Antimicrobial peptide 2K4L disrupts the membrane of multidrug-resistant Acinetobacter baumannii and protects mice against sepsis.

Antimicrobial peptides represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. 2K4L is a rationally-designed analog of a short peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L adopt an α-helical confirm in a membrane-mimetic environment and displayed an improved and broad-spectrum antibacterial activity against sensitive and multidrug-resistant Gram-negative and Gram-positive bacterial strains. Here, the action mechanism of 2K4L on multidrug resistant Acinetobacter baumannii (MRAB) and protection on MRAB-infected mice was investigated. The results demonstrated high bactericidal activity of 2K4L against both a multidrug resistant A. baumannii 0227 strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22934), indicating a potential therapeutic advantage of this peptide. Strong positively-charged residues significantly promoted the electrostatic interaction on 2K4L with lipopolysaccharides (LPS) of the bacterial outer membrane. High hydrophobicity and an α-helical confirm endowed 2K4L remarkably increase the permeability of A. baumannii cytoplasmic membrane by depolarization of membrane potential and disruption of membrane integration, as well as leakage of fluorescein from the liposomes. Additionally, 2K4L at low concentrations inhibited biofilm formation and degraded mature 1-day-old MRAB 0227 biofilms by reducing the expression of biofilm-related genes. In an invasive A. baumannii infection model, 2K4L enhanced the survival of sepsis mice and decreased the production of the proinflammatory cytokines downregulating the phosphorylation level of signaling protein in MAPK and NF-κB signaling pathways, indicating that 2K4L represents a novel therapeutic antibiotic candidate against invasive multidrug-resistant bacterial strain infections.

Membrane mechanism of temporin-1CEc, an antimicrobial peptide isolated from the skin secretions of Rana chensinensis, and its systemic analogs.

Antimicrobial peptides (AMPs) are new and powerful target molecules in the development of new antibacterial agents. Temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis, exhibits low or no antibacterial activity against gram-negative and gram-positive bacteria, which limits its potential therapeutic use; however, it displays low hemolysis to human erythrocytes. Here, a series of temporin-1CEc analogs was designed and synthesized by amino acid residue substitutions based on cationicity, hydrophobicity, amphipathicity and secondary structure to understand the structure-activity relationships of this peptide in depth. The results showed that all of the analogs, except for 2K and 4K, had significantly improved antibacterial activity against the tested standard bacterial strains and multidrug-resistant bacterial strains compared to temporin-1CEc. 2K2L and 2K4L, but not 4K2L and 4K4L, showed the strongest antibacterial activity compared with their parent peptides 2K and 4K, suggesting that peptide hydrophobicity plays a more important role in antibacterial activity than cationicity for this series of AMPs. However, the antibacterial activity of the 6 Trp-containing analogs of 2K4L decreased with a further increase in hydrophobicity based on the results of 2K4L, indicating that it is more important to balance cationicity and hydrophobicity. Moreover, an increase in AMP hydrophobicity led to hemolysis. Notably, all of the peptides adopted α-helical structures in 50% trifluoroethanol/water and 30 mM SDS solutions. 2K2L and 2K4L displayed broad-spectrum antibacterial activity against sensitive and multidrug-resistant bacteria, effectively killing the tested multidrug resistant strain Staphylococcus epidermidis (MRSE1208). 2K2L and 2K4L were able to increase the permeability of the outer and inner membranes by depolarization and disturb the integration of the cytoplasmic membrane of MRSE1208 cells, leading to leakage of its cellular contents. In addition, 2K2L and 2K4L at low concentrations inhibited biofilm formation and degraded mature 1-day-old MRSE1208 biofilms. Notably, 2K2L and 2K4L inhibited the formation of MRSE1208 biofilms at concentrations below its MIC value, suggesting that the peptide may exert an inhibitory effect through not only direct antimicrobial activity but also a biofilm-specific mechanism. Collectively, these results suggest that 2K2L and 2K4L could be effective antibiotics against multidrug-resistant bacterial strains.

Trp-Containing Antibacterial Peptides Impair Quorum Sensing and Biofilm Development in Multidrug-Resistant Pseudomonas aeruginosa and Exhibit Synergistic Effects With Antibiotics.

