Targeting phenylalanine assemblies as a prospective disease-modifying therapy for phenylketonuria.

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment.

Ordered planar plating/stripping enables deep cycling zinc metal batteries.

Metal anodes are emerging as culminating solutions for the development of energy-dense batteries in either aprotic, aqueous, or solid battery configurations. However, unlike traditional intercalation electrodes, the low utilization of "hostless" metal anodes due to the intrinsically disordered plating/stripping impedes their practical applications. Herein, we report ordered planar plating/stripping in a bulk zinc (Zn) anode to achieve an extremely high depth of discharge exceeding 90% with negligible thickness fluctuation and long-term stable cycling. The Zn can be plated/stripped with (0001)Zn preferential orientation throughout the consecutive charge/discharge process, assisted by a self-assembled supramolecular bilayer at the Zn anode-electrolyte interface. Through real-time tracking of the Zn atoms migration, we reveal that the ordered planar plating/stripping is driven by the construction of in-plane Zn─N bindings and the gradient energy landscape at the reaction fronts. The breakthrough results provide alternative insights into the ordered plating/stripping of metal anodes toward rechargeable energy-dense batteries.

Peptide hydrogen-bonded organic frameworks.

Hydrogen-bonded porous frameworks (HPFs) are versatile porous crystalline frameworks with diverse applications. However, designing chiral assemblies or biocompatible materials poses significant challenges. Peptide-based hydrogen-bonded porous frameworks (P-HPFs) are an exciting alternative to conventional HPFs due to their intrinsic chirality, tunability, biocompatibility, and structural diversity. Flexible, ultra-short peptide-based P-HPFs (composed of 3 or fewer amino acids) exhibit adaptable porous topologies that can accommodate a variety of guest molecules and capture hazardous greenhouse gases. Longer, folded peptides present challenges and opportunities in designing P-HPFs. This review highlights recent developments in P-HPFs using ultra-short peptides, folded peptides, and foldamers, showcasing their utility for gas storage, chiral recognition, chiral separation, and medical applications. It also addresses design challenges and future directions in the field.

Co-Assembly of Cancer Drugs with Cyclo-HH Peptides: Insights from Simulations and Experiments.

Peptide-based nanomaterials can serve as promising drug delivery agents, facilitating the release of active pharmaceutical ingredients while reducing the risk of adverse reactions. We previously demonstrated that Cyclo-Histidine-Histidine (Cyclo-HH), co-assembled with cancer drug Epirubicin, zinc, and nitrate ions, can constitute an attractive drug delivery system, combining drug self-encapsulation, enhanced fluorescence, and the ability to transport the drug into cells. Here, we investigated both computationally and experimentally whether Cyclo-HH could co-assemble, in the presence of zinc and nitrate ions, with other cancer drugs with different physicochemical properties. Our studies indicated that Methotrexate, in addition to Epirubicin and its epimer Doxorubicin, and to a lesser extent Mitomycin-C and 5-Fluorouracil, have the capacity to co-assemble with Cyclo-HH, zinc, and nitrate ions, while a significantly lower propensity was observed for Cisplatin. Epirubicin, Doxorubicin, and Methorexate showed improved drug encapsulation and drug release properties, compared to Mitomycin-C and 5-Fluorouracil. We demonstrated the biocompatibility of the co-assembled systems, as well as their ability to intracellularly release the drugs, particularly for Epirubicin, Doxorubicin, and Methorexate. Zinc and nitrate were shown to be important in the co-assembly, coordinating with drugs and/or Cyclo-HH, thereby enabling drug-peptide as well as drug-drug interactions in successfully formed nanocarriers. The insights could be used in the future design of advanced cancer therapeutic systems with improved properties.

Mind your marker: the effect of common auxotrophic markers on complex traits in yeast.

