Effects of Crowding and Cosolutes on Biomolecular Function at Extreme Environmental Conditions.

The extent of the effect of cellular crowding and cosolutes on the functioning of proteins and cells is manifold and includes the stabilization of the biomolecular systems, the excluded volume effect, and the modulation of molecular dynamics. Simultaneously, it is becoming increasingly clear how important it is to take the environment into account if we are to shed light on biological function under various external conditions. Many biosystems thrive under extreme conditions, including the deep sea and subseafloor crust, and can take advantage of some of the effects of crowding. These relationships have been studied in recent years using various biophysical techniques, including neutron and X-ray scattering, calorimetry, FTIR, UV-vis and fluorescence spectroscopies. Combining knowledge of the structure and conformational dynamics of biomolecules under extreme conditions, such as temperature, high hydrostatic pressure, and high salinity, we highlight the importance of considering all results in the context of the environment. Here we discuss crowding and cosolute effects on proteins, nucleic acids, membranes, and live cells and explain how it is possible to experimentally separate crowding-induced effects from other influences. Such findings will contribute to a better understanding of the homeoviscous adaptation of organisms and the limits of life in general.

Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View.

Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.

From a Droplet to a Fibril and from a Fibril to a Droplet: Intertwined Transition Pathways in Highly Dynamic Enzyme-Modulated Peptide-Adenosine Triphosphate Systems.

Many cellular coassemblies of proteins and polynucleotides facilitate liquid-liquid phase separation (LLPS) and the subsequent self-assembly of disease-associated amyloid fibrils within the liquid droplets. Here, we explore the dynamics of coupled phase and conformational transitions of model adenosine triphosphate (ATP)-binding peptides, ACC1-13Kn, consisting of the potent amyloidogenic fragment of insulin's A-chain (ACC1-13) merged with oligolysine segments of various lengths (Kn, n = 16, 24, 40). The self-assembly of ATP-stabilized amyloid fibrils is preceded by LLPS for peptides with sufficiently long oligolysine segments. The two-component droplets and fibrils are in dynamic equilibria with free ATP and monomeric peptides, which makes them susceptible to ATP-hydrolyzing apyrase and ACC1-13Kn-digesting proteinase K. Both enzymes are capable of rapid disassembly of amyloid fibrils, producing either monomers of the peptide (apyrase) or free ATP released together with cleaved-off oligolysine segments (proteinase K). In the latter case, the enzyme-sequestered Kn segments form subsequent droplets with the co-released ATP, resulting in an unusual fibril-to-droplet transition. In support of the highly dynamic nature of the aggregate-monomer equilibria, addition of superstoichiometric amounts of free peptide to the ACC1-13Kn-ATP coaggregate causes its disassembly. Our results show that the droplet state is not merely an intermediate phase on the pathway to the amyloid aggregate but may also constitute the final phase of a complex amyloidogenic protein misfolding scenario rich in highly degraded protein fragments incompetent to transition again into fibrils.

Enabling High Activation of Glucose-6-Phosphate Dehydrogenase Activity Through Liquid Condensate Formation and Compression.

Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

Bacterial model membranes under the harsh subsurface conditions of Mars.

Biomembranes are a key component of all living systems. Most research on membranes is restricted to ambient physiological conditions. However, the influence of extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments, is less well understood. The deep subsurface of Mars is thought to harbour high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments. Here, we investigated the combined effects of high concentrations of Mars-relevant salts, including sodium and magnesium perchlorate and sulphate, and high hydrostatic pressure on the stability, structure, and function of a bacterial model membrane. To this end, several biophysical techniques have been employed, including calorimetry, fluorescence and CD spectroscopy, confocal microscopy, and small-angle X-ray scattering. We demonstrate that sulphate and perchlorate salts affect the properties of the membrane differently, depending on the counterion present (Na+vs. Mg2+). We found that the perchlorates, which are believed to be abundant salts in the Martian environment, induce a more hydrated and less ordered membrane, strongly favouring the physiologically relevant fluid-like phase of the membrane even under high-pressure stress. Moreover, we show that the activity of the phospholipase A2 is strongly modulated by both high pressure and salt. Compellingly, in the presence of the chaotropic perchlorate, the enzymatic reaction proceeded at a reasonable rate even in the presence of condensing Mg2+ and at high pressure, suggesting that bacterial membranes could still persist when challenged to function in such a highly stressed Martian environment.

