Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori.

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

Inhibition of the type IV secretion system from antibiotic-resistant Helicobacter pylori clinical isolates supports the potential of Cagα as an anti-virulence target.

Helicobacter pylori resistance to antibiotics is a growing problem and it increasingly leads to treatment failure. While the bacterium is present worldwide, the severity of clinical outcomes is highly dependent on the geographical origin and genetic characteristics of the strains. One of the major virulence factors identified in H. pylori is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) used to translocate effectors into human cells. Here, we investigated the genetic variability of the cagPAI among 13 antibiotic-resistant H. pylori strains that were isolated from patient biopsies in Québec. Seven of the clinical strains carried the cagPAI, but only four could be readily cultivated under laboratory conditions. We observed variability of the sequences of CagA and CagL proteins that are encoded by the cagPAI. All clinical isolates induce interleukin-8 secretion and morphological changes upon co-incubation with gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cagα protein from the model strain H. pylori 26695, reduces interleukin-8 secretion in one of the clinical isolates. Co-incubation with 1G2 also inhibits the assembly of T4SS pili, suggesting a mechanism for its action on T4SS function.

Fiber Rearrangement and Matrix Compression in Soft Tissues: Multiscale Hypoelasticity and Application to Tendon.

It is widely accepted that the nonlinear macroscopic mechanical behavior of soft tissue is governed by fiber straightening and re-orientation. Here, we provide a quantitative assessment of this phenomenon, by means of a continuum micromechanics approach. Given the negligibly small bending stiffness of crimped fibers, the latter are represented through a number of hypoelastic straight fiber phases with different orientations, being embedded into a hypoelastic matrix phase. The corresponding representative volume element (RVE) hosting these phases is subjected to "macroscopic" strain rates, which are downscaled to fiber and matrix strain rates on the one hand, and to fiber spins on the other hand. This gives quantitative access to the fiber decrimping (or straightening) phenomenon under non-affine conditions, i.e. in the case where the fiber orientations cannot be simply linked to the macroscopic strain state. In the case of tendinous tissue, such an RVE relates to the fascicle material with 50 µm characteristic length, made up of crimped collagen bundles and a gel-type matrix in-between. The fascicles themselves act as parallel fibers in a similar matrix at the scale of a tissue-related RVE with 500 µm characteristic length. As evidenced by a sensitivity analysis and confirmed by various mechanical tests, it is the initial crimping angle which drives both the degree of straightening and the shape of the macroscopic stress-strain curve, while the final linear portion of this curve depends almost exclusively on the collagen bundle elasticity. Our model also reveals the mechanical cooperation of the tissue's key microstructural components: while the fibers carry tensile forces, the matrices undergo hydrostatic pressure.

Use of population PK/PD approach to model the thrombin generation assay: assessment in haemophilia A plasma samples spiked by a TFPI antibody.

The thrombin generation (TG) assay is a well-established tool to capture the clotting potential of any healthy or haemophiliac subject. It measures ex vivo the kinetics of thrombin activation throughout the coagulation. Clinical studies allowed to create two databases gathering the coagulation factor levels and the thrombin generation profile of 40 healthy and 40 haemophiliac A (HA) subjects. Besides, portions of all HA samples were spiked with increasing levels of a TFPI antibody (considered as a possible therapeutic target) and corresponding TG profiles were determined. The non-linear mixed-effect (NLME) modelling aims at describing and explaining the experimentally observed important variability of the TG curves between subjects and the individual effects of spiking with a TFPI antibody. The models consist of an empirical description of the TG kinetics, accounting for an additive residual error and between-subject variability on its parameters. Factor VIII and TFPI were found to significantly explain and reduce the variability of the TG of haemophilia A samples. Besides, the model is shown to correctly reproduce the variability in the response to the ex vivo spiking with the TFPI antibody, by combining the empirical description of TG to a simple Hill equation that accounts for the binding between TFPI and different doses of its antibody. Such models can be useful for clinical practice, with the analysis and comparison of the distributions of TG profiles in healthy and haemophilia populations; and also for research, with the analysis of the effect of TFPI and its neutralization on individual TG profiles.

Implementing a micromechanical model into a finite element code to simulate the mechanical and microstructural response of arteries.

