the effects of neutrophil extracellular traps (NETs) on angiogenesis in vitro and in vivo and the regulatory role of mammalian target of rapamycin (mTOR) activity in it. MethodsThe regulatory role of mTOR in NETs formation was explored. In vitro, human neutrophils were pretreated with rapamycin. NETs formation was measured using immunofluorescence staining of NETs markers, SYTOX Green and PicoGreen after NaOH stimulation. In vivo, mice were treated with rapamycin, and NETs formation in cornea was measured using immunofluorescence staining 7 days after alkali burn. Then, the effects of NETs on angiogenesis were investigated. In vitro, human neutrophils were treated with DNase I or rapamycin. NETs were isolated after NaOH stimulation and the isolated NETs were co-culture with human umbilical vein endothelial cells (HUVECs). HUVECs migration, proliferation, and inflammatory activation were measured. In vivo, mice were injected subconjunctivally with supernatant containing NETs. Corneal neovascularization was visualized by immunofluorescence staining. ResultsNETs structures can be observed in NaOH-stimulated neutrophils and alkali-burned mouse cornea compared with normal group. Treated with rapamycin enhanced NETs formation in response to NaOH management compared with DMSO control in vitro and in vivo. NETs increased the migration, proliferation and inflammatory activation of HUVECs, and subconjunctival injection of NETs promoted inflammatory and angiogenic response in corneal alkali burn model. ConclusionsNETs formation can be triggered by NaOH stimulation. mTOR activity has a negative regulatory effect on NETs formation. NETs promoted angiogenic responses and inflammatory activation of HUVECs and increased corneal neovascularization and inflammatory response.

A novel CD2 staining-based flow cytometric assay for assessment of natural killer cell cytotoxicity.

BackgroundAssessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non‐radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51Chromium‐release assay (CRA) and flow cytometry–based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs.MethodsHere, we report a rapid FCC for quantifying target cell death after co‐incubation with NK cells. In this assay, after 4 hours of NK cell‐target cell co‐incubation, fluorochrome‐conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V‐FITC were further used to detect target cell death in CD2‐negative population. In parallel, both CRA and FCC assay using CFSE/ 7‐AAD were performed to validate the reproducibility and replicability.ResultsWe observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI‐H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2‐based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7‐AAD.ConclusionsWe demonstrated that this CD2‐based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

Abstract 6209: Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages

Abstract Background: Macrophages are multipurpose phagocytes with various functions. Neutrophil extracellular traps (NETs) are web-like structures ensnare microbes which were composed of dsDNA, histones, and antimicrobial peptides. NETs are generally degraded by DNAse and macrophages, however, it is unknown how the degradation of NETs affects the local immune response thereafter. Here, we investigated the interaction between NETs and peripheral blood monocytes (PBMo). Method: 5 × 105 Polymorphonuclear leukocytes(PMNs), PMNs irradiated with UV for 15 minutes (UV-PMN) and PMNs stimulated by PMA for 15min and extensively washed (PMA-PMN) were placed on 6 well plate and cultured for 2 hours and NETs formation was visualized by SYTOX green. Peripheral blood was obtained from healthy volunteers and CD14+ PBMo were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with those PMNs at 37°C in 5% CO2 conditions for 3 days. The differentiation of macrophage was evaluated by the expression of M1/M2 markers by flowcytometry. Cytokine production was evaluated using the Cytokine Array system. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 mAb and the effect on T cell proliferation was evaluated with CFSE dilution assay. Results: PMA-PMN, but not PMN or UV-PMN, produced massive SYTOX(+) NETs within 2 hours. Co-culture of the NETs with PBMo stained with PKH26 showed NETs are phagocytosed by monocytes within 24 hours. PBMo co-cultured with PMA-PMN showed significantly enhanced expression of CD86, and reduced expression of CD163 as compared to those co-cultured with PMNs or UV-PMN. However, the expression of CD86 was suppressed if PMA-PMN were pretreated with 100 u/ml DNase I. The supernatant of the co-culture with PMA-PMN showed markedly increased levels of IL-1β and TNF-α. PBMo co-cultured with PMA-PMN strongly promote the proliferation of autologous T cells. However, the enhanced T cell growth were significantly suppressed by the pretreatment of NETs with DNase I. Conclusion: Monocytes appear to differentiate to proinflammatory macrophages after the phagocytosis of NETs. In tumor-bearing host, NETs are known to be increased in circulating blood, which may promote tumor associated inflammatory process. Citation Format: Akira Saito, Hideyuki Ozawa, Kazuya Takahashi, Yuki Kimura, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Naohiro Sata, Joji Kitayama. Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6209.

