SYTOX Green Research Articles

Antibacterial and antibiofilm features of mutSMAP-18 against Vibrio cholerae

Cholera continues to be a pointed global health issue, prominently in developing nations, where the disease's severe diarrheal symptoms pose substantial public health risks. With the escalating spread of antibiotic resistance among V. cholerae strains, alternative therapeutic approaches are imperative. Antimicrobial peptides are increasingly recognized for their potential, with research focusing on finding the most effective options. We explored the antibacterial and antibiofilm properties of analogues of sheep myeloid antimicrobial peptide-18 (SMAP-18) against V. cholerae in this investigation. Our prior research demonstrated that substituting glycine with alanine at different positions within SMAP-18 altered its structure and antimicrobial activity. Among these altered analogues, our focus was on a mutant variant (mutSMAP-18), characterized by glycine-to-alanine substitutions at positions 2, 7, and 13. Our results indicated that mutSMAP-18 exhibited heightened antimicrobial and antibiofilm activities against V. cholerae compared to SMAP-18. We conducted several mechanistic investigations to check the membrane integrity using DNA-binding dye, SYTOX Green or measuring calcein dye leakage and analyzing flow cytometry by fluorescence-activated cell sorting (FACScan). From these tests, we elucidated that SMAP-18 primarily functions intracellularly, while mutSMAP-18 targets the bacterial membrane. Additionally, scanning electron microscopy (SEM) images illustrated membrane disruption at lower concentrations for mutSMAP-18. Notably, mutSMAP-18 demonstrated significant antibiofilm properties against V. cholerae. Overall, these findings offer valuable perspectives for developing novel antibacterial therapies targeting the pathogenic V. cholerae.

Synergistic effects of oxidative and acid stress on bacterial membranes of Escherichia coli and Staphylococcus simulans

Oxidative stress in combination with acid stress has been shown to inactivate a wide spectrum of microorganisms, including multi-resistant bacteria. This occurs e.g. in phagolysosomes or during treatment by cold atmospheric pressure plasmas (CAP) and possibly depends on the cell membrane. We therefore explored the effects of CAP-generated reactive oxygen and nitrogen species (RONS) on bacterial growth inhibition and membranes in neutral and acidic suspensions. We observed that growth inhibition was most efficient when bacteria were treated by a mix of short and long-lived RONS in an acidic environment. Membrane packing was affected mainly upon contact with short-lived RONS, while also acidity strongly modulated packing. Under these conditions, Gram-negative bacteria displayed large potassium release while SYTOX Green influx remained marginal. Growth inhibition of Gram-negative bacteria correlated well with outer membrane (OM) permeabilization that occurred upon contact with short and/or long-lived RONS in synergy with acidity. In Gram-positive bacteria, CAP impaired membrane potential possibly through pore formation upon contact with short-lived RONS while formation of membrane protein hydroperoxides was probably involved in these effects. In summary, our study provides a wide perspective on understanding inactivation mechanisms of bacteria by RONS in combination with acidity.

Evaluating the Antimicrobial Efficacy of a Designed Synthetic Peptide against Pathogenic Bacteria.

Recent research has focused on discovering peptides that effectively target multi-resistant bacteria while leaving healthy cells unharmed. In this work, we describe the antimicrobial properties of RK8, a peptide composed of eight amino acid residues. Its activity was tested against multidrug-resistant Gram-negative and Gram-positive bacteria. RK8's efficacy in eradicating mature biofilm and increasing membrane permeability was assessed using Sytox Green. Cytotoxicity assays were conducted both in vitro and in vivo models. Circular dichroism analysis revealed that RK8 adopted an extended structure in water and sodium dodecyl sulfate (SDS). RK8 exhibited MICs of 8-64 μM and MBCs of 4-64 μM against various bacteria, with higher effectiveness observed in Staphylococcus aureus (MRSA) and E. coli KPC+ strains than others. Ciprofloxacin and Vancomycin showed varying MIC and MBC values lower than RK8 for Gram-positive bacteria, but competitive for Gram-negative bacteria. The combination of RK8 and ciprofloxacin showed a synergistic effect. The RK8 peptides could reduce 38% of the mature A. baumannii biofilm. Sytox Green reagent achieved 100% membrane permeation of Gram-positive and Gram-negative bacteria. The RK8 peptide did not show cytotoxic effects against murine macrophages (64 μM), erythrocytes (100 μM) or Galleria mellanella larvae (960 μM). In the stability test against peptidases, the RK8 peptide was stable, maintaining around 60% of the molecule intact after 120 min of incubation. These results highlight the potential of RK8 to be a promising strategy for developing a new antimicrobial and antibiofilm agent, inspiring and motivating further research in antimicrobial peptides.

