Multimodal silver-chitosan-acylase nanoparticles inhibit bacterial growth and biofilm formation by Gram-negative Pseudomonas aeruginosa bacterium.

Pseudomonas aeruginosa bacteria originate severe infections in hospitalized patients and those with chronic debilitating diseases leading to increased morbidity and mortality, longer hospitalization and huge financial burden to the healthcare system. The clinical relevance of P. aeruginosa infections is increased by the capability of this bacterium to grow in biofilms and develop multidrug resistant mechanisms that preclude conventional antibiotic treatments. Herein, we engineered novel multimodal nanocomposites that integrate in the same entity antimicrobial silver nanoparticles (NPs), the intrinsically antimicrobial, but biocompatible biopolymer chitosan, and the anti-infective quorum quenching enzyme acylase I. Acylase present in the NPs specifically degraded the signal molecules governing bacterial cell-to-cell communication and inhibited by âˆ¼ 55 % P. aeruginosa biofilm formation, while the silver/chitosan template altered the integrity of bacterial membrane, leading to complete eradication of planktonic bacteria. The innovative combination of multiple bacteria targeting modalities resulted in 100-fold synergistic enhancement of the antimicrobial efficacy of the nanocomposite at lower and non-hazardous towards human skin cells concentrations, compared to the silver/chitosan NPs alone.

Hyaluronic Acid Derivative Molecular Weight-Dependent Synthesis and Antimicrobial Effect of Hybrid Silver Nanoparticles.

Silver nanoparticles (Ag NPs) appeared as promising antimicrobial candidates to face the development of antibiotic resistance. Although reported as toxic towards mammalian cells, their combination with biomolecules have shown reduced toxicity, while maintaining the antimicrobial function. Herein, hyaluronic acid (HA) with low (40 kDa), medium (200 and 600 kDa) and high (2 MDa) molecular weight (Mw) was modified with adipic acid dihydrazide (ADH) and used as reducing and capping agents to synthesise antimicrobial hybrid Ag NPs. The Mw of the polymer played a crucial role in the morphology, size and antibacterial activity of the Ag NPs. The 600 and 200 kDa HA-ADH-Ag NPs were able to reduce the Escherichia coli and Staphylococcus aureus concentration by more than 3 logs, while the 40 kDa NPs reached ~2 logs reduction. The 2 MDa HA-ADH failed to form homogenous NPs with strong bactericidal activity. A mechanistic study of the interaction with a model bacterial membrane using Langmuir isotherms confirmed the greater interaction between bacteria and higher Mw polymers and the effect of the NP's morphology. The nanocomposites low toxicity to human skin cells was demonstrated in vitro, showing more than 90% cell viability after incubation with the NPs.

Lipid artificial tears at a mimetic ocular interface.

We studied the behaviour of three lipid tear products, commercialised by the same brand, as Langmuir films at the air/liquid interface to simulate the ocular environment. No significant differences were observed in the surface behaviour of two of them disclosing the same composition, but commercialised for different applications. The interaction of several subphases, namely sodium chloride, glucose, albumin and lysozyme present in the natural tear, with the lipid films was assessed at room temperature and the temperature of human tear using surface pressure-area isotherms and elastic modulus plots. There is a notable influence of sodium chloride and the proteins albumin and lysozyme on the surface pressure-area isotherm of the lipid Langmuir films. Albumin shifted this isotherm to lower areas while an opposite shift was caused by lysozyme. These studies could be useful for the formulation of new lipid-containing artificial tears, and for increasing the confidence of the customers in commercial eye care formulations.

Interactions of cryoprotective agents with phospholipid membranes - A Langmuir monolayer study.

The influence of four common cryoprotectants (dimethyl sulfoxide, glycerol, ethylene glycol and dimethylformamide) on monolayers of four common phospholipids (DPPC, DOPC, POPC and POPE) have been studied using Langmuir isotherms and monolayer insertion experiments. The cryoprotectant concentrations were chosen to be directly relevant to cryoprotection. We show that DMSO causes an expansion of the DPPC area per lipid (in contrast to previous work at higher concentrations). However, it caused compression for POPC, and had little effect for POPE or DOPC. As most previous studies have involved only DPPC, this highlights the importance of studying different lipid types as these may have a significant effect on the interactions. We show that both ethylene glycol and glycerol cause a small expansion of the monolayer at fixed pressure, implying that they insert into the headgroup regions, regardless of lipid species, and consistent with their ability to penetrate membranes. By contrast, dimethylformamide causes monolayer compression for all lipid species, implying it dehydrates the lipid head groups. Membrane insertion experiments at physiological values of lateral pressure highlight that DPPC is the most difficult lipid to penetrate, implying that the penetrating action of cryoprotectants may only occur for unsaturated phospholipids. Thus, extrapolations of results based solely on the DPPC need to be made with care.