A computational strategy based on the finite element method for simulating the mechanical response of arterial tissues is herein proposed. The adopted constitutive formulation accounts for rotations of the adventitial collagen fibers and introduces parameters which are directly measurable or well established. Moreover, the refined constitutive model is readily utilized in finite element analyses, enabling the simulation of mechanical tests to reveal the influence of microstructural and histological features on macroscopic material behavior. Employing constitutive parameters supported by histological examinations, the results herein validate the model's ability to predict the micro- and macroscopic mechanical behavior, closely matching previously observed experimental findings. Finally, the capabilities of the adopted constitutive description are shown investigating the influence of some collagen disorders on the macroscopic mechanical response of the arterial tissues.

Custom-made macroporous bioceramic implants based on triply-periodic minimal surfaces for bone defects in load-bearing sites.

The architectural features of synthetic bone grafts are key parameters for regulating cell functions and tissue formation for the successful repair of bone defects. In this regard, macroporous structures based on triply-periodic minimal surfaces (TPMS) are considered to have untapped potential. In the present study, custom-made implants based on a gyroid structure, with (GPRC) and without (GP) a cortical-like reinforcement, were specifically designed to fit an intended bone defect in rat femurs. Sintered hydroxyapatite implants were produced using a dedicated additive manufacturing technology and their morphological, physico-chemical and mechanical features were characterized. The implants' integrity and ability to support bone ingrowth were assessed after 4, 6 and 8 weeks of implantation in a 3-mm-long, femoral defect in Lewis rats. GP and GPRC implants were manufactured with comparable macro- to nano-architectures. Cortical-like reinforcement significantly improved implant effective stiffness and resistance to fracture after implantation. This cortical-like reinforcement also concentrated new bone formation in the core of the GPRC implants, without affecting newly formed bone quantity or maturity. This study showed, for the first time, that custom-made TPMS-based bioceramic implants could be produced and successfully implanted in load-bearing sites. Adding a cortical-like reinforcement (GPRC implants) was a relevant solution to improve implant mechanical resistance, and changed osteogenic mechanism compared to the GP implants. STATEMENT OF SIGNIFICANCE: Architectural features are known to be key parameters for successful bone repair using synthetic bioceramic bone graft. So far, conventional manufacturing techniques, lacking reproducibility and complete control of the implant macro-architecture, impeded the exploration of complex architectures, such as triply periodic minimal surfaces (TPMS), which are foreseen to have an unrivaled potential for bone repair. Using a new additive manufacturing process, macroporous TPMS-based bioceramics implants were produced in calcium phosphate, characterized and implanted in a femoral defect in rats. The results showed, for the first time, that such macroporous implants can be successfully implanted in anatomical load-bearing sites when a cortical-like outer shell is added. This outer shell also concentrated new bone formation in the implant center, without affecting new bone quantity or maturity.

Tissue factor pathway inhibitor is the main determinant of thrombin generation in haemophilic patients.

The thrombin generation (TG) assay evaluates haemostatic balance, which is influenced by the levels of many coagulation factors and inhibitors. Our objective was to identify the determinant factors of TG in haemophilia A (HA) and haemophilia B (HB) patients and to compare them to those in healthy controls. Coagulation factor and inhibitor levels, and TG, were measured in platelet-poor plasma from 40 patients with HA, 32 patients with HB and 40 healthy subjects. Data were analysed using multiple regression models. In HA patients, factor VIII was a positive determinant of endogenous thrombin potential (ETP) and peak, whereas tissue factor pathway inhibitor (TFPI) and factor V were negative determinants of ETP and peak. In HB patients, FIX was a positive determinant of ETP and peak, FVII being a positive determinant of peak. Antithrombin and protein S (PS) were negative determinants of ETP while FX was a negative determinant of peak. Above all, in HB patients, TFPI was a negative determinant of ETP and peak. In healthy subjects, FVIII was a positive determinant of ETP and peak, whereas FX and protein S were negative determinants of these parameters. TFPI was not a negative determinant of either peak or ETP. In haemophilic patients, the determinant factors of TG are all implicated in FXa generation and inhibition, the crucial determinant factor being TFPI whatever the type of haemophilia, A or B. These findings contribute to the rationale that recently place TFPI as a target for innovative therapies of haemophilia.

On the Prowl: An In Vivo Method to Identify RNA Partners of a sRNA.