Liquid-Droplet-Mediated ATP-Triggered Amyloidogenic Pathway of Insulin-Derived Chimeric Peptides: Unraveling the Microscopic and Molecular Processes.

Disease-associated progression of protein dysfunction is typically determined by an interplay of transition pathways leading to liquid-liquid phase separation (LLPS) and amyloid fibrils. As LLPS introduces another layer of complexity into fibrillization of metastable proteins, a need for tunable model systems to study these intertwined processes has emerged. Here, we demonstrate the LLPS/fibrillization properties of a family of chimeric peptides, ACC1-13Kn, in which the highly amyloidogenic fragment of insulin (ACC1-13) is merged with oligolysine segments of various lengths (Kn, n = 8, 16, 24, 32, 40). LLPS and fibrillization of ACC1-13Kn are triggered by ATP through Coulombic interactions with Kn fragments. ACC1-13K8 and ACC1-13K16 form fibrils after a short lag phase without any evidence of LLPS. However, in the case of the three longest peptides, ATP triggers instantaneous LLPS followed by the disappearance of droplets occurring in-phase with the formation of amyloid fibrils. The kinetics of the phase transition and the stability of mature co-aggregates are highly sensitive to ionic strength, indicating that electrostatic interactions play a pivotal role in selecting the LLPS-fibrillization transition pathway. Densely packed ionic interactions that characterize ACC1-13Kn-ATP fibrils render them highly sensitive to hydrostatic pressure due to solvent electrostriction, as demonstrated by infrared spectroscopy. Using atomic force microscopy imaging of rapidly frozen samples, we demonstrate that early fibrils form within single liquid droplets, starting at the droplet/bulk interface through the formation of single bent fibers. A hypothetical molecular scenario underlying the emergence of the LLPS-to-fibrils pathway in the ACC1-13Kn-ATP system has been put forward.

Inducin Triggers LC3-Lipidation and ESCRT-Mediated Lysosomal Membrane Repair.

Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.

Alcohol-Induced Conformation Changes and Thermodynamic Signatures in the Binding of Polyphenols to Proline-Rich Salivary Proteins.

The first contact of polyphenols (tannins) with the human body occurs in the mouth, where they are known to interact with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for the development of astringency, but affect also various other biochemical processes. Employing thermodynamic measurements, fluorescence and CD spectroscopy, we investigated the binding process of the prototypical polyphenol ellagic acid (EA) to different IB-PRPs and BSA, also in the presence of ethanol, which is known to influence tannin-protein interactions. Binding of EA to BSA and the small peptide IB7-14 is weak, but very strong to IB9-37. The differences in binding strength and stoichiometry are due to differences in the binding motifs, which also lead to differences in the thermodynamic signatures of the binding process. EA binding to BSA is enthalpy-driven, whereas binding to both IB7-14 and IB9-37 is entropy-driven. The presence of 10 vol.% EtOH, as present in wines, increases the binding constant of EA with BSA and IB7-14 drastically, but not that with IB9-37; however, it changes the binding stoichiometry. These differences can be attributed to the effect of EtOH on the conformation dynamics of the proteins and to changes in hydration properties in alcoholic solution.

High pressure treatment promotes the deteriorating effect of cationic antimicrobial peptides on bacterial membranes.