Interaction of Silver-Lignin Nanoparticles With Mammalian Mimetic Membranes.

Silver nanoparticles (AgNPs) have broad spectrum antibacterial activity, but their toxicity to human cells has raised concerns related to their use as disinfectants or coatings of medically relevant surfaces. To address this issue, NPs comprising intrinsically bactericidal and biocompatible biopolymer and Ag with high antibacterial efficacy against common pathogens and compatibility to human cells have been engineered. However, the reason for their lower toxicity compared to AgNPs has not yet been elucidated. This work studies the in vitro interaction of AgLNPs with model mammalian membranes through two approaches: (i) Langmuir films and (ii) supported planar bilayers studied by quartz crystal microbalance and atomic force spectroscopy. These approaches elucidate the interactions of AgLNPs with the model membranes indicating a prominent effect of the bioresourced lignin to facilitate the binding of AgLNPs to the mammalian membrane, without penetrating through it. This study opens a new avenue for engineering of hybrid antimicrobial biopolymer - Ag or other metal NPs with improved bactericidal effect whereas maintaining good biocompatibility.

Lipid-lipid interactions of Escherichia coli mimetic inner membrane at human physiological temperature.

The current strategies to eradicate bacteria require that the antimicrobial agent either penetrate or disrupt the bacterial membrane. In Escherichia coli (E.coli) as a model of Gram-negative strains, the antimicrobials have to cross two barriers - the outer and the inner membrane being the latter composed by ~ 77% phosphatidylethanolamine (PE), ~ 13% phosphatidylglycerol (PG) and ~ 10% cardiolipin (CL) lipids. Each one of these lipid families shares the same headgroup, but contains acyl chains with varying length and degree of unsaturation. Bacteria adapt their membrane lipid composition and metabolism in response to environmental signals, such as the temperature, resulting in different interactions with exogenous molecules, e.g. antibacterial agents. Herein, bacterial model membranes are prepared to evaluate the lipid-lipid interactions in Langmuir monolayers of binary mixtures at several molar ratios of PE and PG or CL at human physiological temperature (37°C). Both PE:PG and PE:CL monolayers were stable at 37°C and presented higher molecular areas (> 20 Å2/molecule) than at 23°C. However, these lipid mixtures presented liquid-expanded state and rigidity (inverse of the compressibility modulus ~ 90 mN/m) slightly lower than at 23°C. Such athermalicity at biologically relevant temperatures may favour the preservation of the biological functions of E.coli.

New myeloperoxidase detection system based on enzyme-catalysed oxidative synthesis of a dye for paper-based diagnostic devices.

The severity and cost of wound infections strongly demands for simple and fast methods for wound infection determination. Point-of-care testing devices play a crucial role in order to achieve a fast diagnosis and early treatment. Myeloperoxidase (MPO) enzyme, detected in fluids of infected wounds has been postulated as a suitable biomarker for wound diagnostics. Here we present a new system for MPO detection, based on enzyme-catalysed oxidative synthesis of a dye that can be incorporated into paper-based point of care devices. Visual MPO detection has been achieved through the use of phenylenediamine, a common colourless hair dye precursor. MPO oxidation of these compounds yielded bright coloured products distinguishable from the colour of the wound environment. Immobilisation of the MPO substrates on paper strips was achieved through in situ interaction of the oxidised coloured product with branched polyethyleneimine. The colour reaction of the immobilized substrates, detectable by naked eye, responds to the MPO levels present in infected wound fluids revealing an easy system for incorporation of MPO detection in paper based diagnostic devices.

Physical states and thermodynamic properties of model gram-negative bacterial inner membranes.

