Surface integrity modification of CoCrMo alloy by deep rolling in combination with sub-zero cooling as potential implant application.

Alloys made of CoCrMo are well established as implants materials since decades in orthopedic surgery. The good mechanical properties, biocompatibility and especially the corrosion resistance are important rationales for the use of these alloys. Nevertheless, retrieved implants from revision surgery showed the occurrence of abrasion and corrosion. The wear mechanisms and the occurring corrosion processes might be reduced with a functionalization of the surface. The hexagonal phase of the cobalt chromium matrix plays an important role in the surface functionalization. It can be specifically transformed and set during the manufacturing process. One possibility for the induction of the transformation is the use of a deep rolling process in combination with a novel "sub-zero" cooling strategy during machining. The influence of force and temperature during the deep rolling process on the formation of the hexagonal Co-phase is examined in this study. The results from the targeted setting of the hexagonal Co-phase in the subsurface are shown. For this purpose, EBSD studies have been carried out to detect and quantify the proportion of Co-hex phase in the subsurface of the modified alloys. To analyze the mechanical properties, we measured the residual stress and hardness in the near surface layer under conditions close to the application. Furthermore, we performed biological tests to show a potential influence of the modification on the biocompatibility when using the sub-zero cooling approach. We observed no negative effect on the osteoblastic cell line which attached similarly to all tested surfaces. The investigations provide first insights into the potential use of "sub-zero" cooling in modifying orthopedic implant materials, but also the respective limits with regard to the surface functionalization. Deep rolling in combination with an innovative cooling strategy has a great potential to improve the mechanical properties of CoCr28Mo6 wrought alloy, by subsurface hardening and phase transformation.

Flexible Fragment Growing Boosts Potency of Quorum-Sensing Inhibitors against Pseudomonas aeruginosa Virulence.

Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-aminopyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa. We pursue an innovative treatment strategy by interfering with the Pseudomonas quinolone signal (PQS) quorum sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.

Senior driving under the influence: A five-year retrospective study of alcoholized road-users aged 70 and over.

The demographic development in Germany shows a steady increase to senior citizens. The driving suitability of older road-users is of large social and political concern, because awareness and reactivity can be influenced by age-related diseases and potential medication, particularly in combination with the consumption of alcohol. This study provides an overview of senior road-users under the influence of alcohol. Therefore, 404 cases of drunken-driving by road-users aged 70 and over within the purview of the Institute of Legal Medicine at Justus-Liebig-University, Giessen, from the years 2009-2013 were evaluated in retrospect. The vast majority of the drivers were male (88.4% of the cases). Distribution of the blood alcohol concentrations were almost equally in male and female drivers with 62.8% of all cases showing a blood alcohol concentration (BAC) of more than 1.1‰ and 10.9% a BAC of more than 2.0‰. In 48.5% of the cases, drunk driving was associated with an accident. 54.5% of the drivers stated suffering from at least one disease and 60% admitted a regular medication or an intake prior to the incident. Moreover, the collected data includes aspects such as the kind of traffic participation as well as neurological and physiological deficits of the road-users. If accidents were caused by drunk driving, the external circumstances and consequences of these accidents were analyzed, too. The evaluation revealed, that the standard medical examination protocol proved to be improbable to cover polypharmacy and multimorbidity of older alcoholized drivers. So, an evaluation and adaptation of the common medical examination protocol must be considered.

Application of Ultra-Small Micro Grinding and Micro Milling Tools: Possibilities and Limitations.

Current demands for flexible, individual microstructures in high quality result in high requirements for micro tools. As the tool size defines the minimum structure size, ultra-small tools are needed. To achieve tool diameters of 50 µm and lower, we investigate the complete manufacturing chain of micro machining. From the development of the machine tools and components needed to produce and apply the micro tools, the micro tools themselves, as well as the micro machining processes. Machine tools are developed with the possibility of producing the micro geometry (cutting edge design) of micro tools as well as plating processes to produce super abrasive micro grinding tools. Applying these setups, we are able to produce ultra-small micro grinding and micro milling tools with typical diameters of 50 µm and down to 4 µm. However, the application of such tools is very challenging. The article presents possibilities and limitations in manufacturing the micro tools themselves as well as microstructures made with these tools. A special emphasis will be on the influence of the tool substrate in micro milling and grain sizes in micro grinding.

From in vitro to in cellulo: structure-activity relationship of (2-nitrophenyl)methanol derivatives as inhibitors of PqsD in Pseudomonas aeruginosa.

Recent studies have shown that compounds based on a (2-nitrophenyl)methanol scaffold are promising inhibitors of PqsD, a key enzyme of signal molecule biosynthesis in the cell-to-cell communication of Pseudomonas aeruginosa. The most promising molecule displayed anti-biofilm activity and a tight-binding mode of action. Herein, we report on the convenient synthesis and biochemical evaluation of a comprehensive series of (2-nitrophenyl)methanol derivatives. The in vitro potency of these inhibitors against recombinant PqsD as well as the effect of selected compounds on the production of the signal molecules HHQ and PQS in P. aeruginosa were examined. The gathered data allowed the establishment of a structure-activity relationship, which was used to design fluorescent inhibitors, and finally, led to the discovery of (2-nitrophenyl)methanol derivatives with improved in cellulo efficacy providing new perspectives towards the application of PqsD inhibitors as anti-infectives.

