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P. aeruginosa is one of the most common bacteria causing contact lens-related microbial keratitis (CLMK). Previous studies report that disinfecting solutions were ineffective in preventing biofilm formation. Solutions containing novel natural agents may be an excellent alternative for reducing the risk of CLMK. Here, we investigate the disinfecting properties of hydroquinine in combination with multipurpose solutions (MPSs) to prevent P. aeruginosa adhesion and biofilm formation. We examined the antibacterial, anti-adhesion, and anti-biofilm properties of hydroquinine-formulated MPSs compared to MPSs alone. Using RT-qPCR, hydroquinine directly affected the expression levels of adhesion-related genes, namely, cgrC, cheY, cheZ, fimU, and pilV, resulting in reduced adhesion and anti-biofilm formation. Using ISO 14729 stand-alone testing, hydroquinine met the criteria (>99.9% killing at disinfection time) against both P. aeruginosa reference and clinical strains. Using the crystal violet retention assay and FE-SEM, MPSs combined with hydroquinine were effective in inhibiting P. aeruginosa adhesion and destroying preexisting biofilms. This report is the first to highlight the potential utility of hydroquinine-containing formulations as a disinfecting solution for contact lenses, specifically for inhibiting adhesion and destroying biofilm. These findings may aid in the development of novel disinfectants aimed at combating P. aeruginosa, thereby potentially reducing the incidence of CLMK.
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The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as "effluxR detection assay" using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04-34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.
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Tailored porous structures of poly(2-hydroxyethyl methacrylate) (PHEMA) and silk sericin (SS) were used to create porous hydrogel scaffolds using two distinct crosslinking systems. These structures were designed to closely mimic the porous nature of the native extracellular matrix. Conventional free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) was performed in the presence of different concentrations of SS (1.25, 2.50, 5.00% w/v) with two crosslinking systems. A chemical crosslinking system with N'N-methylene bisacrylamide (MBAAm) and a physical crosslinking system with dimethylurea (DMU) were used: C-PHEMA/SS (crosslinked using MBAAm) and C-PHEMA/pC-SS (crosslinked using MBAAm and DMU). The focus of this study was on investigating the impact of these crosslinking methods on various properties of the scaffolds, including pore size, pore characteristics, polymerization time, morphology, molecular interaction, in vitro degradation, thermal properties, and in vitro cytotoxicity. The various crosslinked networks were found to appreciably influence the properties of the scaffolds, especially the pore sizes, in which smaller sizes and higher numbers of pores with high regularity were seen in C-PHEMA/1.25 pC-SS (17 ± 2 µm) than in C-PHEMA/1.25 SS (34 ± 3 µm). Semi-interpenetrating networks were created by crosslinking PHEMA-MBAAm-PHEMA while incorporating free protein molecules of SS within the networks. The additional crosslinking step involving DMU occurred through hydrogen bonding of the -C=O and -N-H groups with the SS, resulting in the simultaneous incorporation of DMU and SS within the PHEMA networks. As a consequence of this process, the scaffold C-PHEMA/pC-SS exhibited smaller pore sizes compared to scaffolds without DMU crosslinking. Moreover, the incorporation of higher loadings of SS led to even smaller pore sizes. Additionally, the gelation time of C-PHEMA/pC-SS was delayed due to the presence of DMU in the crosslinking system. Both porous hydrogel scaffolds, C-PHEMA/pC-SS and PHEMA, were found to be non-cytotoxic to the normal human skin dermal fibroblast cell line (NHDF cells). This promising result indicates that these hydrogel scaffolds have potential for use in tissue engineering applications.
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Hydroquinine has antimicrobial potential with demonstrated activity against several bacteria, including multidrug-resistant (MDR) P. aeruginosa reference strains. Despite this, there is limited evidence confirming the antibacterial activity of hydroquinine against clinical isolates and the underlying mechanism of action. Here, we aimed to investigate the antibacterial effect of hydroquinine in clinical P. aeruginosa strains using phenotypic antimicrobial susceptibility testing and synergistic testing. In addition, we examined the potential inhibitory mechanisms against MDR P. aeruginosa isolates using informatic-driven molecular docking analysis in combination with RT-qPCR. We uncovered that hydroquinine inhibits and kills clinical P. aeruginosa at 2.50 mg/mL (MIC) and 5.00 mg/mL (MBC), respectively. Hydroquinine also showed partial synergistic effects with ceftazidime against clinical MDR P. aeruginosa strains. Using SwissDock, we identified potential interactions between arginine deiminase (ADI)-pathway-related proteins and hydroquinine. Furthermore, using RT-qPCR, we found that hydroquinine directly affects the mRNA expression of arc operon. We demonstrated that the ADI-related genes, including the arginine/ornithine antiporter (arcD) and the three enzymes (arginine deiminase (arcA), ornithine transcarbamylase (arcB), and carbamate kinase (arcC)), were significantly downregulated at a half MIC of hydroquinine. This study is the first report that the ADI-related proteins are potential molecular targets for the inhibitory effect of hydroquinine against clinically isolated MDR P. aeruginosa strains.