Novel antimicrobial agents are focused to interact with the bacterial membrane whose lipid composition (number and position of unsaturations and lipid headgroup) is adapted according to environmental signals. The anticipation of the adapted membrane properties is of high relevance to increase the targetability of such drugs. Herein, natural lipids extracted from Escherichia coli -phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL)- are used to form biomimetic membranes constituted by several PE:lipid ratios using the Langmuir and Langmuir-Blodgett techniques. The use of these techniques and the natural myriad of each lipid structures that constitute the biological E.coli membrane establishes a simple and reproducible model to evaluate the lipid-lipid interactions. PE and PG present similar shape and size, thus establish ideal and fluid -liquid expanded (LE) state - mixtures, whereas the differences between PE and CL motivate the formation of non-ideal and fluid (LE state) mixtures. The same physical state and the minor differences in elasticity (differences in the inverse of the compressibility modulus < 15 mN·m-1) between both systems regardless the PE content in the (PE:lipid) mixture suggest that the changes in the lipid composition influence the membrane proteins function rather than affecting the rigidity of the bacterial membrane.

Metal-Enzyme Nanoaggregates Eradicate Both Gram-Positive and Gram-Negative Bacteria and Their Biofilms.

To palliate the appearance of antimicrobial resistance (AMR), the use of bactericidal agents acting differently than conventional antibiotics and the elimination of bacterial biofilm, are the two most promising strategies. Here, we integrated these two complementary strategies into new antimicrobial metal-enzyme nanoaggregates (NAs) of α-amylase and silver (αAgNAs) that are able to eliminate bacteria and their biofilm. The nanoparticle (NP) synthesis approach applied protein desolvation and laccase-mediated NP stabilization to innovatively produce catalytically active α-amylase nanoparticles (αNPs) for the elimination of the bacterial biofilm. At the same time, αNPs efficiently reduced silver for the incorporation of bactericidal Ag0 and formation of the αAgNAs. The bactericidal and antibiofilm efficacies of αAgNAs were demonstrated by 5.4 and 6.1 log reduction of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, respectively, and more than 80% removal of their biofilms, coupled with high biocompatibility. The biofilm-αAgNA interaction was assessed by quartz crystal microbalance and atomic force microscopy revealing how the degradation of a settled biofilm by αAgNAs caused an increase of the biofilm water content, thus weakening the biofilm surface attachment and facilitating its removal. With the present work, we not only provide a new efficient antimicrobial material to face the AMR threat, but we also envisage that the newly established method for the synthesis of metal-enzyme NAs is potentially transferable to other biocatalysts to expand the enzyme NP toolbox.

Tuning ubiquinone position in biomimetic monolayer membranes.

Artificial lipid bilayers have been extensively studied as models that mimic natural membranes (biomimetic membranes). Several attempts of biomimetic membranes inserting ubiquinone (UQ) have been performed to enlighten which the position of UQ in the lipid layer is, although obtaining contradictory results. In this work, pure components (DPPC and UQ) and DPPC:UQ mixtures have been studied using surface pressure-area isotherms and Langmuir-Blodgett (LB) films of the same compounds have been transferred onto solid substrates being topographically characterized on mica using atomic force microscopy and electrochemically on indium tin oxide slides. DPPC:UQ mixtures present less solid-like physical state than pure DPPC indicating a higher-order degree for the latter. UQ influences considerably DPPC during the fluid state, but it is mainly expelled after the phase transition at [Formula: see text] 26 mN·m^-1 for the 5:1 ratio and at [Formula: see text] 21 mN·m^-1 for lower UQ content. The thermodynamic studies confirm the stability of the DPPC:UQ mixtures before that event, although presenting a non-ideal behaviour. The results indicate that UQ position can be tuned by means of the surface pressure applied to obtain LB films and the UQ initial content. The UQ positions in the biomimetic membrane are distinguished by their formal potential: UQ located in "diving" position with the UQ placed in the DPPC matrix in direct contact with the electrode surface ( -0.04±0.02 V), inserted between lipid chains without contact to the substrate ( 0.00±0.01 V) and parallel to the substrate, above the lipid chains ( 0.09±0.02 V).

Influence of membrane galactolipids and surface pressure on plastoquinone behaviour.