Pseudomonas aeruginosa uses quorum sensing (QS) to control virulence, biofilm formation and antibiotic efflux pump expression. The development of effective small molecules targeting the QS system and biofilm formation represents a novel attractive strategy. In this present study, the effects of a series of Trp-containing peptides on the QS-regulated virulence and biofilm development of multidrug-resistant P. aeruginosa, as well as their synergistic antibacterial activity with three classes of traditional chemical antibiotics were investigated. The results showed that Trp-containing peptides at low concentrations reduced the production of QS-regulated virulence factors by downregulating the gene expression of both the las and rhl systems in the strain MRPA0108. Biofilm formation was inhibited in a concentration-dependent manner, which was associated with extracellular polysaccharide production inhibition by downregulating pelA, algD, and pslA transcription. These changes correlated with alterations in the extracellular production of pseudomonal virulence factors and swarming motility. In addition, the combination of Trp-containing peptides at low concentration with the antibiotics ceftazidime and piperacillin provided synergistic effects. Notably, L11W and L12W showed the highest synergy with ceftazidime and piperacillin. A mechanistic study demonstrated that the Trp-containing peptides, especially L12W, significantly decreased ß-lactamase activity and expression of efflux pump genes OprM, MexX, and MexA, resulting in a reduction in antibiotic efflux from MRPA0108 cells and thus increasing the antibacterial activity of these antibiotics against MRPA0108.

Interactions of Designed Trp-Containing Antimicrobial Peptides with DNA of Multidrug-Resistant Pseudomonas aeruginosa.

To investigate the intracellular mechanisms of seven Trp-containing peptides in clinically isolated multidrug-resistant Pseudomonas aeruginosa (MRPA0108). The results showed that the Trp-containing peptides had high antibacterial activity against the MRPA0108 strain, with minimal inhibitory concentration (MIC) values ranging from 6.25 to 25 µM. The peptides rapidly and completely killed the MRPA0108 at a concentration of 16 × MIC at 60-90 min. The Trp-containing peptides were found to penetrate the bacterial cell membrane and accumulate in the cells. A DNA gel retardation assay indicated that the peptides were able to bind with the genomic DNA of MRPA0108 cells; L5W exhibited a stronger DNA binding ability than that of the other peptides, and the ratio of peptide to DNA was 0.62/1. Next, the UV absorption spectrum of the DNA indicated that L5W interacted with the MRPA0108 genomic DNA and intercalated into the groove of the DNA molecule, resulting in loosening of the double-helical structure of the originally contracted DNA and leading to the occurrence of a hyperchromic effect. The circular dichroism spectrum suggested that I1W and L5W associated with the DNA via a trench combination mode resulting from the compact structure of the DNA double helix and reduction in ππ accumulation between base pairs. Furthermore, real-time quantitative PCR demonstrated that the Trp-containing peptides could downregulate the expression of DNA replication-initiating genes in MRPA0108 cells. MRPA0108 DNA may be a potential active target for the antimicrobial activity of Trp-containing peptides.

Synergistic Antibacterial Activity of Designed Trp-Containing Antibacterial Peptides in Combination With Antibiotics Against Multidrug-Resistant Staphylococcus epidermidis.

Multidrug resistance among various bacterial strains is leading to worldwide resistance to a wide range of antibiotics. Combination therapy involving current antibiotics and other biological or chemical molecules represents an attractive novel strategy. In this study, we investigated the synergistic antibacterial activity of a series of Trp-containing antimicrobial peptides (AMPs) with four classes of traditional chemical antibiotics that are inactive against multidrug-resistant Staphylococcus epidermidis (MRSE) in vitro and in vivo. Among the antibiotics that we studied, penicillin, ampicillin and erythromycin showed a distinct synergistic effect in combination with all of the Trp-containing AMPs, represented by a fractional inhibitory concentration index (FICI) of <0.5. The antibacterial activities were noticeably improved, with 32-to 64-fold reductions in the MIC values for ampicillin and 16- to 32-fold reductions in the MIC values for erythromycin and penicillin. Tetracycline showed synergistic activity with only I1WL5W but additive activity with L11W, L12W, and I4WL5W. Ceftazidime exhibited additive activity with the Trp-containing peptides. In addition, the antibiotics in combination with the peptide significantly inhibited biofilm formation by MRSE 1208. A mechanistic study demonstrated that the Trp-containing peptides, especially I1WL5W and I4WL5W, which contain two tryptophan residues, disrupted bacterial inner and outer membranes, which promoted antibiotic delivery into the cytoplasm and access to cytoplasmic targets; however, L11W and L12W may have increased intracellular antibiotic concentrations by decreasing blaZ, tet(m) and msrA expression. Importantly, strong synergistic activity against the MRSE 1208 strain was observed for the combination of I1WL5W and penicillin in a mouse infection model. Thus, the combination of AMPs and traditional antibiotics could be a promising option for the prevention of acute and chronic infections caused by MRSE.

GPX2 suppression of H2O2 stress regulates cervical cancer metastasis and apoptosis via activation of the ß-catenin-WNT pathway.

