Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease

Escaping undesired gas-phase chemistry: Microwave-driven selectivity enhancement in heterogeneous catalytic reactors

Research in solid-gas heterogeneous catalytic processes is typically aimed toward optimization of catalyst composition to achieve a higher conversion and, especially, a higher selectivity. However, even with the most selective catalysts, an upper limit is found: Above a certain temperature, gas-phase reactions become important and their effects cannot be neglected. Here, we apply a microwave field to a catalyst-support ensemble capable of direct microwave heating (MWH). We have taken extra precautions to ensure that (i) the solid phase is free from significant hot spots and (ii) an accurate estimation of both solid and gas temperatures is obtained. MWH allows operating with a catalyst that is significantly hotter than the surrounding gas, achieving a high conversion on the catalyst while reducing undesired homogeneous reactions. We demonstrate the concept with the CO 2 -mediated oxidative dehydrogenation of isobutane, but it can be applied to any system with significant undesired homogeneous contributions

Isokinetic leg strength and power in elite handball players

Isokinetic strength evaluation of the knee flexion and extension in concentric mode of contraction is an important part of the comprehensive evaluation of athletes. The aims of this study were to evaluate the isokinetic knee peak torque in both the extension and flexion movement in the dominant and non-dominant leg, and the relationship with jumping performance. Twelve elite male handball players from the top Spanish handball division voluntary participated in the study (age 27.68 ± 4.12 years; body mass 92.89 ± 12.34 kg; body height 1.90 ± 0.05 m). The knee extensor and flexor muscle peak torque of each leg were concentrically measured at 60º/s and 180º/s with an isokinetic dynamometer. The Squat Jump and Countermovement Jump were performed on a force platform to determine power and vertical jump height. Non-significant differences were observed between legs in the isokinetic knee extension (dominant= 2.91 ± 0.53 Nm/kg vs non-dominant = 2.70 ± 0.47 Nm/kg at 60º/s; dominant = 1.90 ± 0.31 Nm/kg vs non-dominant = 1.83 ± 0.29 Nm/kg at 180º/s) and flexion peak torques (dominant = 1.76 ± 0.29 Nm/kg vs non-dominant = 1.72 ± 0.39 Nm/kg at 60º/s; dominant = 1.30 ± 0.23 Nm/kg vs non-dominant = 1.27 ± 0.35 Nm/kg at 180º/s). Low and non-significant correlation coefficients were found between the isokinetic peak torques and vertical jumping performance (SJ = 31.21 ± 4.32 cm; CMJ = 35.89 ± 4.20 cm). Similar isokinetic strength was observed between the legs; therefore, no relationship was found between the isokinetic knee flexion and extension peak torques as well as vertical jumping performance in elite handball players.Actividad Física y Deport

Monitoring dexamethasone skin biodistribution with ex vivo MALDI-TOF mass spectrometry imaging and confocal Raman microscopy

Two of the most promising techniques in terms of ex vivo skin imaging and quantifying are confocal Raman microscopy and MALDI-TOF mass spectrometry imaging (MALDI-TOF MSI). Both techniques were set up, and the semiquantitative skin biodistribution of previously developed dexamethasone (DEX) loaded lipomers was compared using Benzalkonium chloride (BAK) as a tracer of the nanoparticles. In MALDI-TOF MSI, DEX was derivatised with GirT (DEX-GirT) and the semiquantitative biodistribution of both DEX-GirT and BAK was successfully obtained. The amount of DEX measured by confocal Raman microscopy was higher than that measured by MALDI-TOF MSI, but MALDI-TOF MSI proved to be a more suitable technique for tracing BAK. An absorption-promoting tendency of DEX loaded in lipomers versus a free-DEX solution was observed in confocal Raman microscopy. The higher spatial resolution of confocal Raman microscopy (350 nm) with respect to MALDI-TOF MSI (50 mu m) allowed to observe specific skin structures like hair follicles. Nevertheless, the faster sampling rate of MALDI-TOF-MSI, permitted the analysis of larger tissue regions. In conclusion, both techniques allowed to simultaneously analyze semiquantitative data together with qualitative images of biodistribution, which is a very helpful tool when designing nanoparticles that accumulate in specific anatomical regions