Yeast cells are extensively used as a key model organism owing to their highly conserved genome, metabolic pathways, and cell biology processes. To assist in genetic engineering and analysis, laboratory yeast strains typically harbor auxotrophic selection markers. When uncompensated, auxotrophic markers cause significant phenotypic bias compared to prototrophic strains and have different combinatorial influences on the metabolic network. Here, we used BY4741, a laboratory strain commonly used as a "wild type" strain in yeast studies, to generate a set of revertant strains, containing all possible combinations of four common auxotrophic markers (leu2∆, ura3∆, his3∆1, met15∆). We examined the effect of the auxotrophic combinations on complex phenotypes such as resistance to rapamycin, acetic acid, and ethanol. Among the markers, we found that leucine auxotrophy most significantly affected the phenotype. We analyzed the phenotypic bias caused by auxotrophy at the genomic level using a prototrophic version of a genome-wide deletion library and a decreased mRNA perturbation (DAmP) library. Prototrophy was found to suppress rapamycin sensitivity in many mutants previously annotated for the phenotype, raising a possible need for reevaluation of the findings in a native metabolic context. These results reveal a significant phenotypic bias caused by common auxotrophic markers and support the use of prototrophic wild-type strains in yeast research.

Preventing biofilm formation and eradicating pathogenic bacteria by Zn doped histidine derived carbon quantum dots.

Bacterial infections are of major medical concern due to antibiotic resistance. Carbon quantum dots (CDs) have emerged as potentially excellent biomaterials for multifunctional applications due to their low toxicity, outstanding water solubility, high fluorescence, and high biocompatibility. All of these properties allow CDs to be exceptional biomaterials for inhibiting the growth of bacteria and stopping biofilm formation due to their strong binding affinity, cell wall penetration, and solubilizing biofilm in water. Here, we describe a strategy for one-pot synthesis of histidine-derived zinc-doped N-doped CDs (Zn-NCDs) by a hydrothermal method for inhibiting the growth of both Gram-positive and Gram-negative bacteria without harming mammalian cells. The NCDs and Zn-NCDs showed uniform sizes (∼6 nm), crystallinity, good photostability, high quantum yield (76%), and long decay time (∼5 ns). We also studied their utilization for live cell bio-imaging and the antimicrobial properties towards the Gram-positive Staphylococcus aureus and the Gram-negative Pseudomonas aeruginosa. Importantly, the Zn-NCDs could penetrate the biofilm and bacterial cell wall to effectively inhibit the growth of bacteria and subsequently inhibit biofilm formation. Thus, the structure, chemical composition, and low toxicity properties of the newly-developed Zn-NCDs exemplify a promising novel method for the preparation of nano-level antibacterial drugs.

High Quantum Yield Amino Acid Carbon Quantum Dots with Unparalleled Refractive Index.

Carbon quantum dots (CQDs) are one of the most promising types of fluorescent nanomaterials due to their exceptional water solubility, excellent optical properties, biocompatibility, chemical inertness, excellent refractive index, and photostability. Nitrogen-containing CQDs, which include amino acid based CQDs, are especially attractive due to their high quantum yield, thermal stability, and potential biomedical applications. Recent studies have attempted to improve the preparation of amino acid based CQDs. However, the highest quantum yield obtained for these dots was only 44%. Furthermore, the refractive indices of amino acid derived CQDs were not determined. Here, we systematically explored the performance of CQDs prepared from all 20 coded amino acids using modified hydrothermal techniques allowing more passivation layers on the surface of the dots to optimize their performance. Intriguingly, we obtained the highest refractive indices ever reported for any CQDs. The values differed among the amino acids, with the highest refractive indices found for positively charged amino acids including arginine-CQDs (∼2.1), histidine-CQDs (∼2.0), and lysine-CQDs (∼1.8). Furthermore, the arginine-CQDs reported here showed a nearly 2-fold increase in the quantum yield (∼86%) and a longer decay time (∼8.0 ns) compared to previous reports. In addition, we also demonstrated that all amino acid based CQD materials displayed excitation-dependent emission profiles (from UV to visible) and were photostable, water-soluble, noncytotoxic, and excellent for high contrast live cell imaging or bioimaging. These results indicate that amino acid based CQD materials are high-refractive-index materials applicable for optoelectronic devices, bioimaging, biosensing, and studying cellular organelles in vivo. This extraordinary RI may be highly useful for exploring cellular elements with different densities.

The von Hippel-Lindau protein forms fibrillar amyloid assemblies that are mitigated by the anti-amyloid molecule Purpurin.