Optimization of anti-virulence PqsR antagonists regarding aqueous solubility and biological properties resulting in new insights in structure-activity relationships.

Increasing antibiotic resistance urgently requires novel therapeutic options to combat bacterial infections. The anti-virulence therapy selectively intervening with pathogenicity without affecting bacterial viability is such a strategy to overcome resistance. We consider the virulence regulator PqsR as an attractive target in the human pathogen Pseudomonas aeruginosa, and recently discovered the first PqsR antagonists, which, however, suffered from poor aqueous solubility. In this work, the antagonists were structurally modified to become more soluble, and their structure-activity as well as structure-property relationships were studied. A novel promising compound with improved solubility and enhanced anti-virulence activity was discovered (IC50: 3.8 µM, pyocyanin). Our findings emphasize the crucial role of substituents at the 3-position and the carbonyl group at the 4-position for ligand-receptor interactions, and illuminate the way for further optimization of PqsR antagonists as anti-virulence agents.

Overcoming the unexpected functional inversion of a PqsR antagonist in Pseudomonas aeruginosa: an in vivo potent antivirulence agent targeting pqs quorum sensing.

The virulence regulator PqsR of Pseudomonas aeruginosa is considered as an attractive target for attenuating the bacterial pathogenicity without eliciting resistance. However, despite efforts and desires, no promising PqsR antagonist has been discovered thus far. Now, a surprising functionality change of a highly affine PqsR antagonist in P. aeruginosa is revealed, which is mediated by a bacterial signal molecule synthase and responsible for low cellular potency. Blockade of the susceptible position led to the discovery of the first antivirulence compound that is potent in vivo and targets PqsR, thus providing a proof of concept for this novel antivirulence therapy.

Discovery and biophysical characterization of 2-amino-oxadiazoles as novel antagonists of PqsR, an important regulator of Pseudomonas aeruginosa virulence.

The human pathogen Pseudomonas aeruginosa employs alkyl quinolones for cell-to-cell communication. The Pseudomonas quinolone signal (PQS) regulates various virulence factors via interaction with the transcriptional regulator PqsR. Therefore, we consider the development of PqsR antagonists a novel strategy to limit the pathogenicity of P. aeruginosa. A fragment identification approach using surface plasmon resonance screening led to the discovery of chemically diverse PqsR ligands. The optimization of the most promising hit (5) resulted in the oxadiazole-2-amine 37 showing pure antagonistic activity in Escherichia coli (EC50 = 7.5 µM) and P. aeruginosa (EC50 = 38.5 µM) reporter gene assays. 37 was able to diminish the production of the PQS precursor HHQ in a PqsH-deficient P. aeruginosa mutant. The level of the major virulence factor pyocyanin was significantly reduced in wild-type P. aeruginosa. In addition, site-directed mutagenesis in combination with isothermal titration calorimetry and NMR INPHARMA experiments revealed that the identified ligands bind to the same site of PqsR by adopting different binding modes. These findings will be utilized in a future fragment-growing approach aiming at novel therapeutic options for the treatment of P. aeruginosa infections.

Identification of small-molecule antagonists of the Pseudomonas aeruginosa transcriptional regulator PqsR: biophysically guided hit discovery and optimization.

The Gram-negative pathogen Pseudomonas aeruginosa produces an intercellular alkyl quinolone signaling molecule, the Pseudomonas quinolone signal. The pqs quorum sensing communication system that is characteristic for P. aeruginosa regulates the production of virulence factors. Therefore, we consider the pqs system a novel target to limit P. aeruginosa pathogenicity. Here, we present small molecules targeting a key player of the pqs system, PqsR. A rational design strategy in combination with surface plasmon resonance biosensor analysis led to the identification of PqsR binders. Determination of thermodynamic binding signatures and functional characterization in E. coli guided the hit optimization, resulting in the potent hydroxamic acid derived PqsR antagonist 11 (IC(50) = 12.5 µM). Remarkably it displayed a comparable potency in P. aeruginosa (IC(50) = 23.6 µM) and reduced the production of the virulence factor pyocyanin. Beyond this, site-directed mutagenesis together with thermodynamic analysis provided insights into the energetic characteristics of protein-ligand interactions. Thus the identified PqsR antagonists are promising scaffolds for further drug design efforts against this important pathogen.

Discovery of antagonists of PqsR, a key player in 2-alkyl-4-quinolone-dependent quorum sensing in Pseudomonas aeruginosa.

The pqs quorum sensing communication system of Pseudomonas aeruginosa controls virulence factor production and is involved in biofilm formation, therefore playing an important role for pathogenicity. In order to attenuate P. aeruginosa pathogenicity, we followed a ligand-based drug design approach and synthesized a series of compounds targeting PqsR, the receptor of the pqs system. In vitro evaluation using a reporter gene assay in Escherichia coli led to the discovery of the first competitive PqsR antagonists, which are highly potent (K(d,app) of compound 20: 7 nM). These antagonists are able to reduce the production of the virulence factor pyocyanin in P. aeruginosa. Our finding offers insights into the ligand-receptor interaction of PqsR and provides a promising starting point for further drug design.