Objectives To describe the effectiveness of secukinumab in the treatment of psoriatic arthritis (PsA) and associated physician satisfaction with secukinumab treatment, in routine clinical practice across five European countries. Methods A retrospective analysis of PsA patients receiving secukinumab for >= 4 months in France, Germany, Italy, Spain and the UK from March to December 2018. Data based on physician-completed questionnaires at initiation of treatment and at the data collection consultation were collected and used to assess effectiveness. Results 572 PsA patients with a mean age of 47.9 years, 57.0% were male, with 5.6% of patients with mild, 55.2% with moderate and 38.1% severe PsA prior to treatment initiation were included. 33.0% of patients received a dosage of 150 mg and 67.0% a dosage of 300 mg secukinumab. Around 84% of patients received secukinumab for 6 months or longer. Symptoms seen at current assessment in over 20% of patients were tender or swollen joints or psoriatic skin lesions. Between initiation of treatment and the current consultation, improvements in skin, joint and overall severity were reported. Physician satisfaction with secukinumab's ability to control disease was very high during the study period, greater than 90%, and was seen irrespective of disease severity at initiation, prior biologic use, treatment duration, time since diagnosis or onset of symptoms, treatment history, and BMI. Conclusion Physicians were satisfied with the ability of secukinumab to control disease and it was effective in the treatment of PsA patients in routine clinical settings.

Objective To assess the cost-effectiveness of pazopanib versus sunitinib as a first-line treatment for patients with metastatic renal cell carcinoma (mRCC) from an Italian National Health Service perspective, considering the evolving Italian landscape in terms of new reimbursement agreements trend. Methods This analysis is an update of the previously published cost-effectiveness analysis to incorporate recent 2019 costs and additional changes regarding drug discounting. A partitioned-survival analysis model with three different health states (progression-free survival, post-progression survival, and dead) was utilized. Outcomes included progression-free life years, post-progression life years, overall life years, quality-adjusted life years (QALYs), and costs calculated for both treatments. Cost-effectiveness was assessed in terms of incremental costs per QALY gained and the net monetary benefit (NMB) of pazopanib versus sunitinib. In the base case analysis, a time horizon of 5 years was used and future costs and QALYs were discounted at a 3% annual discount rate. An impact of methodological and parameter uncertainly on base case results was evaluated using probabilistic and deterministic sensitivity analyses. Results In the base case, pazopanib had higher QALYs (+0.060) at lower costs (-euro5,857) versus sunitinib, hence it dominated sunitinib. At willingness-to-pay thresholds of euro30,000 and euro50,000 per QALY, the NMB with pazopanib were euro7,647 and euro8,841 per patient, respectively, versus sunitinib. The probability that pazopanib is cost-effective versus sunitinib was estimated to be 97.5% at a cost-effectiveness threshold of euro20,000, 95.4% at a threshold of euro30,000, and 90.2% at a threshold of euro50,000 per QALY. Cost-effectiveness results were robust to changes in key parameter values and assumptions as demonstrated by deterministic sensitivity analyses. Conclusions Pazopanib is likely to represent a cost-effective treatment option compared with sunitinib as a first-line treatment for patients with metastatic RCC in Italy.

Significance Genome sequencing of a multidrug-resistant clinical isolate of Salmonella Typhimurium from a patient that failed ciprofloxacin therapy revealed a mutation in the efflux pump gene, acrB . Computational modelling revealed that the G288D substitution changed the binding of drugs to the distal binding pocket of AcrB. The mutation was recreated in an unrelated Salmonella strain and also in Escherichia coli ; in both species the efflux of ciprofloxacin was increased by the mutation, explaining its resistant phenotype. This is the first time a substitution within an efflux pump protein has been shown to cause drug resistance. Importantly, the finding that one amino acid change can cause resistance to some drugs, but susceptibility to others, informs those developing new antibiotics.

Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.