Sodium Acetate Enhances Neutrophil Extracellular Trap Formation via Histone Acetylation Pathway in Neutrophil-like HL-60 Cells.

Neutrophil extracellular trap formation has been identified as a new cell death mediator, termed NETosis, which is distinct from apoptosis and necrosis. NETs capture foreign substances, such as bacteria, by releasing DNA into the extracellular environment, and have been associated with inflammatory diseases and altered immune responses. Short-chain fatty acids, such as acetate, are produced by the gut microbiota and reportedly enhance innate immune responses; however, the underlying molecular mechanisms remain unclear. Here, we investigated the effects of sodium acetate, which has the highest SCFA concentration in the blood and gastrointestinal tract, on NETosis by focusing on the mechanisms associated with histone acetylation in neutrophil-like HL-60 cells. Sodium acetate enhanced NETosis, as shown by fluorescence staining with SYTOX green, and the effect was directly proportional to the treatment duration (16-24 h). Moreover, the addition of sodium acetate significantly enhanced the acetylation of Ace-H3, H3K9ace, and H3K14ace. Sodium acetate-induced histone acetylation rapidly decreased upon stimulation with the calcium ionophore A23187, whereas histone citrullination markedly increased. These results demonstrate that sodium acetate induces NETosis via histone acetylation in neutrophil-like HL-60 cells, providing new insights into the therapeutic effects based on the innate immunity-enhancing effect of dietary fiber.

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-α and IFN-γ, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

GGPPS Negatively Regulates the Formation of Neutrophil Extracellular Traps in Lipopolysaccharide-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are life-threatening diseases. Neutrophil extracellular traps (NETs) play a key role in lung damage. Geranylgeranyl diphosphate synthase (GGPPS) is associated with the development of inflammatory diseases. We aimed to explore the role of GGPPS in NETs formation in ARDS/ALI. First, lung pathological changes in lipopolysaccharide (LPS)-induced ALI mice after myeloid-specific GGPPS deletion were evaluated. The level of NETs formation was analyzed by immunofluorescence, PicoGreen assay and Western blotting. Next, we determined the role of GGPPS in NETs formation and underlying mechanisms using immunofluorescence, flow cytometry, DCFH-DA, and SYTOX GREEN staining in vitro. Finally, the correlation between GGPPS expression incirculating neutrophils and dsDNA levels in plasma was evaluated. Myeloid-specific GGPPS deletion mice showed increased NETs deposition in lung tissue and aggravated histopathological damage of lung tissue. In vitro, GGPPS deficiency in neutrophils resulted in increased NETs formation by Phorbol-12-myristate-13-acetate (PMA), which was reversed by Geranylgeranyl diphosphate (GGPP). In addition, inhibitors blocking protein kinase C (PKC) and NADPH-oxidase (NOX) decreased NETs formation induced by GGPPS deletion. Importantly, GGPPS expression in circulating neutrophils was decreased in ARDS patients compared with the healthy control, and the level of dsDNA in plasma of ARDS patients was negatively correlated with the GGPPS expression. Taken together, GGPPS deficiency in neutrophils aggravates LPS-induced lung injury by promoting NETs formation via PKC/NOX signaling. Thus, neutrophil GGPPS could be a key therapeutic target for ARDS.

Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability

The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.

Antibacterial Activity and Action Mechanism of Bacteriocin Paracin wx7 as a Selective Biopreservative against Vancomycin-Resistant Enterococcus faecalis in Fresh-Cut Lettuce.

Fresh-cut vegetables are widely consumed, but there is no food preservative available to selectively inhibit vancomycin-resistant E. faecalis, which is a serious health menace in fresh-cut vegetables. To develop a promising food biopreservative, a bacteriocin, paracin wx7, was synthesized, showing selective inhibition against E. faecalis with MIC values of 4-8 μM. It showed instant bactericidal mode within 1 h at high concentrations with concomitant cell lysis against vancomycin-resistant E. faecalis. Its lethal effect was visualized in a dose-dependent manner by PI/SYTO9 staining observation. The results of an in vivo control experiment carried out on E. faecalis in fresh-cut lettuce showed that 99.97% of vancomycin-resistant E. faecalis were dead after 64 μM paracin wx7 treatment for 7 days without influencing total bacteria. Further, the action mechanism of paracin wx7 was investigated. Confocal microscopy showed that paracin wx7 was located both on the cell envelope and in cytoplasm. For the cell envelope, the studies of membrane permeability using SYTOX Green dyeing and DNA leakage revealed that paracin wx7 damaged the membrane integrity of E. faecalis. Simultaneously, it exhibited membrane depolarization after analysis using DiSC3(5). Damage to the cell envelope resulted in cell deformation observed by scanning electron microscopy. On entering the cytoplasm, the paracin wx7 induced the production of endogenous reactive oxygen species.

Abstract 2136: The Effects Of Dual Antiplatelet Therapy On Neutrophil Function

Introduction: Dual anti-platelet therapy (DAPT) is routinely administered to inhibit platelet aggregation in acute myocardial infarction (AMI) and, in doing so, reduces the risk of recurrent adverse cardiovascular events. Alongside platelets, neutrophils are also key mediators of inflammation during AMI, both secreting cytokines and chemokines and producing Neutrophil Extracellular Traps (NETs). While DAPT is known to mediate off-target anti-inflammatory effects, the extent to which DAPT affects neutrophil function is not well understood. Objective: To investigate the effects of DAPT on neutrophil-mediated inflammation. Methods: Neutrophils were isolated from whole blood of ten healthy subjects pre- and post-DAPT (ticagrelor and aspirin) treatment and NET formation in response to classical NET-inducing agonists (PMA and ionomycin) was measured by SYTOX Green fluorescence and MPO activity. Cytokine and chemokine production following stimulation with an activating agonist (FSL-1) was measured by cytometric bead array and reported as median fluorescence intensity (MFI). DAPT was effective in inhibiting platelet aggregation in response to AA and ADP. Results: NET formation in response to ionomycin, but not PMA, was greater in DAPT-treated neutrophils compared to treatment-naïve neutrophils (69.9 % ± 30.66 SEM). Levels of inflammatory mediators were also markedly different pre- and post- DAPT treatment. For example, MFI of IL-8 was decreased by 18% ± 9.97 SEM post-DAPT compared with pre-DAPT MFI, while MFI of IL-12p and IFN-α were significantly increased (32.54% ± 12.12 SEM, 58.68% ± 18.72 SEM respectively) compared to pre-DAPT. Conclusion: DAPT primes neutrophils for inducing some, but not all, NETotic pathways and releasing some, but not all, inflammatory mediators. This suggests that our primary strategy for inhibiting platelet aggregation in AMI does not provide broad off-target anti-inflammatory effects on neutrophils. In fact, DAPT may encourage a pro-inflammatory neutrophil phenotype.

Antibacterial activity of closantel against methicillin-resistant Staphylococcus aureus and itsbiofilm.