Assuntos
Anti-Infecciosos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Simulação de Acoplamento Molecular , Genes Bacterianos , Anti-Infecciosos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Testes de Sensibilidade Microbiana , Arginina/metabolismoRESUMO
This work was concerned with the fabrication of a porous hydrogel system suitable for medium to heavy-exudating wounds where traditional hydrogels cannot be used. The hydrogels were based on 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPs). In order to produce the porous structure, additional components were added (acid, blowing agent, foam stabilizer). Manuka honey (MH) was also incorporated at concentrations of 1 and 10% w/w. The hydrogel samples were characterized for morphology via scanning electron microscopy, mechanical rheology, swelling using a gravimetric method, surface absorption, and cell cytotoxicity. The results confirmed the formation of porous hydrogels (PH) with pore sizes ranging from ~50-110 µm. The swelling performance showed that the non-porous hydrogel (NPH) swelled to ~2000%, while PH weight increased ~5000%. Additionally, the use of a surface absorption technique showed that the PH absorbed 10 µL in <3000 ms, and NPH absorbed <1 µL over the same time. Incorporating MH the enhanced gel appearance and mechanical properties, including smaller pores and linear swelling. In summary, the PH produced in this study had excellent swelling performance with rapid absorption of surface liquid. Therefore, these materials have the potential to expand the applicability of hydrogels to a range of wound types, as they can both donate and absorb fluid.
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Hydroquinine is an organic alkaloid compound that exhibits antimicrobial activity against several bacterial strains including strains of both drug-sensitive and multidrug-resistant P. aeruginosa. Despite this, the effects of hydroquinine on virulence factors in P. aeruginosa have not yet been characterized. We therefore aimed to uncover the mechanism of P. aeruginosa hydroquinine-sensitivity using high-throughput transcriptomic analysis. We further confirmed whether hydroquinine inhibits specific virulence factors using RT-qPCR and phenotypic analysis. At half the minimum inhibitory concentration (MIC) of hydroquinine (1.250 mg/mL), 254 genes were differentially expressed (97 downregulated and 157 upregulated). We found that flagellar-related genes were downregulated by between −2.93 and −2.18 Log2-fold change. These genes were consistent with the analysis of gene ontology and KEGG pathway. Further validation by RT-qPCR showed that hydroquinine significantly suppressed expression of the flagellar-related genes. By analyzing cellular phenotypes, P. aeruginosa treated with ½MIC of hydroquinine exhibited inhibition of motility (30−54% reduction) and pyocyanin production (~25−27% reduction) and impaired biofilm formation (~57−87% reduction). These findings suggest that hydroquinine possesses anti-virulence factors, through diminishing flagellar, pyocyanin and biofilm formation.
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Hydroquinine is an organic compound that is closely related to quinine-derivative drugs and contains anti-malarial and anti-arrhythmia activities. It has been also found in abundance in some natural extracts that possess antibacterial properties. However, there is little evidence demonstrating the antibacterial effect of hydroquinine. Therefore, we aimed to investigate the antibacterial properties of hydroquinine using broth microdilution methods. In addition, we evaluated the transcriptional responses of P. aeruginosa to hydroquinine-induced stress using RNA sequencing with transcriptomic analysis and validated the results using PCR-based methods. The MIC and MBC values of hydroquinine against all eight bacterial strains investigated ranged from 650 to 2500 and from 1250 to 5000 µg/mL, respectively. Transcriptomic analysis demonstrated that RND efflux pump transcripts were overexpressed (4.90−9.47 Log2 fold change). Using mRT-dPCR and RT-qPCR, we identified that mRNA levels of mexD and mexY genes were overexpressed in response to just half the MIC of hydroquinine in P. aeruginosa. In conclusion, we uncover the antimicrobial potential of hydroquinine as well as identify changes in gene expression that may contribute to bacterial resistance. Further work will be required to explore the efficacy and potential use of hydroquinine in the clinic.