The cell envelope of the Gram negative opportunistic pathogen Pseudomonas aeruginosa is poorly permeable to many classes of hydrophilic molecules including antibiotics due to the presence of the narrow and selective porins. Here we focused on one of the narrow-channel porins, that is, OprP, which is responsible for the high-affinity uptake of phosphate ions. Its two central binding sites for phosphate contain a number of positively charged amino acids together with a single negatively charged residue (D94). The presence of this negatively charged residue in a binding site for negatively charged phosphate ions is highly surprising due to the potentially reduced binding affinity. The goal of this study was to better understand the role of D94 in phosphate binding, selectivity, and transport using a combination of mutagenesis, electrophysiology, and free-energy calculations. The presence of a negatively charged residue in the binding site is critical for this specific porin OprP as emphasized by the evolutionary conservation of such negatively charged residue in the binding site of several anion-selective porins. Mutations of D94 in OprP to any positively charged or neutral residue increased the binding affinity of phosphate for OprP. Detailed analysis indicated that this anionic residue in the phosphate binding site of OprP, despite its negative charge, maintained energetically favorable phosphate binding sites in the central region of the channel and at the same time decreased residence time thus preventing excessively strong binding of phosphate that would oppose phosphate flux through the channel. Intriguingly mutations of D94 to positively charged residues, lysine and arginine, resulted in very different binding affinities and free energy profiles, indicating the importance of side chain conformations of these positively charged residues in phosphate binding to OprP.

The outer membrane porin OprP of Pseudomonas aeruginosa is a highly phosphate-selective channel. It is induced under the condition of phosphate starvation and facilitates the high-affinity uptake of phosphate ions across the outer membrane of bacteria [1]. An investigation of the structure-function relationship of OprP is required to understand the anion and phosphate selectivity of this porin in particular and to expand the present understanding of ion selectivity of different channels in general. To this end, we investigated the wild-type OprP and several important mutants of OprP to decode the phosphate selectivity of the channel [2, 3]. Mutants helped to probe the individual contribution of important residues toward the selectivity of OprP. Both electrophysiological bilayer measurements and free-energy molecular dynamics (MD) simulations were carried out to monitor the change in ion selectivity and phosphate binding affinity of various mutants compared to wild-type OprP. Results obtained from MD simulations were in qualitative agreement with experiments and complemented experimental observations by providing atomistic details regarding function and dynamics of OprP. Molecular details learned from such studies could be exploited to engineer the channel for various applications [4, 5]. [1] R. E. W. Hancock, K. Poole, R. Benz, J. Bacteriol. 150, 730-738 (1982). [2] N. Modi, R. Benz, R. E. W. Hancock, U. Kleinekathöfer, J. Phys. Chem. Lett. 3, 3639-3645 (2012). [3] N. Modi, I. Bárcena-Uribarri, M. Bains, R. Benz, R. E. W. Hancock, U. Kleinekathöfer, Biochemistry. 52, 5522-5532 (2013). [4] P. Pongprayoon, O. Beckstein, M. S. P. Sansom, J. Phys. Chem. B. 116, 462-268 (2011). [5] N. Modi, M. Winterhalter, and U. Kleinekathöfer, Nanoscale 4, 6166-6180 (2012).

Ion transport through membrane proteins and nanopores is a process of significant importance which has implications ranging from controlling various biological processes to applications in the field of nanoanalytics and stochastic sensing. Therefore it is imperative to probe the behavior and dynamics of ions in nanoscale confinements provided by membrane proteins. The research work reported in this thesis is aimed at understanding ion transport processes, namely ion selectivity and ion conductance, through bacterial outer membrane porins using molecular dynamics simulations. The major focus of this thesis is to probe the phosphate selectivity of the OprP porin from the bacterium Pseudomonas aeruginosa. The protein OprP is induced in the outer membrane of bacteria under conditions of phosphate starvation and is responsible for the high-affinity uptake of phosphate ions under such circumstances. Free-energy molecular dynamics simulations revealed atomic details leading to the phosphate selectivity of the channel. To further understand the phosphate selectivity of OprP and underlying structure-function relationships, several important residues of OprP have been mutated. Such studies on the mutant OprP channels have enabled us to probe the relative contributions of the residues and their properties, namely charge, size, the ability to desolvate the permeating ion etc., in assigning the phosphate selectivity to OprP. Moreover, the findings obtained for the phosphate selectivity of OprP were further extended to probe the diphosphate selectivity of OprO, a homologous porin of OprP with a high sequence and structural similarity. In silico double mutants of OprP and OprO demonstrated a trend to interchange the phosphate selectivity of OprP and the diphosphate selectivity of OprO. The other focus of the thesis is to decipher the ion conductance properties through the OmpF and NanC porins from Escherichia coli. A particular kind of bulky ions, i.e., ionic liquids have been investigated with respect to their temperature-dependent pore conductance properties through OmpF. Such ionic liquids can improve the time-resolution of electrophysiological measurements and may be useful in various biosensing applications. Applied-field simulations revealed the importance of a particular orientation of the permeating ion to be able to pass through the pore. In case of NanC, an asymmetric distribution of charged residues inside the pore was found to be responsible for an asymmetric conductance property and a weak anion selectivity of the porin. In addition, mutants of OmpF and NanC have been generated to modify ion conductance properties of these porins. The findings presented in this thesis enhance the atomistic and functional understanding of ion transport processes through bacterial outer membrane porins in particular and various other membrane proteins in general. Molecular details obtained from such studies can be further exploited to engineer the ion transport properties through nanopores to achieve diverse possible applications, e.g., the design of ion-specific sieves and sensing of biological agents.