Gold Nanoclusters as an Antibacterial Alternative Against Clostridium difficile.

BackgroundClostridium difficile infection (CDI) has become one of the most important factors threatening human health, and about 20–30% antibiotic-related diarrhea cases and almost all pseudomembranous enteritis cases are related to CDI. The high recurrence of Clostridium difficile (C. difficile) and the emergence of drug resistance make clinical treatment of CDI difficult. Therefore, there is an urgent need to develop a non-antibiotic-alternative therapy against CDI. Gold nanoclusters (AuNCs) can better interact with bacteria due to its ultrasmall size. The aim of the present study was to explore whether AuNCs could be used as an antibacterial agent to kill C. difficile.MethodsAuNCs and C. difficile were co-cultivated in an anaerobic atmosphere to evaluate the bactericidal effect of AuNCs. The bacterial growth rate was estimated by using two concentrations of AuNCs (50 and 100 μM). The damage of AuNCs to C. difficile is detected by SYTOX Green staining methods and SEM image analysis. The mechanism of AuNCs on C. difficile was explored by reactive oxygen species (ROS) detection. The toxic effect of AuNCs on human cells was evaluated by MTT method.ResultsAuNCs (100 μM) killed C. difficile drastically. AuNCs increased the release of ROS by about 5 fold and destroyed the membrane integrity of C. difficile cells without causing significant toxic effect on human cells.ConclusionAuNCs showed great potential as an alternative to traditional antibiotics in killing C. difficile and may prove to be an alternative to treat CDI.

Characterization of Five Novel Anti-MRSA Compounds Identified Using a Whole-Animal Caenorhabditis elegans/Galleria mellonella Sequential-Screening Approach.

There is a significant need to combat the growing challenge of antibacterial drug resistance. We have previously developed a whole-animal dual-screening platform that first used the nematode Caenorhabditis elegans, to identify low-toxicity antibacterial hits in a high-throughput format. The hits were then evaluated in the wax moth caterpillar Galleria mellonella infection model to confirm efficacy and low toxicity at a whole animal level. This multi-host approach is a powerful tool for revealing compounds that show antibacterial effects and relatively low toxicity at the whole organism level. This paper reports the use of the multi-host approach to identify and validate five new anti-staphylococcal compounds: (1) 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol(PPT), (2) (1S,2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylate dihydrochloride(NNC), (3) 4,5,6,7-tetrabromobenzotriazole (TBB), (4) 3-[2-[2-chloro-4-[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl] benzoic acid(GW4064), and (5) N-(cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino] benzamide(PD198306). The compounds reduced the severity of methicillin-resistant Staphylococcus aureus (MRSA, strain MW2) infections in both C. elegans and G. mellonella and showed minimal inhibitory concentrations (MICs) in the range of 2–8 µg/mL. Compounds NNC, PPT, and TBB permeabilized MRSA-MW2 cells to SYTOX green, suggesting that they target bacterial membranes. Compound TBB showed synergistic activity with doxycycline and oxacillin against MRSA-MW2, and compounds PPT, NNC, GW4064, and PD198306 synergized with doxycycline, polymyxin-B, gentamicin, and erythromycin, respectively. The study demonstrates the utility of the multi-host approach with follow-up hit characterization for prioritizing anti-MRSA compounds for further evaluation.