In this work biomimetic monolayers of a MGDG, monogalactosyldiacylglycerol, and DGDG, digalactosyldiacylglycerol mixture (MD), in a ratio close to that of the thylakoid membranes of oxygenic photosynthetic organisms, have been prepared. The lipid mixture incorporates plastoquinone-9 (PQ), that is the electron and proton shuttle of the photosynthetic reaction centres. The MD:PQ mixtures have been firstly studied using surface pressure-area isotherms. Langmuir-Blodgett (LB) films of those mixtures have been transferred onto a substrate forming a monolayer that mimics one of the bilayer sides of the thylakoid membranes. These monolayers have been characterized topographically and electrochemically. The results show the influence of PQ in the MD matrix and its partial expulsion when increasing the surface pressure, obtaining two main PQ positions in the MD matrix. The calculated apparent electron transfer rate constants indicate a different kinetic control for the reduction and the oxidation of the PQ/PQH2 couple, being kRapp(I)=0.7·10(-6)s(-1), kRapp(II)=2.2·10(-9)s(-1), kOapp(I)=7.4·10(-4)s(-1) and kOapp(II)=5.2·10(-5)s(-1), respectively. The comparison of the different galactolipid:PQ systems that our group has studied is also presented, concluding that the PQ position in the galactolipid matrix can be tuned according to several controlled variables.

Escherichia coli and Pseudomonas aeruginosa eradication by nano-penicillin G.

The transformation of penicillin G into nano/micro-sized spheres (nanopenicillin) using sonochemical technology was explored as a novel tool for the eradication of Gram-negative bacteria and their biofilms. Known by its effectiveness only against Gram-positive microorganisms, the penicillin G spherization boosted the inhibition of the Gram-negative Pseudomonas aeruginosa 10-fold (from 0.3 to 3.0 log-reduction) and additionally induced 1.2 log-reduction of Escherichia coli growth. The efficient penetration of the spheres within a Langmuir monolayer sustained the theory that nanopenicillin is able to cross the membrane and reach the periplasmic space in Gram-negative bacteria where they inhibit the ß-lactam targets: the transferases that build the bacteria cell wall. Moreover, it considerably suppressed the growth of both bacterial biofilms on a medically relevant polystyrene surface, leaving majority of the adhered cells dead compared to the treatment with the non-processed penicillin G. Importantly, nanopenicillin was found innocuous towards human fibroblasts at the antibacterial-effective concentrations.

Monogalactosyldiacylglycerol and digalactosyldiacylglycerol role, physical states, applications and biomimetic monolayer films.

The relevance of biomimetic membranes using galactolipids has not been expressed in any extensive experimental study of these lipids. Thus, on the one hand, we present an in-depth article about the presence and role of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) in thylakoid membranes, their physical states and their applications. On the other hand, we use the Langmuir and Langmuir-Blodgett (LB) techniques to prepare biomimetic monolayers of saturated galactolipids MGDG, DGDG and MGDG:DGDG 2:1 mixture (MD)--biological ratio--. These monolayers are studied using surface pressure-area isotherms and their data are processed to enlighten their physical states and mixing behaviour. These monolayers, once transferred to a solid substrate at several surface pressures are topographically studied on mica using atomic force microscopy (AFM) and using cyclic voltammetry for studying the electrochemical behaviour of the monolayers once transferred to indium-tin oxide (ITO), which has good optical and electrical properties. Moreover, MD presents other differences in comparison with its pure components that are explained by the presence of different kinds of galactosyl headgroups that restrict the optimal orientation of the MGDG headgroups.

Biomimetic monolayer films of monogalactosyldiacylglycerol incorporating plastoquinone.

Photosynthetic organisms use light to convert the inorganic matter in organic one. Photosynthetic process consists on several steps, and one of them involves plastoquinone (PQ) that acts as electron and proton shuttle between photosystem II and cytochrome. We prepared membranes that mimic the characteristics and composition of natural photosynthetic membranes and we characterized them using several techniques in order to obtain both the PQ molecules disposition in the membrane and their electrochemical behavior. The selected lipid was monogalactosyldiacylglycerol (MGDG) that represents the 50% of the lipid content of the thylakoid membrane. Both MGDG and PQ, and the MGDG:PQ mixtures have been studied using surface pressure-area isotherms and the presence of PQ alters the physical state and compactness of the MGDG matrix. Langmuir-Blodgett (LB) films have been obtained by transferring a monolayer that mimics half of the bilayer of a biological membrane. The AFM topographical characterization of the monolayers on mica indicates the presence of differentiated domains, corresponding to different physical states linked to the influence of the PQ content. Moreover, the electrochemical behavior of the monolayers has been studied when transferred on ITO, observing one main electrochemical process that is due to the diving position of PQ molecules in the lipid matrix.

