scholarly journals Novel Detection Strategy To Rapidly Evaluate the Efficacy of Antichlamydial Agents

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Yan Zhang ◽  
Yuqi Xian ◽  
Leiqiong Gao ◽  
Hiba Elaasar ◽  
Yao Wang ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Green Fluorescence Protein ◽  
Proof Of Concept ◽  
Reporter System ◽  
Content Type ◽  
Gfp Reporter ◽  
Fluorescence Protein ◽  
Novel Strategy ◽  
The Impact ◽  
Antimicrobial Effectiveness

ABSTRACT Chlamydia trachomatis infections present a major heath burden worldwide. The conventional method used to detect C. trachomatis is laborious. In the present study, a novel strategy was utilized to evaluate the impact of antimicrobial agents on the growth of C. trachomatis and its expression of ompA promoter-driven green fluorescence protein (GFP). We demonstrate that this GFP reporter system gives a robust fluorescent display of C. trachomatis growth in human cervical epithelial cells and, further, that GFP production directly correlates to changes in ompA expression following sufficient exposure to antimicrobials. Validation with azithromycin, the first-line macrolide drug used for the treatment of C. trachomatis infection, highlights the advantages of this method over the traditional method because of its simplicity and versatility. The results indicate both that ompA is highly responsive to antimicrobials targeting the transcription and translation of C. trachomatis and that there is a correlation between changing GFP levels and C. trachomatis growth. This proof-of-concept study also reveals that the ompA-GFP system can be easily adapted to rapidly assess antimicrobial effectiveness in a high-throughput format.

scholarly journals BrpA Is Involved in Regulation of Cell Envelope Stress Responses in Streptococcus mutans

2012 ◽  
Vol 78 (8) ◽  
pp. 2914-2922 ◽  
Author(s):  
J. P. Bitoun ◽  
S. Liao ◽  
X. Yao ◽  
S.-J. Ahn ◽  
R. Isoda ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Stress Responses ◽  
Antimicrobial Agents ◽  
Galleria Mellonella ◽  
Cell Envelope ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Lipid Ii ◽  
The Impact

ABSTRACTPrevious studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation byStreptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression ofbrpAis regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In aGalleria mellonella(wax worm) model, BrpA deficiency was shown to diminish the virulence ofS. mutansOMZ175, which, unlikeS. mutansUA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain ofS. mutans.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals A Mechano-Activated Cell Reporter System as a Proxy for Flow-Dependent Endothelial Atheroprotection

2018 ◽  
Vol 23 (8) ◽  
pp. 869-876
Author(s):  
Bendix R. Slegtenhorst ◽  
Oscar R. Fajardo Ramirez ◽  
Yuzhi Zhang ◽  
Zahra Dhanerawala ◽  
Stefan G. Tullius ◽  
...  
Keyword(s):  
Vascular Endothelium ◽  
Critical Role ◽  
Green Fluorescence Protein ◽  
Dependent Manner ◽  
Reporter System ◽  
Health And Disease ◽  
Fluorescence Protein ◽  
Biomechanical Stimuli ◽  
Dose Dependent

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the “pharmacomimetics of atheroprotective flow” on the vascular endothelium.

scholarly journals Apotransferrin in Combination with Ciprofloxacin Slows Bacterial Replication, Prevents Resistance Amplification, and Increases Antimicrobial Regimen Effect

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Paul G. Ambrose ◽  
Brian D. VanScoy ◽  
Brian M. Luna ◽  
Jun Yan ◽  
Amber Ulhaq ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Bactericidal Effect ◽  
Bacterial Density ◽  
Infection Model ◽  
Time Curve ◽  
Content Type ◽  
Wide Range ◽  
Bacterial Replication ◽  
The Impact

ABSTRACT There has been renewed interest in combining traditional small-molecule antimicrobial agents with nontraditional therapies to potentiate antimicrobial effects. Apotransferrin, which decreases iron availability to microbes, is one such approach. We conducted a 48-h one-compartment in vitro infection model to explore the impact of apotransferrin on the bactericidal activity of ciprofloxacin. The challenge panel included four Klebsiella pneumoniae isolates with ciprofloxacin MIC values ranging from 0.08 to 32 mg/liter. Each challenge isolate was subjected to an ineffective ciprofloxacin monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC ratio] ranging from 0.19 to 96.6) with and without apotransferrin. As expected, the no-treatment and apotransferrin control arms showed unaltered prototypical logarithmic bacterial growth. We identified relationships between exposure and change in bacterial density for ciprofloxacin alone (R2 = 0.64) and ciprofloxacin in combination with apotransferrin (R2 = 0.84). Addition of apotransferrin to ciprofloxacin enabled a remarkable reduction in bacterial density across a wide range of ciprofloxacin exposures. For instance, at a ciprofloxacin AUC/MIC ratio of 20, ciprofloxacin monotherapy resulted in nearly 2 log10 CFU increase in bacterial density, while the combination of apotransferrin and ciprofloxacin resulted in 2 log10 CFU reduction in bacterial density. Furthermore, addition of apotransferrin significantly reduced the emergence of ciprofloxacin-resistant subpopulations compared to monotherapy. These data demonstrate that decreasing the rate of bacterial replication with apotransferrin in combination with antimicrobial therapy represents an opportunity to increase the magnitude of the bactericidal effect and to suppress the growth rate of drug-resistant subpopulations.