BACKGROUND: Increasing evidence suggests that glutathione peroxidase 2 (GPX2) plays important roles in the tumorigenesis and progression of various human cancers, such as colorectal carcinomas and lung adenocarcinomas. However, the role of GPX2 in cervical cancer is unclear. In this study, we identified the role of GPX2 in cervical cancer tissues and cell lines. MATERIALS AND METHODS: The basal mRNA and protein expression of GPX2 in cervical cancer cells and a series of key molecules in the epithelial to mesenchymal transition (EMT) and WNT/ß-catenin pathways were examined via real time fluorescence quantitative PCR (qRT-PCR) and Western blot assays. The biological phenotype of the cervical cancer cell lines was detected by the cloning formation and transwell assays, and intracellular reactive oxygen species (ROS) levels were detected by flow cytometry. Finally, the GPX2 expression level in 100 clinical cervical tissues was examined by immunohistochemistry. RESULTS: We found that GPX2 was highly expressed in cervical cancer tissues compared to normal individuals and promoted the proliferation and metastasis of cervical cancer cells, and this promotion correlated with the activation of EMT and WNT/ß-catenin signaling in vitro. GPX2 was determined to reduce apoptotic damage by reducing hydroperoxides. According to the characteristics and verification of GPX2, this series of phenotypes is clearly related to oxidative stress in cells. Furthermore, we verified that GPX2 was highly expressed in cervical cancer tissues and promoted the metastasis of cervical cancer. CONCLUSION: In summary, we found that GPX2 was highly expressed in cervical cancer cells and promoted the proliferation and metastasis of cervical cancer by affecting oxidative stress. Our study provides a new target for the clinical treatment of cervical cancer.

Inflammatory anemia-associated parameters are related to 28-day mortality in patients with sepsis admitted to the ICU: a preliminary observational study.

BACKGROUND: Anemia is one of the most common complications of sepsis. Sepsis-related anemia is associated mainly with inflammation. We aimed to observe the changes in the inflammatory anemia-associated parameters of patients with sepsis in the early stage of intensive care unit (ICU) admission and to evaluate their association with 28-day mortality. METHODS: A total of 198 patients with sepsis were divided into survivor (n = 110) and non-survivor (n = 88) groups on the basis of 28-day survival. Healthy volunteers (n = 20) were enrolled as a control group. Plasma levels of iron, ferritin, erythropoietin (EPO), soluble transferrin receptor (sTfR), hepcidin, interleukin-6 (IL-6), hemoglobin and the red blood cell distribution width (RDW) were measured on days 1, 3 and 7 of ICU admission. Clinical data and laboratory findings were collected, and the Sequential Organ Failure Assessment (SOFA) score was calculated. RESULTS: Patients with sepsis showed significant decreases in hemoglobin, plasma iron and sTfR/log ferritin and significant increases in plasma EPO, sTfR, hepcidin, ferritin and IL-6 on days 1, 3 and 7 of ICU admission compared with healthy volunteers. Hemoglobin was correlated negatively with plasma IL-6 and hepcidin. In patients with sepsis, non-survivors had significantly lower plasma iron, EPO and sTfR/log ferritin, but higher plasma hepcidin, ferritin and IL-6 than survivors on days 1, 3 and 7 of ICU admission. Plasma EPO, hepcidin, ferritin, IL-6, sTfR/log ferritin, the RDW and SOFA score were associated significantly with 28-day mortality but to a varying extent. In particular, in predicting 28-day mortality, plasma hepcidin had an area under the receiver operating curve of 0.808 and 87.3% specificity, which was the highest among the inflammatory anemia-associated parameters tested. CONCLUSIONS: Inflammatory anemia-associated parameters changed significantly in patients with sepsis in the first week of ICU admission. Plasma EPO, hepcidin, ferritin, IL-6, sTfR/log ferritin, the RDW and SOFA score were associated significantly with 28-day mortality. Plasma hepcidin might have a superior predictive value, with high specificity, compared with other inflammatory anemia-associated parameters for 28-day mortality of sepsis patients in the ICU.

High-dose OxyContin to treat pain associated with bone metastasis in patients with small-cell lung cancer: a case study report.

Pain management is an important topic that has received extensive attention from clinical practitioners. Nearly all patients with malignant tumors suffer pain at the advanced stage of their disease. Oxycodone is a first-line choice for treating moderate-to-severe cancer-related pain, and OxyContin, a controlled-release oxycodone hydrochloride tablet, is internationally recognized as a safe and effective opioid analgesic. OxyContin has the characteristics of both immediate release and sustained release, with a time to onset and peak similar to those of immediate-release morphine. It acts on both µ and κ receptors and has been shown to be effective in treating different types of pain, especially neuropathic pain, theoretically without a dose cap. However, the dose is limited in clinical applications due to various factors that are likely to affect its analgesic effect and reduce patient quality of life. Cooperation with a patient's family members is required during the treatment of cancer pain. Chronic cancer pain has a long disease course, which could easily cause complex psychological symptoms due to their important role in the pain experience. Pain is controllable, and patients have a right to not experience pain. An optimal living state can be achieved through collaboration between physicians and patients. Rational personalized treatment of cancer pain can improve patient quality of life, relieve pain, and help prolong patient survival. This article reports the treatment procedure and adverse reactions in a patient who was treated with high-dose OxyContin, with the aim of providing a reference for other clinical practitioners.