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Bombyx mori silk fibroin (SF), from Nangnoi Srisaket 1 Thai strain, has shown potential for various biomedical applications such as wound dressing, a vascular patch, bone substitutes, and controlled release systems. The hemocompatibility of this SF is one of the important characteristics that have impacts on such applications. In this study, the hemocompatibility of Thai SF was investigated and its improvement by low molecular weight heparin (LMWH) immobilization was demonstrated. Endothelial cell proliferation on the SF and LMWH immobilized SF (Hep/SF) samples with or without fibroblast growth factor-2 (FGF-2) was also evaluated. According to hemocompatibility evaluation, Thai SF did not accelerate clotting time, excess stimulate complement and leukocyte activation, and was considered a non-hemolysis material compared to the negative control PTFE sheet. Platelet adhesion of SF film was comparable to that of the PTFE sheet. For hemocompatibility enhancement, LMWH was immobilized successfully and could improve the surface hydrophilicity of SF films. The Hep/SF films demonstrated prolonged clotting time and slightly lower complement and leukocyte activation. However, the Hep/SF films could not suppress platelet adhesion. The Hep/SF films demonstrated endothelial cell proliferation enhancement, particularly with FGF-2 addition. This study provides fundamental information for the further development of Thai SF as a hemocompatible biomaterial.
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PURPOSE: The purposes of this study were to use speckle tracking echocardiography to confirm the influence of obesity on cardiac functions and to assess their relationships with leptin and uric acid levels in obese adolescents. METHODS: Eighty-one participants aged 16-19 years were recruited and classified as either non-obese (n = 30) or obese (n = 51). Global longitudinal strain (GLS), leptin and uric acid levels for each group were assessed and compared. The data from obese participants were then compared based on their leptin levels and analyzed for correlation using regression analysis. RESULTS: The obese group had significantly lower absolute GLS compared to the non-obese group (19.10 ± 0.30 versus 21.10 ± 0.30%, p < 0.001). In obese group, subclinical cardiac dysfunction was worse in the hyperleptinemic group than that of the normoleptinemic group (p = 0.03). Multivariate regression analysis showed that leptin and triglyceride levels were negatively associated with absolute GLS. Leptin could predict the absolute GLS with ß = -0.35 (p = 0.02). CONCLUSION: Subclinical left ventricular systolic dysfunction was found in obese adolescents, while GLS was worse in the hyperleptinemic subjects. Leptin, but not uric acid, levels were associated with a worsening of GLS.
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Regulation of protein synthesis is crucial for cells to maintain viability and to prevent unscheduled proliferation that could lead to tumorigenesis. Exposure to stress results in stalling of translation, with many translation initiation factors, ribosomal subunits and mRNAs being sequestered into stress granules or P bodies. This allows the re-programming of the translation machinery. Many aspects of translation are regulated by post-translational modification. Several proteomic screens have identified translation initiation factors as targets for sumoylation, although in many cases the role of this modification has not been determined. We show here that eIF4A2 is modified by SUMO, with sumoylation occurring on a single residue (K226). We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol. In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules. Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Estresse Fisiológico , Sumoilação , Sequência de Aminoácidos , Arsenitos/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos da radiação , Fator de Iniciação 4A em Eucariotos/química , Células HeLa , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Mutação/genética , Radiação Ionizante , Esteróis/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/efeitos da radiação , Sumoilação/efeitos dos fármacos , Sumoilação/efeitos da radiaçãoRESUMO
SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively.
Assuntos
Endopeptidases/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sumoilação/fisiologia , Células HeLa , Humanos , Schizosaccharomyces/metabolismoRESUMO
Regulation of protein synthesis is of fundamental importance to cells. It has a critical role in the control of gene expression, and consequently cell growth and proliferation. The importance of this control is supported by the fact that protein synthesis is frequently upregulated in tumor cells. The major point at which regulation occurs is the initiation stage. Initiation of translation involves the interaction of several proteins to form the eIF4F complex, the recognition of the mRNA by this complex, and the subsequent recruitment of the 40S ribosomal subunit to the mRNA. This results in the formation of the 48S complex that then scans the mRNA for the start codon, engages the methionyl-tRNA and eventually forms the mature 80S ribosome which is elongation-competent. Formation of the 48S complex is regulated by the availability of individual initiation factors and through specific protein-protein interactions. Both of these events can be regulated by post-translational modification by ubiquitin or Ubls (ubiquitin-like modifiers) such as SUMO or ISG15. We provide here a summary of translation initiation factors that are modified by ubiquitin or Ubls and, where they have been studied in detail, describe the role of these modifications and their effects on regulating protein synthesis.
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The present study was undertaken to assess the feasibility of whole dried blood spots to identify alpha-thalassemia-1 by PCR technique. Logistical constraints associated with sample collection and processing make the measurement of alpha-thalassemia-1 at community laboratories difficult. Dried blood spot samples provide a relatively convenient and minimally invasive means for collecting and transporting samples. We present Whatman 3 MM paper for spotting blood samples used for the first time for alpha-thalassemia-1 detection. Measurements of fresh EDTA whole blood and Whatman 3 MM dried blood spots prepared from venous blood were performed in 60 subjects. Whatman 3 MM dried blood spots results were precise and reliable. There was no actual difference in comparison to fresh EDTA whole blood samples. Whatman 3 MM dried blood spots for alpha-thalassemia-1 detection using the PCR technique showed no changes for 6 months.