AFM in peak force mode applied to worn siloxane-hydrogel contact lenses.

The objective of this work is to apply Atomic Force Microscopy in Peak Force mode to obtain topographic characteristics (mean roughness, root-mean-square roughness, skewness and kurtosis) and mechanical characteristics (adhesion, elastic modulus) of Siloxane-Hydrogel Soft Contact Lenses (CLs) of two different materials, Lotrafilcon B of Air Optix (AO) and Asmofilcon A of PremiO (P), after use (worn CLs). Thus, the results obtained with both materials will be compared, as well as the changes produced by the wear at a nanoscopic level. The results show significant changes in the topographic and mechanical characteristics of the CLs, at a nanoscopic level, due to wear. The AO CL show values of the topographic parameters lower than those of the P CL after wear, which correlates with a better comfort qualification given to the former by the wearers. A significant correlation has also been obtained between the adhesion values found after the use of the CLs with tear quality tests, both break-up-time and Schirmer.

Electrochemistry of LB films of mixed MGDG:UQ on ITO.

The electrochemical behaviour of biomimetic monolayers of monogalactosyldiacylglycerol (MGDG) incorporating ubiquinone-10 (UQ) has been investigated. MGDG is the principal component in the thylakoid membrane and UQ seems a good substitute for plastoquinone-9, involved in photosynthesis chain. The monolayers have been performed using the Langmuir and Langmuir-Blodgett (LB) techniques and the redox behaviour of the LB films, transferred at several surface pressures on a glass covered with indium-tin oxide (ITO), has been characterized by cyclic voltammetry. The cyclic voltammograms show that UQ molecules present two redox processes (I and II) at high UQ content and high surface pressures, and only one redox process (I) at low UQ content and low surface pressures. The apparent rate constants calculated for processes I and II indicate a different kinetic control for the reduction and the oxidation of UQ/UQH2 redox couple, being k(Rapp)(I) = 2.2 · 10(-5) s(-1), k(Rapp)(II) = 5.1 · 10(-14) k(Oapp)(I) = 3.3 · 10(-3) s(-1) and k(Oapp)(II) = 6.1 · 10(-6) s(-1), respectively. The correlation of the redox response with the physical states of the LB films allows determining the positions of the UQ molecules in the biomimetic monolayer, which change with the surface pressure and the UQ content. These positions are known as diving and swimming.

Biomimetic monolayer films of digalactosyldiacylglycerol incorporating plastoquinone.

The photosynthesis is the process used by plants and bacteria cells to convert inorganic matter in organic thanks to the light energy. This process consist on several steps, being one of them the electronic transport from the photosystem II to the cytochrome thanks to plastoquinone-9 (PQ). Here we prepare membranes that mimic the characteristics and composition of natural photosynthetic cell membranes and we characterize them in order to obtain the PQ molecules position in the membrane and their electrochemical behaviour. The selected galactolipid is digalactosyldiacylglycerol (DGDG) that represents the 30% of the thylakoid membrane lipid content. The results obtained are worthful for several science fields due to the relevance of galactolipids as anti-algal, anti-viral, anti-tumor and anti-inflammatory agents and the antioxidant and free radical scavenger properties of prenylquinones. Both pure components (DGDG and PQ) and the DGDG:PQ mixtures have been studied using surface pressure-area isotherms. These isotherms give information about the film stability and indicate the thermodynamic behaviour of the mixture and their physical state. The Langmuir-Blodgett (LB) film has been transferred forming a monolayer that mimics the bottom layer of the biological membranes. This monolayer on mica has been topographically characterized using AFM and both the height and the physical state that they present have been obtained. Moreover, these monolayers have been transferred onto ITO that is a hydrophilic substrate with good optical and electrical features, so that, it is suitable for studying the electrochemical behaviour of these systems and it is a good candidate for energy producing devices.