scholarly journals The Sociomicrobiology of Antivirulence Drug Resistance: a Proof of Concept

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Brett Mellbye ◽  
Martin Schuster
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Social Interactions ◽  
Proof Of Concept ◽  
Wild Type ◽  
Content Type ◽  
Future Studies ◽  
Cooperative Behaviors ◽  
The Impact ◽  
Virulence Factor Production ◽  
Proof Of Principle

ABSTRACTAntivirulence drugs disarm rather than kill pathogens and are thought to alleviate the problem of resistance, although there is no evidence to support this notion. Quorum sensing (QS) often controls cooperative virulence factor production and is therefore an attractive antivirulence target, for which inhibitors (QSI) have been developed. We designed a proof-of-principle experiment to investigate the impact of bacterial social interactions on the evolution of QSI resistance. We coculturedPseudomonas aeruginosaQS-deficient mutants with small proportions of the QS-proficient wild type, which in the absence of QSI mimic QSI-sensitive and -resistant variants, respectively. We employed two different QS-dependent nutrients that are degraded by extracellular (public) and cell-associated (private) enzymes. QS mutants (QSI-sensitive mimics) behaved as social cheaters that delayed population growth and prevented enrichment of wild-type cooperators (QSI-resistant mimics) only when nutrient acquisition was public, suggesting that QSI resistance would not spread. This highlights the potential for antivirulence strategies that target cooperative behaviors and provides a conceptual framework for future studies.

scholarly journals Generation of a GFP Reporter Akabane Virus with Enhanced Fluorescence Intensity by Modification of Artificial Ambisense S Genome

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 634
Author(s):  
Akiko Takenaka-Uema ◽  
Shin Murakami ◽  
Nanako Ushio ◽  
Tomoya Kobayashi-Kitamura ◽  
Masashi Uema ◽  
...  
Keyword(s):  
Imaging Study ◽  
Green Fluorescence Protein ◽  
Histopathological Study ◽  
Akabane Virus ◽  
Gfp Reporter ◽  
The Central Nervous System ◽  
Infected Cells ◽  
Fluorescence Protein

We previously generated a recombinant reporter Akabane virus expressing enhanced green fluorescence protein (eGFP-AKAV), with an artificial S genome encoding eGFP in the ambisense RNA. Although the eGFP-AKAV was able to detect infected cells in in vivo histopathological study, its fluorescent signal was too weak to apply to in vivo imaging study. Here, we successfully generated a modified reporter, eGFP/38-AKAV, with 38-nucleotide deletion of the internal region of the 5′ untranslated region of S RNA. The eGFP/38-AKAV expressed higher intensity of eGFP fluorescence both in vitro and in vivo than the original eGFP-AKAV did. In addition, eGFP/38-AKAV was pathogenic in mice at a comparable level to that in wild-type AKAV. In the mice infected with eGFP/38-AKAV, the fluorescent signals, i.e., the virus-infected cells, were detected in the central nervous system using the whole-organ imaging. Our findings indicate that eGFP/38-AKAV could be used as a powerful tool to help elucidate the dynamics of AKAV in vivo.

scholarly journals Impact of Penicillin Nonsusceptibility on Clinical Outcomes of Patients with Nonmeningeal Streptococcus pneumoniae Bacteremia in the Era of the 2008 Clinical and Laboratory Standards Institute Penicillin Breakpoints

2012 ◽  
Vol 56 (9) ◽  
pp. 4650-4655 ◽  
Author(s):  
Seong-Ho Choi ◽  
Jin-Won Chung ◽  
Heungsup Sung ◽  
Mi-Na Kim ◽  
Sung-Han Kim ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Clinical Outcomes ◽  
Antimicrobial Agents ◽  
Wide Spectrum ◽  
Median Number ◽  
Clinical Impact ◽  
Content Type ◽  
Duration Of Hospital Stay ◽  
Independent Risk Factors ◽  
The Impact