The antimicrobial resistance of Staphylococcus aureus (S. aureus) has become a challenge in the treatment of infectious diseases. It is of great clinical value to discovery effective antimicrobial agents against multi-drug resistant S. aureus and its biofilms. This study aims to explore the antibacterial activity of the antiparasitic drug closantel against methicillin-resistant S. aureus and its biofilms through drug repurposing. The sensitivity of S. aureus to closantel was assessed using microbroth dilution and disk diffusion methods. The bacteriostatic and bactericidal activities of closantel were determined by time-kill curves and colony count. Scanning electron microscopy combined with SYTOX Green and DiSC3(5) fluorescence probes were used to study the bactericidal mechanism of closantel. The influence of resistance was assessed by continuous exposure to sub-inhibitory concentrations of closantel. The anti-biofilm activity was evaluated using 96-well plates and crystal violet staining, and cytotoxicity was measured using the CCK-8 assay. The minimal inhibitory concentration (MIC) of closantel for both methicillin-sensitive and methicillin-resistant S. aureus ranged from 0.125 to 1.000 μg/mL. Disk diffusion tests showed that 80 μg of closantel created an inhibition zone, which increased in diameter with higher drug amounts. Sub-inhibitory concentrations (0.031 μg/mL) of closantel significantly inhibited S. aureus proliferation, reducing bacterial turbidity from 0.26±0.00 to 0.11±0.01 (t=16.06, P<0.001), with stronger inhibition at higher concentrations. Closantel at 0.25×MIC inhibited S. aureus proliferation for 12 hours, while 1×MIC inhibited it for over 24 hours, with the number of viable bacteria decreasing as the drug concentration increased. Mechanistic studies indicated that closantel effectively disrupted the integrity of S. aureus cell membranes, significantly increasing SYTOX Green and DiSC3(5) fluorescence intensity. Even after 25 days of continuous exposure to sub-inhibitory concentrations of closantel, no resistance developed. Closantel at 0.0625 μg/mL significantly inhibited biofilm formation, reducing it from 1.29±0.16 to 0.62±0.04 (t=11.62, P<0.001), showing a clear dose-dependent effect. Closantel at 2 μg/mL also significantly eradicated established biofilms, reducing biofilm mass from 1.62±0.34 to 0.51±0.39 (t=4.84, P<0.01). Additionally, closantel exhibited extremely low cytotoxicity, with half-maximal lethal concentrations for HepG2 liver cancer cells and normal LO2 liver cells both exceeding 64 μg/mL. Closantel exhibits strong antibacterial activity against S. aureus and its biofilm with low cytotoxicity against human cells, making it a promising candidate for new therapeutic strategies against S. aureus-related infections.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Differentiation and quantification of extracellular polymeric substances from microalgae and bacteria in the mixed culture

Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (<4.5 %) for the entire range of M:B ratios. EPS concentrations in bacteria were larger than in microalgae: 42.8 ± 0.4 mg COD/g TSS versus 9.19 ± 0.38 mg COD/g TSS. These findings document sensitive and accurate methods to extract and quantify EPS from microalgal-bacteria aggregates.

Imbalance of programmed cell death patterns mediated by dendritic cell subsets in systemic lupus erythematosus and lupus nephritis.

Abnormal programmed cell death in immune cells is associated with autoimmune diseases, but the patterns of programmed cell death in systemic lupus erythematosus (SLE) and especially lupus nephritis (LN) remain unclear. This study aims to explore the association between SLE, LN, and immune cell death patterns. Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 μmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 μmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group. Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group. This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.