The outer membrane porin OprP of Pseudomonas aeruginosa forms a highly specific phosphate selective channel. This channel is responsible for the high-affinity uptake of phosphate ions into the periplasmic space of the bacteria. A detailed investigation of the structure-function relationship of OprP is inevitable to decipher the anion and phosphate selectivity of this porin in particular and to broaden the present understanding of the ion selectivity of different channels. To this end we investigated the role of the central arginine of OprP, R133, in terms of its effects in selectivity and ion transport properties of the pore. Electrophysiological bilayer measurements and free-energy molecular dynamics simulations were carried out to probe the transport of different ions through various R133 mutants. For these mutants, the change in phosphate binding specificity, ion conduction, and anion selectivity was determined and compared to previous molecular dynamic calculations and electrophysiological measurements with wild-type OprP. Molecular analysis revealed a rather particular role of arginine 133 and its charge, while at the same time this residue together with the network of other residues, namely, D94 and Y114, has the ability to dehydrate the permeating ion. These very specific features govern the ion selectivity of OprP.

N-acetylneuraminic acid-inducible channel (NanC) is an outer membrane channel of Escherichia coli . This porin folds as a 12-stranded β-barrel leading to a tubular shape. Electrophysiological experiments have revealed an asymmetric conductance with respect to the direction of the applied voltage and a weak anion selectivity of the channel. To this end, we performed all-atom molecular dynamics (MD) simulations to decipher the ion transport properties of the NanC channel. Concentration-dependent applied-field MD simulations recover the asymmetric conductance property and the anion selectivity of the channel in agreement with experiments. Further molecular analysis revealed the role of the asymmetric charge distribution inside the channel as the basis of the asymmetry in conductance. In addition, the particular distribution of charged residues at the inner channel walls leads to a faster permeation of Cl(-) ions compared to K(+) ions resulting in the anion selectivity of NanC. These findings are well supported by position-dependent diffusion coefficients and potential of mean force profiles derived from unbiased MD simulations. Taking one step further, we were able to engineer the NanC channel in silico by mutations leading to enhanced asymmetric conductances and anion selectivities. The E186Q mutant, for example, changes NanC into a decent molecular diode with an ionic current ratio of about 3:1 for opposite bias voltages.

We investigated translocation of cationic peptides through nanochannels derived from the Gram-positive bacterium Nocardia farcinica at the single-molecule level. The two subunits NfpA and NfpB form a hetero-oligomeric cation selective channel. On the basis of amino acid comparison we performed homology modeling and obtained a channel structurally related to MspA of Mycobacterium smegmatis. The quantitative single-molecule measurements provide an insight into transport processes of solutes through nanochannels. High-resolution ion conductance measurements in the presence of peptides of different charge and length revealed the kinetics of peptide binding. The observed asymmetry in peptide binding kinetics indicated a unidirectional channel insertion in the lipid bilayer. In the case of cationic peptides, the external voltage acts as a driving force that promotes the interaction of the peptide with the channel surface. At low voltage, the peptide just binds to the channel, whereas at higher voltage, the force is strong enough to pull the peptide across the channel. This allows distinguishing quantitatively between peptide binding and translocation through the channel.

Nanoscale pores are ubiquitous in biological systems while artificial nanopores are being fabricated for an increasing number of applications. Biological pores are responsible for the transport of various ions and substrates between the different compartments of biological systems separated by membranes while artificial pores are aimed at emulating such transport properties. As an experimental method, electrophysiology has proven to be an important nano-analytical tool for the study of substrate transport through nanopores utilizing ion current measurements as a probe for the detection. Independent of the pore type, i.e., biological or synthetic, and objective of the study, i.e., to model cellular processes of ion transport or electrophysiological experiments, it has become increasingly important to understand the dynamics of ions in nanoscale confinements. To this end, numerical simulations have established themselves as an indispensable tool to decipher ion transport processes through biological as well as artificial nanopores. This article provides an overview of different theoretical and computational methods to study ion transport in general and to calculate ion conductance in particular. Potential new improvements in the existing methods and their applications are highlighted wherever applicable. Moreover, representative examples are given describing the ion transport through biological and synthetic nanopores as well as the high selectivity of ion channels. Special emphasis is placed on the usage of molecular dynamics simulations which already have demonstrated their potential to unravel ion transport properties at an atomic level.

The permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. An adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. The temperature-dependent transport of the ionic liquid 1-butyl-3-methyl-imidazolium chloride (BMIM-Cl) in aqueous solution is studied theoretically and experimentally. Using molecular dynamics simulations and ion-conductance measurements, the transport is examined in bulk as well as through a biological nanopore, OmpF and its mutant D113A. This investigation is motivated by the observation that aqueous solutions of BMIM-Cl drastically reduce the translocation speed of DNA or antibiotics through nanopores in electrophysiological measurements. This makes BMIM-Cl an interesting alternative salt to improve the time resolution. In line with previous investigations of simple salts, the size of the ions and their orientation adds another important degree of freedom to the ion transport, thereby slowing the transport through nanopores. An excellent agreement between theory and conductance measurements is obtained for wild type OmpF and a reasonable agreement for the mutant. Moreover, all-atom simulations allow an atomistic analysis revealing molecular details of the rate-limiting ion interactions with the channel. [1] Mahendran KR et al, J. Phys: Condens. Matter 22 (2010) 454131. [2] Niraj Modi, Pratik Raj Singh et al, J. Phys. Chem. Lett. 2 (2011) 2331-36.

Ion selectivity of transport systems is an essential property of membranes from living organisms. These entities are used to regulate multifarious biological processes by virtue of selective participation of specific ions in transport processes. To understand this process, we studied the phosphate selectivity of the OprP porin from Pseudomonas aeruginosa using all-atom free-energy molecular dynamics simulations. These calculations were performed to define the energetics of phosphate, sulfate, chloride, and potassium ion transport through OprP. Atomic-level analysis revealed that the overall electrostatic environment of the channel was responsible for the anion selectivity of the channel, whereas the particular balance of interactions between the permeating ions and water as well as channel residues drove the selectivity between different anions. The selectivity of OprP is discussed in light of well-studied ion channels that are highly selective for potassium or chloride.

The temperature-dependent transport of the ionic liquid 1-butyl-3-methylimidazolium chloride (BMIM-Cl) in aqueous solution is studied theoretically and experimentally. Using molecular dynamics simulations and ion-conductance measurements, the transport is examined in bulk as well as through a biological nanopore, that is, OmpF and its mutant D113A. This investigation is motivated by the observation that aqueous solutions of BMIM-Cl drastically reduce the translocation speed of DNA or antibiotics through nanopores in electrophysiological measurements. This makes BMIM-Cl an interesting alternative salt to improve the time resolution. In line with previous investigations of simple salts, the size of the ions and their orientation adds another important degree of freedom to the ion transport, thereby slowing the transport through nanopores. An excellent agreement between theory and conductance measurements is obtained for wild type OmpF and a reasonable agreement for the mutant. Moreover, all-atom simulations allow an atomistic analysis revealing molecular details of the rate-limiting ion interactions with the channel.

The permeation of water soluble molecules across cell membranes is controlled by channel-forming proteins and, in particular, the channel surface determines the selectivity. An adequate method to study the properties of these channels is electrophysiology and, in particular, analyzing the ion current fluctuation in the presence of permeating solutes. Ion current fluctuation analysis provides information on possible interactions of solutes with the channel surface. Due to the limited time resolution, fast permeation events are not visible using standard techniques. Here, we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. Although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. Molecular details of solute permeation can be revealed by combining electrophysiology and all-atom computer modeling; these methods include ion conductance, selectivity, ion pair formation, and rate limiting interactions of the solute with the channel walls during permeation.

The cytoskeletal protein, FtsZ plays a pivotal role in prokaryotic cell division and is present in majority of the bacterial species. In recent years, inhibitors of FtsZ have been identified that may function as lead compounds for the development of novel antimicrobials. It has been found that curcumin, the main bioactive component of Curcuma longa, inhibits Bacillus subtilis and Escherichia coli growth by inhibiting FtsZ assembly. Though it is experimentally established that curcumin inhibits FtsZ polymerization, the binding site of curcumin in FtsZ is not known. In this study, interaction of curcumin with catalytic core domain of E. coli and B. subtilis FtsZ was investigated using computational docking.