Mechanisms of antimicrobial and antiendotoxin activities of a triazine-based amphipathic polymer.

TZP4 is a triazine-based amphipathic polymer designed to mimic the amphipathic structure found in antimicrobial peptides. TZP4 showed potent antimicrobial activity comparable to melittin against antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. TZP4 showed high resistance to proteolytic degradation and low tendency to develop drug resistance. The results from membrane depolarization, SYTOX Green uptake, flow cytometry, and gel retardation revealed that the mechanism of antimicrobial action of TZP4 involved an intracellular target rather than the bacterial cell membrane. Furthermore, TZP4 suppressed the messenger RNA levels of inducible nitric oxide synthase and tumor necrosis factor-α (TNF-α) and inhibited the release of nitric oxide and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BODIPY-TR-cadaverine displacement and dissociation of fluorescein isothiocyanate (FITC)-labeled LPS assays revealed that TZP4 strongly bound to LPS and disaggregated the LPS oligomers. Flow cytometric analysis demonstrated that TZP4 inhibits the binding of FITC-conjugated LPS to RAW264.7 cells. These observations indicate that TZP4 may exert its antiendotoxic activity by directly binding with LPS and inhibiting the interaction between LPS and CD14+ cells. Collectively, TZP4 is a promising drug candidate for the treatment of endotoxic shock and sepsis caused by Gram-negative bacterial infections.

Differential lethal action of C17:2 and C17:0 anacardic acid derivatives in Trypanosoma cruzi – A mechanistic study

The n-hexane extract from leaves of Schinus terebinthifolius (Anacardiaceae) induced 100% of death of trypomastigote forms of T. cruzi at 300 μg/mL and was subjected to a bioactivity-guided fractionation to afford a C17:2 derivative of anacardic acid [6-(8′Z,11′Z)-heptadecadienyl-salicylic acid, 1]. Additionally, compound 1 was subjected to hydrogenation procedures to afford a C17:0 derivative (6-heptadecanyl-salicylic acid, 1a). Compounds 1 and 1a were effective in killing trypomastigote forms of T. cruzi with IC50 values of 8.3 and 9.0 μM, respectively, while a related compound, salicylic acid, was inactive. Furthermore, no cytotoxicity was observed for the highest tested concentration (CC50 > 200 µM) for all evaluated compounds. Due to the promising results, the mechanism of parasite death was investigated for compounds 1 and 1a using flow cytometry and spectrofluorimetry. The cell membrane permeability assay with SYTOX Green indicated that compound 1 significantly altered this parameter after 40 min of incubation, while compound 1a caused no alteration. Considering that the hydrogenation rendered a differential cellular target in parasites, additional assays were performed with 1a. Despite no permeabilization of the plasma membrane, compound 1a induced depolarization of the electric potential after two hours of incubation. The mitochondria of the parasite were also affected by compound 1a, with depolarization of the mitochondrial membrane potential, and reduction of reactive oxygen species (ROS) levels. The Ca2+ levels were not affected during the time of incubation. Considering that the mitochondrion is a single organelle in Trypanosoma cruzi for ATP generation, compounds affecting the bioenergetic system are of interest for drug discovery against Trypanosomatids.