The helical structure that cationic antimicrobial peptides (cAMPs) adopt upon interaction with membranes is key to their activity. We show that a high hydrostatic pressure not only increases the propensity of cAMPs to adopt a helical conformation in the presence of bacterial lipid bilayer membranes, but also in bulk solution, and the effect on bacterial membranes persists even up to 10 kbar. Therefore, high-pressure treatment could boost cAMP activity in high-pressure food processing to extend the shelf-life of food.

Hidden intermediates in Mango III RNA aptamer folding revealed by pressure perturbation.

Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways. Here we use pressure as an alternative variable to probe the suboptimal states of the Mango III aptamer with both fluorescence and NMR spectroscopy approaches. At moderate KCl concentrations, increasing pressure disrupted the G-quadruplex structure of the Mango III RNA and led to an intermediate with lower fluorescence. These observations indicate the existence of suboptimal RNA structural states that still bind the TO1-biotin fluorophore and moderately enhance fluorescence. At higher KCl concentration as well, the intermediate fluorescence state was populated at high pressure, but the G-quadruplex remained stable at high pressure, supporting the notion of parallel folding and/or binding pathways. These results demonstrate the usefulness of pressure for characterizing RNA folding intermediates.

The N-terminal domain of the prion protein is required and sufficient for liquid-liquid phase separation: A crucial role of the Aß-binding domain.

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aß oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aß-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aß-binding domain in this process.

Modulation of the Conformational Space of SARS-CoV-2 RNA Quadruplex RG-1 by Cellular Components and the Amyloidogenic Peptides α-Synuclein and hIAPP.

Given the emergence of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which particularly threatens older people with comorbidities such as diabetes mellitus and dementia, understanding the relationship between Covid-19 and other diseases is an important factor for treatment. Possible targets for medical intervention include G-quadruplexes (G4Qs) and their protein interaction partners. We investigated the stability and conformational space of the RG-1 RNA-G-quadruplex of the SARS-CoV-2 N-gene in the presence of salts, cosolutes, crowders and intrinsically disordered peptides, focusing on α-Synuclein and the human islet amyloid polypeptide, which are involved in Parkinson's disease (PD) and type-II diabetes mellitus (T2DM), respectively. We found that the conformational dynamics of the RG-1 G4Q is strongly affected by the various solution conditions. Further, the amyloidogenic peptides were found to strongly modulate the conformational equilibrium of the RG-1. Considerable changes are observed with respect to their interaction with human telomeric G4Qs, which adopt different topologies. These results may therefore shed more light on the relationship between PD as well as T2DM and the SARS-CoV-2 disease and their molecular underpinnings. Since dysregulation of G4Q formation by rationally designed targeting compounds affects the control of cellular processes, this study should contribute to the development of specific ligands for intervention.

Suppression of Liquid-Liquid Phase Separation and Aggregation of Antibodies by Modest Pressure Application.

The high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid-liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity.

Deep sea osmolytes in action: their effect on protein-ligand binding under high pressure stress.

Because organisms living in the deep sea and in the sub-seafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding reactions, must be adjusted to keep the associated volume changes low in order to function efficiently. Almost all organisms use organic cosolvents (osmolytes) to protect their cells from adverse environmental conditions. They counteract osmotic imbalance, stabilize the structure of proteins and maintain their function. We studied the binding properties of the prototypical ligand proflavine to two serum proteins with different binding pockets, BSA and HSA, in the presence of two prominent osmolytes, trimethylamine-N-oxide (TMAO) and glycine betaine (GB). TMAO and GB play an important role in the regulation and adaptation of life in deep-sea organisms. To this end, pressure dependent fluorescence spectroscopy was applied, supplemented by circular dichroism (CD) spectroscopy and computer modeling studies. The pressure-dependent measurements were also performed to investigate the intimate nature of the complex formation in relation to hydration and packing changes caused by the presence of the osmolytes. We show that TMAO and GB are able to modulate the ligand binding process in specific ways. Depending on the chemical make-up of the protein's binding pocket and thus the thermodynamic forces driving the binding process, there are osmolytes with specific interaction sites and binding strengths with water that are able to mediate efficient ligand binding even under external stress conditions. In the binding of proflavine to BSA and HSA, the addition of both compatible osmolytes leads to an increase in the binding constant upon pressurization, with TMAO being the most efficient, rendering the binding process also insensitive to pressurization even up to 2 kbar as the volume change remains close to zero. This effect can be corroborated by the effects the cosolvents impose on the strength and dynamics of hydration water as well as on the conformational dynamics of the protein.