Bacterial cells dispose of numerous strategies to regulate gene expression. Small regulatory RNAs (sRNA) are pervasive molecules that allow gene expression regulation with exquisite precision. These molecules can bind mRNAs and negatively or positively modify their stability and interfere with translation. However, many features of sRNAs render identification of new targets or RNA interacting partners increasingly complex. In this chapter, we present a detailed procedure of MAPS, an in vivo technique based on the copurification of any type of RNA bound to an MS2-tagged sRNA. By focusing on the interaction between two RNAs rather than the outcome of this interaction, MAPS has proven useful in identifying unprecedented sRNA-RNA interactions. Below, we describe how to prepare MAPS samples and how to analyze RNA sequencing data files to determine enrichment ratios of different RNAs in an experimental condition vs a control condition. MAPS can be applied to most sRNAs of Escherichia coli and Salmonella spp., and can be easily optimized to more distant bacterial species.

Evaluation and Calibration of In Silico Models of Thrombin Generation Using Experimental Data from Healthy and Haemophilic Subjects.

The coagulation cascade comprises numerous chemical reactions between many proteins, that finally lead to the formation of a clot to stop bleeding. Many numerical models have attempted to translate understanding of this cascade into mathematical equations that simulate the chain reactions. However, their predictions have not been validated against clinical data stemming from patients. In this paper, we propose an extensive validation of five available models, by comparing in healthy and haemophilic subjects, thrombin generation measured in vitro to thrombin generation predicted by the models in silico. In order to render the models more predictive, we calibrated the models to have an acceptable agreement between the experimental and estimated data. Optimization processes based on genetic algorithms were developed to search for those calibrated kinetic parameters. Our results show that the thrombin generation kinetics are so complex that they cannot be predicted by a unique set of kinetic parameters for all patients: the calibration of only three parameters in a subject-specific way allows reaching good model estimations for different experimental conditions realized on the same patient.

Kinematics of collagen fibers in carotid arteries under tension-inflation loading.

Biomechanics of the extracellular matrix in arteries determines their macroscopic mechanical behavior. In particular, the distribution of collagen fibers and bundles plays a significant role. Experimental data showed that, in most arterial walls, there are preferred fiber directions. However, the realignment of collagen fibers during tissue deformation is still controversial: whilst authors claim that fibers should undergo affine deformations, others showed the contrary. In order to have an insight about this important question of affine deformations at the microscopic scale, we measured the realignment of collagen fibers in the adventitia layer of carotid arteries using multiphoton microscopy combined with an unprecedented Fourier based method. We compared the realignment for two types of macroscopic loading applied on arterial segments: axial tension under constant pressure (scenario 1) and inflation under constant axial length (scenario 2). Results showed that, although the tissue underwent macroscopic stretches beyond 1.5 in the circumferential direction, fiber directions remained unchanged during scenario 2 loading. Conversely, fibers strongly realigned along the axis direction for scenario 1 loading. In both cases, the motion of collagen fibers did not satisfy affine deformations, with a significant difference between both cases: affine predictions strongly under-estimated fiber reorientations in uniaxial tension and over-estimated fiber reorientations during inflation at constant length. Finally, we explained this specific kinematics of collagen fibers by the complex tension-compression interactions between very stiff collagen fibers and compliant surrounding proteins. A tensegrity representation of the extracellular matrix in the adventitia taking into account these interactions was proposed to model the motion of collagen fibers during tissue deformation.

A comprehensive study of layer-specific morphological changes in the microstructure of carotid arteries under uniaxial load.

The load bearing properties of large blood vessels are principally conferred by collagen and elastin networks and their microstructural organization plays an important role in the outcomes of various arterial pathologies. In particular, these fibrous networks are able to rearrange and reorient spatially during mechanical deformations. In this study, we investigate for the first time whether these well-known morphological rearrangements are the same across the whole thickness of blood vessels, and subsequently if the underlying mechanisms that govern these rearrangements can be predicted using affine kinematics. To this aim, we submitted rabbit carotid samples to uniaxial load in three distinct deformation directions, while recording live images of the 3D microstructure using multiphoton microscopy. Our results show that the observed realignment of collagen and elastin in the media layer, along with elastin of the adventitia layer, remained limited to small angles that can be predicted by affine kinematics. We show also that collagen bundles of fibers in the adventitia layer behaved in significantly different fashion. They showed a remarkable capacity to realign in the direction of the load, whatever the loading direction. Measured reorientation angles of the fibers were significantly higher than affine predictions. This remarkable property of collagen bundles in the adventitia was never observed before, it shows that the medium surrounding collagen in the adventitia undergoes complex deformations challenging traditional hyperelastic models based on mixture theories. STATEMENT OF SIGNIFICANCE: The biomechanical properties of arteries are conferred by the rearrangement under load of the collagen and elastin fibers making up the arterial microstructure. Their kinematics under deformation is not yet characterized for all fiber networks. In this respect we have submitted samples of arterial tissue to uniaxial tension, simultaneously to confocal imaging of their microstructure. Our method allowed identifying for the first time the remarkable ability of adventitial collagen fibers to reorient in the direction of the load, achieving reorientation rotations that exceeded those predicted by affine kinematics, while all other networks followed the affine kinematics. Our results highlight new properties of the microstructure, which might play a role in the outcomes of vascular pathologies like aneurysms.