Discovery of a novel class of rosmarinic acid derivatives as antibacterial agents: Synthesis, structure-activity relationship and mechanism of action

Twenty-seven rosmarinic acid derivatives were synthesized, among which compound RA-N8 exhibited the most potent antibacterial ability. The minimum inhibition concentration of RA-N8 against both S. aureus (ATCC 29213) and MRSA (ATCC BAA41 and ATCC 43300) was found to be 6 μg/mL, and RA-N8 killed E. coli (ATCC 25922) at 3 μg/mL in the presence of polymyxin B nonapeptide (PMBN) which increased the permeability of E. coli. RA-N8 exhibited a weak hemolytic effect at the minimum inhibitory concentration. SYTOX Green assay, SEM, and LIVE/DEAD fluorescence staining assay proved that the mode of action of RA-N8 is targeting bacterial cell membranes. Furthermore, no resistance in wildtype S. aureus developed after incubation with RA-N8 for 20 passages. Cytotoxicity studies further demonstrated that RA-N8 is non-toxic to the human normal cell line (HFF1). RA-N8 also exerted potent inhibitory ability against biofilm formation of S. aureus and even collapsed the shaped biofilm.

Abstract 1491: The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host

Abstract Background: Diabetes is associated with increased risk of cancer development and progression. Recent studies have shown that neutrophils in diabetic patients are prone to forming neutrophil extracellular traps (NETs) which have been reported to capture circulating cancer cells, leading to the promotion of liver and lung metastasis. This study aims to clarify the causative relationship between NETs and the peritoneal metastasis (PM) in diabetic condition. Methods: Outcome of the diabetic patients who received curative gastrectomy for gastric cancer from 2010 to 2021 in our institute was comparatively investigated with those without diabetes. Neutrophils were isolated from the bone marrow from C57BL/JHamSlc-ob/ob mouse (type 2 diabetes model) and wild type mouse using Ly6G-based positive immunomagnetic selection. The neutrophils were stimulated with 100nM of PMA for 4 hours and NETs formation was evaluated by the addition of Sytox Green under fluorescent microscope. A mouse gastric cancer cell, YTN16P (5 × 105 cells) was intraperitoneally (IP) injected, the number of metastatic nodules on mesentery was comparatively evaluated two weeks later. Results: Disease-free survival (p=0.004, HR=1.65) and overall survival (p=0.035, HR=1.46) of 203 diabetic patients were significantly worse those of 975 non-diabetic patients. Neutrophils isolated from ob/ob mice produced markedly increased NETs than that from wild-type mice. NETs produced by neutrophils obtained from the peritoneal cavity of ob/ob mice after IP injection of a 9% casein solution effectively captured many YTN16P in vitro. The concentration of double strand DNA (dsDNA) in plasma in ob/ob mice compared with their counterparts (676ng/mL vs 590ng/mL, p=0.036, n=3). The number of PM in ob/ob mice was significantly more than that in wild-type controls (39.1 vs 15.5, p=0.027, n=10). Conclusion: Enhanced NETs formation in diabetic conditions may be associated with an increased risk of PM in gastric cancer. Citation Format: Rei Takahashi, Hideyuki Ohzawa, Yuki Kaneko, Misaki Matsumiya, Yurie Futoh, Kohei Tamura, Kazuya Takahashi, Yuki Kimura, Akira Saito, Hideyo Miyato, Naohiro Sata, Joji Kitayama. The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1491.

NETosis Secondary to the Use of Levamisole-Adulterated Cocaine: A Likely Underlying Mechanism of Vasculopathy.