P0340CYPD DEFICIENCY AMELIORATES AUTOIMMUNE VASCULITIS IN MOUSE MODEL VIA THE INHIBITION OF PROGRAMMED NECROSIS

Abstract Background and Aims Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a necrotizing vasculitis that affects systemic organs. Although the standard immunosuppressive therapy in AAV could control disease activity, these existing treatments have no effect to regulate the necrosis, which could cause the persistent organ damage. ANCA binds to neutrophils, leading to neutrophil extracellular (NETs) formation. The formed NETs induce vascular injury and become auto-antigens against ANCA, resulting in an auto-amplification loop of necrosis and inflammation. NETs work as innate immunity and simultaneously undergo programmed cell necrosis. However, the signaling pathway of ANCA-mediated NETs remains elusive. Cyclophilin D (CypD) regulates program necrosis via the opening of mitochondrial permeability transition pores (mPTP) with the consequence of mitochondrial ROS release. The Inhibition or genetic deletion of CypD has been reportedly to protect organ damage in brain and kidney ischemia-reperfusion injury mice. Herein, we hypothesized that CypD would be involved in ANCA-mediated NETs pathway and the pathogenesis of AAV. Method Animal experiment; Autoimmune vasculitis was induced in 10 week-old CypD(+/+) or CypD deficient (-/-) mice by the intravenous injection with anti-mouse MPO serum. At 24 hours after injection, we evaluated the renal and lung involvements. The severity of glomerulonephritis was assessed by the levels of BUN, haematuria, histological findings and TUNEL positivity. The lung injury was evaluated by histopathological alveolar haemorrhage scores and TUNEL positivity. The mPTP related necrosis in tissues was identified by immunostaining against cytchrome C. The NETs-forming neutrophils in peripheral bloods were detected as Sytox Green (SG)-positive neutrophils by flow cytometry (FCM). In vitro experiment; We investigated whether CypD inhibitor and genetic ablation suppresses ANCA-mediated NETs. Healthy human neutrophils were incubated with ANCA-IgG derived from AAV patients with pre-treatment of CypD inhibitor (cyclosporine A) or PBS for 4 hours. Next, WT or CypD (-/-) mice neutrophils were cultured with anti mouse MPO antibody for 4 hours. The NETs induction was assessed as Sytox green positive neutrophil using FCM and live imaging. Results The amount of haematuria, histological severity including glomerulonephritis and pulmonary haemorrhage in CypD(-/-) mice significantly decreased compared to those in CypD(+/+) mice. Furthermore, TUNEL and cytochrome C positivity of tissues in CypD(-/-) mice was lower than that of CypD(+/+) mice, respectively. The level of BUN in AAV-induced CypD (-/-) mice was significantly lower than that of AAV-induced CypD(+/+) mice (92.4 ± 41.8 vs 36.8 ± 21.5 mg/ dL, p <0.05). FCM analysis showed that circulating NETs and mitochondrial ROS in CypD(-/-) mice were lower than those in CypD(+/+) mice (NETs; 4.7 ± 2.4 vs 7.5 ± 2.6 %, p <0.05, ROS; 1.9 ± 0.38 vs 4.7 ± 0.78%, p <0.05). In vitro experiments, CypD inhibitor suppressed ANCA-induced NETs formation (Control; 0.45 ± 0.14 vs CypD inhibitor; 0.16 ± 0.04%, p <0.05). In addition, CypD(-/-) mice neutrophils-treated with ANCA showed lower NETs induction rate compared to CypD(+/+) neutrophils (CypD+/+: 4.27 ± 2.09 vs CypD-/-: 0.63 ± 0.35%, p <0.05). Conclusion Our data indicate that the inhibition of CypD might ameliorate autoimmune vasculitis via the suppression of programmed NETs formation.

Proadrenomedullin N-terminal 20 peptide (PAMP) and its C-terminal 12-residue peptide, PAMP(9–20): Cell selectivity and antimicrobial mechanism