ABSTRACTTo investigate the impact of penicillin nonsusceptibility on clinical outcomes of patients with nonmeningealStreptococcus pneumoniaebacteremia (SPB), a retrospective cohort study was performed. The characteristics of 39 patients with penicillin-nonsusceptible SPB (PNSPB) were compared to those of a group of age- and sex-matched patients (n= 78) with penicillin-susceptible SPB (PSSPB). Susceptibility to penicillin was redetermined by using the revised Clinical and Laboratory Standards Institute (CLSI) penicillin breakpoints in CLSI document M100-S18. Although the PNSPB group tended to have more serious initial manifestations than the PSSPB group, the two groups did not differ significantly in terms of their 30-day mortality rates (30.8% versus 23.1%;P= 0.37) or the duration of hospital stay (median number of days, 14 versus 12;P= 0.89). Broad-spectrum antimicrobial agents, such as extended-spectrum cephalosporins, vancomycin, and carbapenem, were frequently used in both the PNSPB and PSSPB groups. Multivariate analysis revealed that ceftriaxone nonsusceptibility (adjusted odds ratio [aOR] = 4.88; 95% confidence interval [CI] = 1.07 to 22.27;P= 0.041) was one of the independent risk factors for 30-day mortality. Thus, when the 2008 CLSI penicillin breakpoints are applied and the current clinical practice of using wide-spectrum empirical antimicrobial agents is pursued, fatal outcomes in patients with nonmeningeal SPB that can be attributed to penicillin nonsusceptibility are likely to be rare. Further studies that examine the clinical impact of ceftriaxone nonsusceptibility in nonmningeal SPB may be warranted.

Open innovation in the new context of proof of concepts: evidence from Italy

2020 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Mariacarmela Passarelli ◽  
Giovanni Catello Landi ◽  
Alfio Cariola ◽  
Mauro Sciarelli
Keyword(s):  
Empirical Analysis ◽  
Development Process ◽  
Positive Impact ◽  
Early Stage ◽  
Development Project ◽  
Proof Of Concept ◽  
Content Type ◽  
Main Factors ◽  
The Impact ◽  
The Empirical Analysis

PurposeThe paper aims to advance knowledge by investigating the main factors that impact on innovation through the co-development process between researchers and firms at the very early stage of proof of concept.Design/methodology/approachThe authors developed an empirical analysis on the proof of concept network project, through a mixed empirical analysis. They explored the main factors that affect the enactment of the co-development process and tested the impact of such factors on the probability for partners to enact a co-development project and generate innovation.FindingsFrom the quantitative analysis comes out that the trust of the research team into the potentiality of the technology, the commitment of researchers concerning the scalability of technology and the IP value issued by external experts have a positive impact on the probability to create a match among partners and generate innovation.Research limitations/implicationsEven if all the population of technologies (108) considered in the project implementation are analyzed, the development of the empirical analysis on a specific project within a single country represents a limitation. Future analysis will concentrate on a larger panel of proof of concept experience across Europe.Practical implicationsThe success of a co-development process between researchers and companies at the embryonic phase of the technology considers the opportunity to exploit the technologies into real products for the market.Originality/valueThis is an empirical analysis of the first Italian proof of concept implementation that deeply investigates which critical factors can enable innovation by enacting a co-development process between researchers and small and medium-sized enterprises (SMEs).

scholarly journals Monitoring F1651P-Like Fimbria Expression at the Single-Cell Level Reveals a Highly Heterogeneous Phenotype

2015 ◽  
Vol 83 (5) ◽  
pp. 1929-1939 ◽  
Author(s):  
Richard Graveline ◽  
Rémi Lavoie ◽  
Philippe Garneau ◽  
France Daigle ◽  
Serge Sénéchal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Intensity ◽  
Reporter System ◽  
Clonal Population ◽  
Content Type ◽  
Unpredictable Environments ◽  
Gfp Reporter ◽  
High Level ◽  
Large Repertoire

F1651and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors. They play a key role in establishing disease caused by extraintestinal pathogenicEscherichia coli(ExPEC) strains in animals and humans. Both F1651and Pap are under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Using the Gfp reporter system, we monitoredin vitrothe level of fluorescence intensity corresponding to the F1651and Pap fimbrial synthesis. Monitoring individualEscherichia colicells by flow cytometry and by real-time fluorescence microscopy, we identified cells associated with a low or high level of fluorescence intensity and a large amount of cells with partial levels of fluorescence, mostly present in the F1651system. This mixed population identified through fluorescence intensity could be attributed to the high switching rate previously observed in F1651-positive bacteria. The fimbrial heterogeneous phenotype for these ExPEC could represent increased fitness in unpredictable environments. Our study illustrates that within the large repertoire of fimbrial variants such as the well-characterized Pap, F1651is an exquisite example of regulatory expression that arms the bacterium with strategies for surviving in more than one particular environment.

scholarly journals A Novel Gene, ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae

2010 ◽  
Vol 9 (10) ◽  
pp. 1495-1503 ◽  
Author(s):  
Kyunghun Min ◽  
Jungkwan Lee ◽  
Jin-Cheol Kim ◽  
Sang Gyu Kim ◽  
Young Ho Kim ◽  
...  
Keyword(s):  
Crop Residues ◽  
Turgor Pressure ◽  
Green Fluorescence Protein ◽  
Dispersal Distance ◽  
Gibberella Zeae ◽  
Multiple Roles ◽  
Head Blight ◽  
Content Type ◽  
Novel Gene ◽  
Fluorescence Protein

ABSTRACT Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.

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