Patient-specific fracture risk assessment of vertebrae: A multiscale approach coupling X-ray physics and continuum micromechanics.

While in clinical settings, bone mineral density measured by computed tomography (CT) remains the key indicator for bone fracture risk, there is an ongoing quest for more engineering mechanics-based approaches for safety analyses of the skeleton. This calls for determination of suitable material properties from respective CT data, where the traditional approach consists of regression analyses between attenuation-related grey values and mechanical properties. We here present a physics-oriented approach, considering that elasticity and strength of bone tissue originate from the material microstructure and the mechanical properties of its elementary components. Firstly, we reconstruct the linear relation between the clinically accessible grey values making up a CT, and the X-ray attenuation coefficients quantifying the intensity losses from which the image is actually reconstructed. Therefore, we combine X-ray attenuation averaging at different length scales and over different tissues, with recently identified 'universal' composition characteristics of the latter. This gives access to both the normally non-disclosed X-ray energy employed in the CT-device and to in vivo patient-specific and location-specific bone composition variables, such as voxel-specific mass density, as well as collagen and mineral contents. The latter feed an experimentally validated multiscale elastoplastic model based on the hierarchical organization of bone. Corresponding elasticity maps across the organ enter a finite element simulation of a typical load case, and the resulting stress states are increased in a proportional fashion, so as to check the safety against ultimate material failure. In the young patient investigated, even normal physiological loading is probable to already imply plastic events associated with the hydrated mineral crystals in the bone ultrastructure, while the safety factor against failure is still as high as five. Copyright © 2016 John Wiley & Sons, Ltd.

A multiscale poromicromechanical approach to wave propagation and attenuation in bone.

Ultrasonics is an important diagnostic tool for bone diseases, as it allows for non-invasive assessment of bone tissue quality through mass density-elasticity relationships. The latter are, however, quite complex for fluid-filled porous media, which motivates us to develop a rigorous multiscale poromicrodynamics approach valid across the great variety of different bone tissues. Multiscale momentum and mass balance, as well as kinematics of a hierarchical double porous medium, together with Darcy's law for fluid flow and micro-poro-elasticity for the solid phase of bone, give access to the so-called dispersion relation, linking the complex wave numbers to corresponding wave frequencies. Experimentally validated results show that 2.25 MHz acoustical signals transmit healthy cortical bone (exhibiting a low vascular porosity) only in the form of fast waves, agreeing very well with experimental data, while both fast and slow waves transmit highly osteoporotic as well as trabecular bone (exhibiting a large vascular porosity). While velocities and wavelengths of both fast and slow waves, as well as attenuation lengths of slow waves, are always monotonously increasing with the permeability of the bone sample, the attenuation length of fast waves shows a minimum when considered as function of the permeability.

Mineralization-driven bone tissue evolution follows from fluid-to-solid phase transformations in closed thermodynamic systems.

The fundamental mechanisms that govern bone mineralization have been fairly well evidenced by means of experimental research. However, rules for the evolution of the volume and composition of the bone tissue compartments (such as the mineralized collagen fibrils and the extrafibrillar space in between) have not been provided yet. As an original contribution to this open question, we here test whether mineralizing bone tissue can be represented as a thermodynamically closed system, where crystals precipitate from an ionic solution, while the masses of the fibrillar and extrafibrillar bone tissue compartments are preserved. When translating, based on various experimental and theoretical findings, this mass conservation proposition into diffraction-mass density relations, the latter are remarkably well confirmed by independent experimental data from various sources. Resulting shrinkage and composition rules are deemed beneficial for further progress in bone materials science and biomedical engineering.

Fibrillar structure and elasticity of hydrating collagen: a quantitative multiscale approach.