Proadrenomedullin N-terminal 20 peptide (PAMP) is a regulatory peptide that is found in various cell types. It is involved in many biological activities and is rich in basic and hydrophobic amino acids, a common feature of antimicrobial peptides (AMPs). In this study, the cell selectivity and antimicrobial mechanism of PAMP and its C-terminal peptide, PAMP(9–20), were investigated. PAMP and PAMP(9–20) displayed potent antimicrobial activity (minimum inhibitory concentration: 4–32 μM) against standard bacterial strains, but showed no hemolytic activity even at the highest tested concentration of 256 μM. PAMP(9–20) showed 2- to 4-fold increase in antimicrobial activity against gram-negative bacteria compared to PAMP. Cytoplasmic membrane depolarization, leakage of calcein dye from membrane mimic liposomes, SYTOX Green uptake, membrane permeabilization, and flow cytometry studies indicated that the major target of PAMP and PAMP(9–20) is not the microbial cell membrane. Interestingly, laser-scanning confocal microscopy demonstrated that FITC-labeled PAMP and PAMP(9–20) enter the cytoplasm of Escherichia coli similar to buforin-2, and gel retardation assay indicated that PAMP and PAMP(9–20) effectively bind to bacterial DNA. These results suggest that the intracellular target mechanism is responsible for the antimicrobial action of PAMP and PAMP(9–20). Collectively, PAMP and PAMP(9–20) could be considered promising candidates for the development of new antimicrobial agents.

Seasonal variation in estuarine phytoplankton viability and its relationship with carbon dynamics in the Baltic Sea

Cell death drives the magnitude and community composition of phytoplankton and can result in the conversion of particulate organic carbon to dissolved organic carbon (DOC), thereby affecting carbon cycling in the aquatic food web. We used a membrane integrity probe (Sytox Green) to study the seasonal variation in the percentage of viable cells in the phytoplankton population in an estuary in the northern Baltic Sea for 21 months. The associated dissolved and particulate organic matter concentrations were also studied. The viable fraction of phytoplankton cells varied from < 20% to almost 100%, with an average of 62%. Viability was highest when a single phytoplankton group (diatoms or dinoflagellates) dominated the community. Viability of sinking phytoplankton cells, including some motile species, was in general as high as in surface water. Changes in viability were not closely related to nutrient concentrations, virus-like particle abundance, seawater temperature or salinity. There was a weak but significant negative correlation between viability and DOC, although at this location, the DOC pool was mainly influenced by the inflow of riverine water. This study demonstrates that cell viability, and its relationship with carbon export, is highly variable in the complex microbial populations common within estuarine and coastal marine ecosystems.

Cathelicidin-DM is an Antimicrobial Peptide from Duttaphrynus melanostictus and Has Wound-Healing Therapeutic Potential.

Antimicrobial peptides (AMPs) are a class of templates with application potential for drug development. Amphibians are important sources of AMPs. Duttaphrynus melanostictus is the main source of traditional Chinese medicine “Chansu”, which has anti-infection effect while without a clear mechanism. This study aimed to find the cathelicidin peptide in D. melanostictus and then investigate the activity in vivo and in vitro, and an AMP-encoding gene (cathelicidin-DM, GenBank: KJ820824.1) was obtained from the constructed cDNA library of D. melanostictus. The MIC test and SYTOX Green uptake were used for the evaluation of the bactericidal capacity and mechanisms. The serum stability tests were used for the evaluation of the application potential. The skin wound infection model and in vivo imaging were used for in vitro application of possibility evaluation. The results showed that cathelicidin-DM was a 37 amino acid AMP with good bactericidal ability, which was similar to melittin: both can kill bacteria within 15 min. Moreover, cathelicidin-DM exhibits good therapeutic potential in the mouse wound infection model, and it can be enriched to the site of infection for treatment. Thus, cathelicidin-DM could be a new template for antimicrobial drug development given its good antibacterial activity in vivo and in vitro.