The von Hippel-Lindau protein (pVHL) is a tumor suppressor involved in oxygen regulation via dynamic nucleocytoplasmic shuttling. It plays a crucial role in cell survival by degrading hypoxia-inducible factors (HIFs). Mutations in the VHL gene cause angiogenic tumors, characterized as VHL syndrome. However, aggressive tumors involving wild-type pVHL have also been described but the underlying mechanism remains to be revealed. We have previously shown that pVHL possesses several short amyloid-forming motifs, making it aggregation-prone. In this study, using a series of biophysical assays, we demonstrated that a pVHL-derived fragment (pVHL104-140) that harbors the nuclear export motif and HIF binding site, forms amyloid-like fibrillar structures in vitro by following secondary-nucleation-based kinetics. The peptide also formed amyloids at acidic pH that mimics the tumor microenvironment. We, subsequently, validated the amyloid formation by pVHL in vitro. Using the Curli-dependent amyloid generator (C-DAG) expression system, we confirmed the amyloidogenesis of pVHL in bacterial cells. The pVHL amyloids are an attractive target for therapeutics of the VHL syndrome. Accordingly, we demonstrated in vitro that Purpurin is a potent inhibitor of pVHL fibrillation. The amyloidogenic behavior of wild-type pVHL and its inhibition provide novel insights into the molecular underpinning of the VHL syndrome and its possible treatment.

Bioinspired Amino Acid Based Materials in Bionanotechnology: From Minimalistic Building Blocks and Assembly Mechanism to Applications.

Inspired by natural hierarchical self-assembly of proteins and peptides, amino acids, as the basic building units, have been shown to self-assemble to form highly ordered structures through supramolecular interactions. The fabrication of functional biomaterials comprised of extremely simple biomolecules has gained increasing interest due to the advantages of biocompatibility, easy functionalization, and structural modularity. In particular, amino acid based assemblies have shown attractive physical characteristics for various bionanotechnology applications. Herein, we propose a review paper to summarize the design strategies as well as research advances of amino acid based supramolecular assemblies as smart functional materials. We first briefly introduce bioinspired reductionist design strategies and assembly mechanism for amino acid based molecular assembly materials through noncovalent interactions in condensed states, including self-assembly, metal ion mediated coordination assembly, and coassembly. In the following part, we provide an overview of the properties and functions of amino acid based materials toward applications in nanotechnology and biomedicine. Finally, we give an overview of the remaining challenges and future perspectives on the fabrication of amino acid based supramolecular biomaterials with desired properties. We believe that this review will promote the prosperous development of innovative bioinspired functional materials formed by minimalistic building blocks.

Molecular Engineering of Ordered Piezoelectric Sulfonic Acid-Containing Assemblies.

Sulfonic acid-containing bioorganic monomers with wide molecular designability and abundant hydrogen bonding sites hold great potential to design diverse functional biocrystals but have so far not been explored for piezoelectric energy harvesting applications due to the lack of strategies to break the centrosymmetry of their assemblies. Here, a significant molecular packing transformation from centrosymmetric into non-centrosymmetric conformation by the addition of an amide terminus in the sulfonic acid-containing bioorganic molecule is demonstrated, allowing a high electromechanical response. The amide-functionalized molecule self-assembles into a polar supramolecular parallel ß-sheet-like structure with a high longitudinal piezoelectric coefficient d11 = 15.9 pm V-1 that produces the maximal open-circuit voltage of >1 V and the maximal power of 18 nW in nanogenerator devices pioneered. By contrast, molecules containing an amino or a cyclohexyl terminus assemble into highly symmetric 3D hydrogen bonding diamondoid-like networks or 2D double layer structures that show tunable morphologies, thermostability, and mechanical properties but non-piezoelectricity. This work not only presents a facile approach to achieving symmetry transformation of bioorganic assemblies but also demonstrates the terminal group and the property correlation for tailor-made design of high-performance piezoelectric biomaterials.

Investigating and Modeling the Factors That Affect Genetic Circuit Performance.