Since 2010, several cases of a new vasculopathy induced by the use of levamisole-adulterated cocaine (LAC) have been reported. This vasculopathy is characterized by retiform purpura, earlobe necrosis, multisystem compromise, and multiple autoantibodies. Given its similarity to antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, LAC-associated vasculopathy is postulated to be mediated by pathophysiologic processes resulting from neutrophil cell death by NETosis, a phenomenon previously described in ANCA vasculitis. This study tries to establish the presence of NETosis induced by cocaine, levamisole, or both. Methodology. Neutrophils were isolated from the peripheral blood of healthy controls by Ficoll-Hystopaque density gradient centrifugation followed by dextran sedimentation. Cell viability and purity were evaluated by flow cytometry after staining with PI/DiOC6 and labeling with fluorescent anti-CD45/anti-CD3 monoclonal antibodies (mAbs), respectively. Neutrophils were exposed to levamisole, cocaine, a cocaine-levamisole mixture, and sera pools from healthy controls and patients with LAC-associated vasculopathy. NETosis was then assessed by flow cytometry after staining cells with Sytox Green, Hoechst-33342, and fluorescent antineutrophil elastase (NE) and antimyeloperoxidase (MPO) mAbs. In addition, NETosis was morphologically confirmed by fluorescence microscopy. Proinflammatory cytokine levels in culture supernatants and reactive oxygen species (ROS) synthesis were determined by flow cytometry. The involvement of calcium and muscarinic receptors in cell death induction was evaluated in parallel experiments carried out in the presence of 1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid (BAPTA) and hyoscine butylbromide (HBB), their respective inhibitors. Cocaine, levamisole, and a cocaine-levamisole mixture induced neutrophil cell death. DNA/MPO extrusion and cell morphology patterns were consistent with NETosis. Neither proinflammatory cytokines nor ROS behaved as proNETotic factors. Preliminary results suggested that muscarinic receptors and calcium-dependent signals were involved in LAC-induced NETosis. Cocaine, levamisole, and a cocaine-levamisole mixture can induce NETosis through mechanisms involving muscarinic receptors and calcium-dependent pathways.

Computational analysis of curcumin-mediated alleviation of inflammation in periodontitis patients with experimental validation in mice.

Using network pharmacology and experimental validation to explore the therapeutic efficacy and mechanism of curcumin (Cur) in periodontitis treatment. Network pharmacology was utilized to predict target gene interactions of Cur-Periodontitis. Molecular docking was used to investigate the binding affinity of Cur for the predicted targets. A mouse model with ligature-induced periodontitis (LIP) was used to verify the therapeutic effect of Cur. Microcomputed tomography (micro-CT) was used to evaluate alveolar bone resorption, while western blotting, haematoxylin-eosin staining and immunohistochemistry were used to analyse the change in immunopathology. SYTOX Green staining was used to assess the in vitro effect of Cur in a mouse bone marrow-isolated neutrophil model exposed to lipopolysaccharide. Network pharmacology identified 114 potential target genes. Enrichment analysis showed that Cur can modulate the production of neutrophil extracellular traps (NETs). Molecular docking experiments suggested that Cur effectively binds to neutrophil elastase (ELANE), peptidylarginine deiminase 4 (PAD4) and cathepsin G, three enzymes involved in NETs. In LIP mice, Cur alleviated alveolar bone resorption and reduced the expression of ELANE and PAD4 in a time-dependent but dose-independent manner. Cur can directly inhibit NET formation in the cell model. Our research suggested that Cur may alleviate experimental periodontitis by inhibiting NET formation.

LC-AMP-F1 Derived from the Venom of the Wolf Spider Lycosa coelestis, Exhibits Antimicrobial and Antibiofilm Activities.

In recent years, there has been a growing interest in antimicrobial peptides as innovative antimicrobial agents for combating drug-resistant bacterial infections, particularly in the fields of biofilm control and eradication. In the present study, a novel cationic antimicrobial peptide, named LC-AMP-F1, was derived from the cDNA library of the Lycosa coelestis venom gland. The sequence, physicochemical properties and secondary structure of LC-AMP-F1 were predicted and studied. LC-AMP-F1 was tested for stability, cytotoxicity, drug resistance, antibacterial activity, and antibiofilm activity in vitro compared with melittin, a well-studied antimicrobial peptide. The findings indicated that LC-AMP-F1 exhibited inhibitory effects on the growth of various bacteria, including five strains of multidrug-resistant bacteria commonly found in clinical settings. Additionally, LC-AMP-F1 demonstrated effective inhibition of biofilm formation and disruption of mature biofilms. Furthermore, LC-AMP-F1 exhibited favorable stability, minimal hemolytic activity, and low toxicity towards different types of eukaryotic cells. Also, it was found that the combination of LC-AMP-F1 with conventional antibiotics exhibited either synergistic or additive therapeutic benefits. Concerning the antibacterial mechanism, scanning electron microscopy and SYTOX Green staining results showed that LC-AMP-F1 increased cell membrane permeability and swiftly disrupted bacterial cell membranes to exert its antibacterial effects. In summary, the findings and studies facilitated the development and clinical application of novel antimicrobial agents.