Peptidyl-prolyl isomerase and the biological activities of recombinant protein cyclophilin from Pyropia yezoensis (PyCyp)

Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the pPROEX-HTA expression vector. The plasmid was transformed into BL21 Escherichia coli by high efficiency transformation. Recombinant protein was expressed using 0.1 mM IPTG and the fusion protein was purified by affinity column chromatography. The His-tag was removed by TEV protease. The recombinant protein was further purified on a HiPrep Sephacryl S-200 HR column and by reversed-phase high performance liquid chromatography with a Sep-pak plus C18 column. Purified cyclophilin was characterized by a variety of analytical methods and analyzed for its peptidyl-prolyl isomerase activity. Our recombinant PyCyp was shown to catalyze cis-trans isomerization. PyCyp was also evaluated for antimicrobial activity against both Gram-positive and Gram-negative bacteria cultures and showed significant antibacterial activity against tested pathogens. PyCyp was shown to permeabilize bacterial membranes as evidenced by increased fluorescence intensity in SYTOX Green uptake assays with Staphylococcus aureus. The radical scavenging activity of PyCyp increased in a dose-dependent manner, indicating significant antioxidant activity. This study provides information for the development of therapeutic proteins from marine algae.

Flow Cytometry Analysis of Anastasis using CellTrace Far Red (CTFR) cell tracker in Cultured Mammalian T Lymphocytes

Anastasis is a cellular recovery process that rescues apoptotic cells from the brink of death. Our lab has developed a model for studying anastasis using cultured mouse HT2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Within 24 hours of IL2 deprivation, HT2 cells display characteristic changes underlying apoptosis, including cellular condensation, membrane blebbing and fragmentation into apoptotic bodies, phosphatidylserine externalization, caspase 3 activation as well as G1 cell cycle arrest. HT2 cells can subsequently be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry in conjunction with CellTrace Far Red (CTFR) and SYTOX Green (live/dead indicator) to track the kinetics of anastasis and re‐entry of viable apoptotic cells into the cell cycle. HT2 lymphocytes continuously maintained in IL2 comprised a homogeneous, viable population of CTFR+ cells which decreased in fluorescence intensity with each subsequent cell cycle for up to 96 hours post‐CTFR labelling. To investigate apoptosis reversal, HT2 cells were labeled with CTFR, deprived of IL2 for 24 hours and then rescued with IL2 re‐addition for periods of 24, 48, and 72 hours. Our findings indicate that viable, CTFR+ HT2 cells gradually resumed cell cycle entry over the 72‐hour rescue period. As caspase activation represents a cellular marker of early‐mid stage apoptotic cells, we also investigated caspase 3/7 activity in viable CTFR+ cells using the Nucview‐488 fluorescent caspase 3 substrate. Three distinct populations of caspase‐high, medium and low, viable CTFR+ HT2 cells were observed at 24 hours following IL2 deprivation. IL2‐rescued cells subsequently segregated into caspase‐high and low populations, ultimately merging into a single, caspase‐low population by 72 hours post‐rescue. Collectively, our findings suggest that CTFR tracking can be used successfully as a tool to follow the kinetics of apoptosis reversal in IL2‐dependent mouse HT2 lymphocytes, and is a promising model for the study of the mechanistic basis of anastasis.Support or Funding InformationUnion College Faculty and Student Research Grants

Chromatin extracellular trap release in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)