Sonochemically processed cationic nanocapsules: efficient antimicrobials with membrane disturbing capacity.

Bacterial-mediated diseases are a major healthcare concern worldwide due to the rapid spread of antibiotic-resistant bacteria. One strategy to manage the bacterial infections while avoiding the emergence of resistant strains implies specific targeting and disruption of bacteria membranes. This work evaluates the potential of nanostructured biopolymer derivatives, nanocapsules (NCs), to disrupt the bacteria cell walls and effectively kill planktonic microorganisms. Two biopolymers, chitosan and cellulose, were chemically modified to synthesize derivatives with improved cationic character (thiolated chitosan and aminocellulose) prior to their processing into nanocapsules via a one-step sonochemical process. The interactions of NCs, displaying an average size of around 250 nm, with bacteria membrane were evaluated using two membrane models: Langmuir monolayers and liposome bilayers composed of a l-α-phosphatidylglycerol phospholipid extracted from Escherichia coli. NCs possessed improved membrane disturbing capacity in comparison to the nonprocessed biopolymer derivatives, by drastically increasing the monolayer fluidity and inducing more than 50% leakage of a dye inserted in the bilayered liposomes. In addition, membrane disturbance was directly proportional to the NCs cationic charge. Whereas evidence showed that thiolated chitosan and aminocellulose interacted with the bacteria membrane through a "carpet model", the NCs were found to induce larger surface defects and high local perturbance through a "detergent model". Importantly, the degree of disruption caused by the biopolymer derivatives and NCs correlated well with the antimicrobial capacity against Escherichia coli, selectively killing bacteria cells without imparting toxicity to human fibroblasts.

Incorporation of ubiquinone in supported lipid bilayers on ITO.

Ubiquinone (UQ) is one of the main electron and proton shuttle molecules in biological systems, and dipalmitoylphosphatidylcholine (DPPC) is one of the most used model lipids. Supported planar bilayers (SPBs) are extensively accepted as biological model membranes. In this study, SPBs have been deposited on ITO, which is a semiconductor with good electrical and optical features. Specifically, topographic atomic force microscopy (AFM) images and force curves have been performed on SPBs with several DPPC:UQ ratios to study the location and the interaction of UQ in the SPB. Additionally, cyclic voltammetry has been used to understand the electrochemical behavior of DPPC:UQ SPBs. Obtained results show that, in our case, UQ is placed in two main different positions in SPBs. First, between the DPPC hydrophobic chains, fact that originates a decrease in the breakthrough force of the bilayer, and the second between the two leaflets that form the SPBs. This second position occurs when increasing the UQ content, fact that eventually forms UQ aggregates at high concentrations. The formation of aggregates produces an expansion of the SPB average height and a bimodal distribution of the breakthrough force. The voltammetric response of UQ depends on its position on the bilayer.

Improved conformational stability of the visual G protein-coupled receptor rhodopsin by specific interaction with docosahexaenoic acid phospholipid.

Rhodopsin is the photoreceptor located in the rod cells of the retina. It has seven transmembrane helices and is a prototypic member of the G protein-coupled receptor superfamily. The structures and functions of these receptors are clearly affected by the lipid composition of the cell membrane, and their study in a purified recombinant form is usually performed in detergent solution. There is a need to study these receptors in a physiologically relevant environment because the lipid environment is known to have an important effect on their function. In this work, rhodopsin reconstituted in docosahexaenoic acid (DHA) liposomes is shown to have more thermal stability than when it is solubilised with the neutral detergent dodecyl maltoside. Moreover, the specific interaction between rhodopsin and DHA was followed by means of Langmuir experiments with insertion of rhodopsin into lipid monolayers; this showed high affinity for the lipid-receptor interaction. Furthermore, fluorescence spectroscopy measurements indicate that the conformation of opsin obtained after photobleaching is preserved in DHA-containing liposomes, thereby allowing retinal to re-enter the binding pocket even long after bleaching. Overall, our results demonstrate that liposomes of this specific lipid provide a more stable environment for ground-state inactive rhodopsin in the dark, than dodecyl maltoside detergent, and that this lipid can also preserve the native correctly folded ligand-free opsin conformation obtained after illumination. This strategy will be used in further studies on mutations of rhodopsin associated with congenital retinopathies.