Fighting Microbial Infections from Escherichia coli O157:H7: The Combined Use of Three Essential Oils of the Cymbopogon Genus and a Derivative of Esculentin-1a Peptide.

The absence of effective therapy against Escherichia coli O157:H7 infections has led to the need to develop new antimicrobial agents. As the use of synergistic combinations of natural antimicrobial compounds is growing as a new weapon in the fight against multidrug-resistant bacteria, here, we have tested new synergistic combinations of natural agents. Notably, we investigated a possible synergistic effect of combinations of essential oils and natural peptides to counteract the formation of biofilm. We chose three essential oils (i.e., Cymbopogon citratus, C. flexuosus and C. martinii) and one peptide already studied in our previous works. We determined the fractional inhibitory concentration (FIC) by analyzing the combination of the peptide derived from esculentin-1a, Esc(1-21), with the three essential oils. We also studied the effects of combinations by time-kill curves, scanning electron microscopy on biofilm and Sytox Green on cell membrane permeability. Finally, we analyzed the expression of different genes implicated in motility, biofilm formation and stress responses. The results showed a different pattern of gene expression in bacteria treated with the mixtures compared to those treated with the peptide or the single C. citratus essential oil. In conclusion, we demonstrated that the three essential oils used in combination with the peptide showed synergy against the E. coli O157:H7, proving attractive as an alternative strategy against E. coli pathogen infections.

Extracellular DNA level as an indicator of the activity of inflammatory reactions in patients with rheumatoid arthritis and asthma

BACKGROUND: We performed a clinical and laboratory examination of patients with rheumatoid arthritis and bronchial asthma. Nosologies were selected to examine extracellular DNA changes in the blood during immunopathological processes based on generally accepted views on the role of helper balance in disease pathogenesis. The degree of disease activity in rheumatoid arthritis primarily depends on the severity of inflammatory changes in the joints; therefore, it is closely associated with a shift in the balance of helpers toward the Th1. According to today’s concepts, the pathogenesis of asthma is determined by the intensive production of specific antibodies, which are controlled by activated Th2 lymphocytes. AIM: To study extracellular DNA changes in the blood of patients during immunopathological processes depending on the Th1/Th2 balance ratio and compare these data with the parameters of standard laboratory tests and indicators of neutrophil activities and their ability to netosis. METHODS: Extracellular DNA concentration was determined using the Quant-ITTM PicoGreen fluorescent reagent for double-stranded DNA. Neutrophils isolated in a double-density gradient of ficoll–urografin were cultured with or without the addition of phorbol myristate acetate followed by Sytox Green. RESULTS: The studied immunopathological conditions had significantly decreased extracellular DNA levels in the plasma of patients with asthma, which sharply contrasted with its increase in patients with arthritis. Laboratory parameters confirmed the presence of local inflammation in patients with asthma without obvious symptoms of a systemic inflammatory response, while in patients with arthritis, inflammatory changes in the joints were accompanied by an increased C-reactive protein level, indicating a systemic reaction of the body in response to inflammatory stimuli. CONCLUSIONS: Thus, the state of the Th1/Th2 balance is assumed as one of the significant regulators that determined the extracellular DNA concentration in the blood. According to the proposed hypothesis, the shift in this ratio toward the predominance of Th1 cells should promote the development of inflammatory processes in the body and increase the amount of extracellular DNA, while a shift in the balance of helpers toward the dominance of Th2 lymphocytes should suppress these processes and consequently decrease the extracellular DNA in the blood.