The C-terminus of the GKY20 antimicrobial peptide, derived from human thrombin, plays a key role in its membrane perturbation capability.

Previously, we characterized in detail the mechanism of action of the antimicrobial peptide GKY20, showing that it selectively perturbs the bacterial-like membrane employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. Here, we used a combination of biophysical techniques to similarly characterize the antimicrobial activity as well as the membrane perturbing capability of GKY10, a much shorter version of the GKY20 peptide. GKY10 is only half of the parent peptide and consists of the last 10 amino acids (starting from the C-terminus) of the full-length peptide. Despite a large difference in length, we found that GKY10, like the parent peptide, retains the ability to adopt a helical structure and to induce lipid segregation upon membrane binding. Overall, our results suggest that the amino acid sequence of GKY10 is responsible for most of the observed behaviors of GKY20. Our results shed further light on the mechanism of action of the full-length peptide and provide useful information for the design and development of new peptides that serve as antimicrobial agents.

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex.

Previous studies suggest that berberine, an isoquinoline alkaloid, has antiviral potential and is a possible therapeutic candidate against SARS-CoV-2. The molecular underpinnings of its action are still unknown. Potential targets include quadruplexes (G4Q) in the viral genome as they play a key role in modulating the biological activity of viruses. While several DNA-G4Q structures and their binding properties have been elucidated, RNA-G4Qs such as RG-1 of the N-gene of SARS-CoV-2 are less explored. Using biophysical techniques, the berberine binding thermodynamics and the associated conformational and hydration changes of RG-1 could be characterized and compared with human telomeric DNA-G4Q 22AG. Berberine can interact with both quadruplexes. Substantial changes were observed in the interaction of berberine with 22AG and RG-1, which adopt different topologies that can also change upon ligand binding. The strength of interaction and the thermodynamic signatures were found to dependent not only on the initial conformation of the quadruplex, but also on the type of salt present in solution. Since berberine has shown promise as a G-quadruplex stabilizer that can modulate viral gene expression, this study may also contribute to the development of optimized ligands that can discriminate between binding to DNA and RNA G-quadruplexes.

Biomolecular Condensates under Extreme Martian Salt Conditions.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

Towards DNA-Encoded Micellar Chemistry: DNA-Micelle Association and Environment Sensitivity of Catalysis.

The development of DNA-compatible reaction methodologies is a central theme to advance DNA-encoded screening library technology. Recently, we were able to show that sulfonic acid-functionalized block copolymer micelles facilitated Brønsted acid-promoted reactions such as the Povarov reaction on DNA-coupled starting materials with minimal DNA degradation. Here, the impact of polymer composition on micelle shape, and reaction conversion was investigated. A dozen sulfonic acid-functionalized block copolymers of different molar mass and composition were prepared by RAFT polymerization and were tested in the Povarov reaction, removal of the Boc protective group, and the Biginelli reaction. The results showed trends in the polymer structure-micellar catalytic activity relationship. For instance, micelles composed of block copolymers with shorter acrylate ester chains formed smaller particles and tended to provide faster reaction kinetics. Moreover, fluorescence quenching experiments as well as circular dichroism spectroscopy showed that DNA-oligomer-conjugates, although highly water-soluble, accumulated very effectively in the micellar compartments, which is a prerequisite for carrying out a DNA-encoded reaction in the presence of polymer micelles.

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.