It is well known that hydration of collagenous tissues leads to their swelling, as well as to softening of their elastic behavior. However, it is much less clear which microstructural and micromechanical "rules" are involved in this process. Here, we develop a theoretical approach cast in analytical mathematical formulations, which is experimentally validated by a wealth of independent tests on collagenous tissues, such as X-ray diffraction, vacuum drying, mass measurements, and Brillouin light scattering. The overall emerging picture is the following: air-drying leaves water only in the gap zones between the triple-helical collagen molecules; upon re-hydration, the extrafibrillar space is established at volumes directly proportional to the hydration-induced swelling of the (micro) fibrils, until the maximum equatorial distance between the long collagen molecules is reached. Thereafter, the volume of the fibrils stays constant, and only the extrafibrillar volume continues to grow. At all these hydration stages, the elastic behavior is governed by the same, hydration-invariant mechanical interaction pattern of only two, interpenetrating mechanical phases: transversely isotropic molecular collagen and isotropic water (or empty pores in the vacuum-dried case).

5% lidocaine medicated plaster in elderly patients with postherpetic neuralgia: results of a compassionate use programme in France.

BACKGROUND: Postherpetic neuralgia (PHN) is a common, debilitating complication of herpes zoster that has a major impact on patients' quality of life. PHN prevalence increases with advancing age. One treatment option is the topical analgesic 5% lidocaine (lignocaine) medicated plaster (Versatis®), which has been proven to be efficacious and well tolerated in a number of randomized clinical studies. OBJECTIVE: The aim of this analysis was to assess the use of the lidocaine medicated plaster under clinical practice conditions in a patient population whose previous PHN treatment with antidepressant and/or antiepileptic agents was inadequate or was not tolerated, or for whom such treatment was contraindicated or not recommended. METHODS: This was a prospective, multicentre, non-interventional observation conducted in private and public health centres in France under a compassionate use programme (CUP). To obtain this new - and, at the time, unauthorized - PHN treatment alternative, physicians (in accordance with French guidelines) had to complete standardized case report forms for each patient before his/her inclusion in the CUP. As it was a CUP and therefore a non-interventional observation, returning documented information on follow-up visits to the medication provider was voluntary, and only a limited number of physicians returned completed forms. Documentation was, however, mandatory for adverse events (AEs) occurrence. Depending on the size of the painful skin area, up to three lidocaine plasters daily were applied for a maximum of 12 hours with plaster-free intervals of at least 12 hours. The study assessed changes in the prescription of concomitant PHN medication from the start of lidocaine plaster treatment to the last follow-up visit, both in terms of the sum of all concomitant PHN treatments and stratified by type of treatment: antiepileptic drugs, tricyclic antidepressants (TCAs), serotonin reuptake inhibitors (SRIs), classical analgesics (classified as step 1, 2 or 3 according to the WHO cancer pain ladder), transcutaneous electrical nerve stimulation, and others (mainly NSAIDs). AEs were monitored for safety. RESULTS: A total of 625 patients were included in the CUP and permitted to receive lidocaine plaster treatment. Physicians returned 273 documented follow-up visit report forms. The mean ± SD CUP duration (i.e. duration of lidocaine plaster treatment) was 2.4 ± 2.5 months (median 1 month). Efficacy was assessed in the group of patients with documented follow-up visits (n = 273; mean ± SD age 73.6 ± 11.2 years), of whom 184 were aged ≥70 years (elderly efficacy population). The safety analysis included 625 patients (mean ± SD age 73.2 ± 11.9 years). Lidocaine plaster treatment resulted in a significant mean reduction of one concomitant PHN treatment per patient in the overall efficacy population analysed at the end of the observation (p < 0.001). In both populations (overall efficacy and elderly efficacy population), significantly fewer patients received TCAs (p = 0.003 and p = 0.001, respectively), step 3 analgesics (p = 0.001 and p = 0.005, respectively), and other miscellaneous treatments (p < 0.001 for both populations); there was also a significant reduction in the proportion of patients who took step 2 analgesics (p = 0.009) in the overall efficacy group. AEs (mainly related to local plaster application) were documented for 2.6% of the patients in the safety population; none were considered serious. CONCLUSIONS: In day-to-day clinical practice management of PHN, treatment with the 5% lidocaine medicated plaster permitted a significant quantitative reduction in concomitant treatments for neuropathic pain in the overall efficacy population. In the subgroup aged ≥70 years, the quantitative reduction was non-significant. However, in both populations, 5% lidocaine medicated plaster reduced use of TCAs and step 3 analgesics. An improved polymedication status and good tolerability in this likely multimorbid age group indicate that the plaster is a new therapeutic alternative for patients suffering from PHN in France.