Differential Calculi on Commutative Algebras

A differential calculus on an associative algebra A is an algebraic analogue of the calculus of differential forms on a smooth manifold. It supplies A with a structure on which dynamics and field theory can be formulated to some extent in very much the same way we are used to from the geometrical arena underlying classical physical theories and models. In previous work, certain differential calculi on a commutative algebra exhibited relations with lattice structures, stochastics, and parametrized quantum theories. This motivated the present systematic investigation of differential calculi on commutative and associative algebras. Various results about their structure are obtained. In particular, it is shown that there is a correspondence between first order differential calculi on such an algebra and commutative and associative products in the space of 1-forms. An example of such a product is provided by the Ito calculus of stochastic differentials. For the case where the algebra A is freely generated by `coordinates' x^i, i=1,...,n, we study calculi for which the differentials dx^i constitute a basis of the space of 1-forms (as a left A-module). These may be regarded as `deformations' of the ordinary differential calculus on R^n. For n < 4 a classification of all (orbits under the general linear group of) such calculi with `constant structure functions' is presented. We analyse whether these calculi are reducible (i.e., a skew tensor product of lower-dimensional calculi) or whether they are the extension (as defined in this article) of a one dimension lower calculus. Furthermore, generalizations to arbitrary n are obtained for all these calculi.Comment: 33 pages, LaTeX. Revision: A remark about a quasilattice and Penrose tiling was incorrect in the first version of the paper (p. 14

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

18 pags., 6 figs., 1 tab. -- Open Access funded by Creative Commons Atribution Licence 4.0Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.This work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from INSTRUCT (“Innovative EM/NMR approach for the characterization of the drug target ClpP APPID: 301“), FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank the ESRF for beamtime at ID30A and ID23-1. Funding: This work was supported by Spanish Ministerio de Economia y Competitividad (BFU2016-78232-P) and Instituto de Salud Carlos III co-funded by European Union (PI15/00663 and PI18/00349, ERDF/ ESF, “Investing in your future”). This work was financially supported by the European Research Council (ERC-Stg-2012-311318 to P.S.). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017)

Thermal liquid biopsy (TLB) focused on benign and premalignant pancreatic cyst diagnosis

Background: Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology. Methods: In this work, twenty patients (12 males and 8 females, average ages 62) diagnosed with a pancreatic cyst benign (10) and premalignant (10) cyst lesions were recruited, and biological samples were obtained during the endoscopic ultrasonography procedure. Results: Proteomic content of cyst liquid samples was studied and several common proteins in the different groups were identified. TLB cyst liquid profiles reflected protein content. Also, TLB serum score was able to discriminate between healthy and cysts patients (71% sensitivity and 98% specificity) and between benign and premalignant cysts (75% sensitivity and 67% specificity). Conclusions: TLB analysis of plasmatic serum sample, a quick, simple and non-invasive technique that can be easily implemented, reports valuable information on the observed pancreatic lesion. These preliminary results set the basis for a larger study to refine TLB serum score and move closer to the clinical application of TLB providing useful information to the gastroenterologist during patient diagnosis

Fiber-optic lossy mode resonance sensors

In the last 4 years, experimental evidences about the potential use of optical sensors based on Lossy Mode Resonances (LMR) have been presented in the literature. These LMR sensors have some similarities with Surface Plasmon Resonance (SPR) sensors, the gold standard in label-free, real-time biomolecular interaction analysis. In these new LMR sensors, if the non-metallic nanocladding of an optical waveguide fulfills the conditions explained in this work, coupling of light to the cladding modes happens at certain resonance wavelengths, which enables the use of LMR devices as refractometers and opens the door to diverse applications such as in biology and proteomics research. These highly sensitive refractometers have already shown sensitivities higher than 20,000 nm/RIU or 5x10-7 RIU and, given the youth of this field, it is expected to achieve even better values

Optical sensors based on lossy-mode resonances

Lossy-mode resonance (LMR)–based optical sensing technology has emerged in the last two decades as a nanotechnological platform with very interesting and promising properties. LMR complements the metallic materials typically used in surface plasmon resonance (SPR)–based sensors, with metallic oxides and polymers. In addition, it enables one to tune the position of the resonance in the optical spectrum, to excite the resonance with both transverse electric (TE) and transverse magnetic (TM) polarized light, and to generate multiple resonances. The domains of application are numerous: as sensors for detection of refractive indices voltage, pH, humidity, chemical species, and antigens, as well as biosensors. This review will discuss the bases of this relatively new technology and will show the main contributions that have permitted the optimization of its performance to the point that the question arises as to whether LMR–based optical sensors could become the sensing platform of the near future