Neutrophils release nuclear chromatin decorated with antimicrobial proteins into the extracellular milieu as an innate immune defence mechanism to counter invading microbes. These chromatin structures, called extracellular traps (ETs) and released by a process called NETosis, have been detected in mammals, certain invertebrates and some fish species, including fathead minnow, zebrafish, common carp, turbot, sole and barramundi. However, there have been no previous studies of ETs in the Salmonidae. ETs are released in response to chemical and biological stimuli, but observations from different fish species are inconsistent, particularly regarding the potency of various inducers and inhibitors. Thus, this present study aimed to describe ET release in a salmonid (rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)) and uncover the inducers and inhibitors that can control this response. Highly enriched suspensions of polymorphonuclear cells (PMNs; mainly neutrophils) were prepared from head kidney tissues by a triple-layer Percoll gradient technique. ET structures were visualised in PMN-enriched suspensions through staining of the chromatin with nucleic acid-specific dyes and immunocytochemical probing of characteristic proteins expected to decorate the structure. ET release was quantified after incubation with inducers and inhibitors known to affect this response in other organisms. Structures resembling ETs stained positively with SYTOX Green (a stain specific for nucleic acid) while immunocytochemistry was used to detect neutrophil elastase, myeloperoxidase and H2A histone in the structures, which are diagnostic proteinaceous markers of ETs. Consistent with other studies on mammals and some fish species, calcium ionophore and flagellin were potent inducers of ETs, while cytochalasin D inhibited NETosis. Phorbol 12-myristate 13-acetate (PMA), used commonly to induce ETs, exerted only weak stimulatory activity, while heat-killed bacteria and lipopolysaccharide did not induce ET release. Unexpectedly, the ET-inhibitor diphenyleneiodonium chloride acted as an inducer of ET release, an observation not reported elsewhere. Taken together, these data confirm for the first time that ETs are released by salmonid PMNs and compounds useful for manipulating NETosis were identified, thus providing a platform for further studies to explore the role of this mechanism in fish immunity. This new knowledge provides a foundation for translation to farm settings, since manipulation of the innate immune response offers a potential alternative to the use of antibiotics to mitigate against microbial infections, particularly for pathogens where protection by vaccination has yet to be realised.

A Versatile Liquid Culture Method to Control the &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Development of Shoot and Root Apical Meristems of Bamboo Plants

We focus on controlling morphological and histochemical responses of the shoot apical meristem (SAM) and root apical meristem (RAM) of bamboo node by using a simple and versatile liquid culture system. First, nodes of 11 different bamboo species that belong to seven major bamboo genera (Bambusa, Dendrocalamus, Phyllostachys, Tetragonocalamus, Chimonobambusa, Pleioblastus, and Sasa) were cultured using 2 mL per well of a liquid medium in a 6-well microplate to form a small-scale liquid culture environment (SLCE). The dormant lateral buds of all bamboo nodes resumed expanding and elongating within 7 days in the SLCE. The dormant and active lateral buds were sectioned longitudinally and stained with Sytox green (SG) to monitor mitotic activity and counterstained with safranin (SF) to detect the inward region of the SAM region. Further, mitotic activity was calculated using a digital imaging analysis, which showed an increase of up to 1.2- to 3.8-fold in terms of the SG/SF ratio after 7 days in the culture. Moreover, we used in vitro node cultures of two typical bamboo species, the sympodial clump-forming type (Bambusa multiplex Raeush, Bm) and the monopodial single culm-forming type (Phyllostachys meyeri McClure, Pm), and noted the following: 1) since gradual white-to-green tinge shoots were observed, we investigated the relation between color variation in the outer regions of culm and node tissues and their suitability as explants. By checking the autofluorescence property of whole shoots under LED 365 nm illumination with an RGB (red, green, and blue) digital imaging analysis using ImageJ software, we specified the color variation of explants as the relative intensity of the blue value. 2) Since the obtained shoots of a 1-month-old culture box showed growth variation, we distinguished shoot types based on plant height, i.e., short (less than 5 cm), medium (ca. 5 - 10 cm), and tall (more than 10 cm). Tall shoots that have ca. 5 nodes on average were suitable for explant. 3) Three types of node portions—the first node (the base node near a rhizome tissue), middle nodes (upper nodes near the 1st node), and the top meristem—were independently cultured in the SLCE, and it was found that the first node showed the best growth performance. 4) By culturing the first node in the SLCE system, we performed a quick survey during the 3 weeks in the culture and found that a combination of 10 μM benzyl adenine and 3 μM thidiazuron was effective for in vitro SAM development, while the addition of 2, 4-D was effective for promoting in vitro RAM development. 5) The detailed autofluorescence properties of the outer regions of culm and node tissues were also identified using an inverted fluorescent microscope under B- and U-excitation lights with RGB and HSB (hue, saturation, and brightness) digital imaging analysis.