Over the past 2 decades, synthetic biology has yielded ever more complex genetic circuits that are able to perform sophisticated functions in response to specific signals. Yet, genetic circuits are not immediately transferable to an outside-the-lab setting where their performance is highly compromised. We propose introducing a broader test step to the design-build-test-learn workflow to include factors that might contribute to unexpected genetic circuit performance. As a proof of concept, we have designed and evaluated a genetic circuit in various temperatures, inducer concentrations, nonsterilized soil exposure, and bacterial growth stages. We determined that the circuit's performance is dramatically altered when these factors differ from the optimal lab conditions. We observed significant changes in the time for signal detection as well as signal intensity when the genetic circuit was tested under nonoptimal lab conditions. As a learning effort, we then proceeded to generate model predictions in untested conditions, which is currently lacking in synthetic biology application design. Furthermore, broader test and learn steps uncovered a negative correlation between the time it takes for a gate to turn ON and the bacterial growth phases. As the synthetic biology discipline transitions from proof-of-concept genetic programs to appropriate and safe application implementations, more emphasis on test and learn steps (i.e., characterizing parts and circuits for a broad range of conditions) will provide missing insights on genetic circuit behavior outside the lab.

Branched-Chain Amino Acid Assembly into Amyloid-like Fibrils Provides a New Paradigm for Maple Syrup Urine Disease Pathology.

Inborn error of metabolism disorders (IEMs) are a family of diseases resulting from single-gene mutations that lead to the accumulation of metabolites that are usually toxic or interfere with normal cell function. The etiological link between metabolic alteration and the symptoms of IEMs is still elusive. Several metabolites, which accumulate in IEMs, were shown to self-assemble to form ordered structures. These structures display the same biophysical, biochemical, and biological characteristics as proteinaceous amyloid fibrils. Here, we have demonstrated, for the first time, the ability of each of the branched-chain amino acids (BCAAs) that accumulate in maple syrup urine disease (MSUD) to self-assemble into amyloid-like fibrils depicted by characteristic morphology, binding to indicative amyloid-specific dyes and dose-dependent cytotoxicity by a late apoptosis mechanism. We could also detect the presence of the assemblies in living cells. In addition, by employing several in vitro techniques, we demonstrated the ability of known polyphenols to inhibit the formation of the BCAA fibrils. Our study implies that BCAAs possess a pathological role in MSUD, extends the paradigm-shifting concept regarding the toxicity of metabolite amyloid-like structures, and suggests new pathological targets that may lead to highly needed novel therapeutic opportunities for this orphan disease.

Spin-induced electron transmission through metal-organic chiral crystals.

Metal-organic Co(II)-phenylalanine crystals were studied and were found to possess magnetic properties and long-range spin transport. Magnetic measurements confirmed that in the crystals there are antiferromagnetic interactions between Co(II) and the lattice. The metal-organic crystals (MOCs) also present the chirality-induced spin selectivity (CISS) effect at room temperature. A long-range spin polarization is observed using a magnetic conductive-probe atomic force microscope. The spin polarization is found to be in the range of 35-45%.

Optimization Strategies for Mass Spectrometry-Based Untargeted Metabolomics Analysis of Small Polar Molecules in Human Plasma.

The untargeted approach to mass spectrometry-based metabolomics has a wide potential to investigate health and disease states, identify new biomarkers for diseases, and elucidate metabolic pathways. All this holds great promise for many applications in biological and chemical research. However, the complexity of instrumental parameters on advanced hybrid mass spectrometers can make the optimization of the analytical method immensely challenging. Here, we report a strategy to optimize the selected settings of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for untargeted metabolomics studies of human plasma, as a sample matrix. Specifically, we evaluated the effects of the reconstitution solvent in the sample preparation procedure, the injection volume employed, and different mass spectrometry-related operating parameters including mass range, the number of data-dependent fragmentation scans, collision energy mode, duration of dynamic exclusion time, and mass resolution settings on the metabolomics data quality and output. This study highlights key instrumental variables influencing the detection of metabolites along with suggested settings for the IQ-X tribrid system and proposes a new methodological framework to ensure increased metabolome coverage.

Perspectives on recent advancements in energy harvesting, sensing and bio-medical applications of piezoelectric gels.

The development of next-generation bioelectronics, as well as the powering of consumer and medical devices, require power sources that are soft, flexible, extensible, and even biocompatible. Traditional energy storage devices (typically, batteries and supercapacitors) are rigid, unrecyclable, offer short-lifetime, contain hazardous chemicals and possess poor biocompatibility, hindering their utilization in wearable electronics. Therefore, there is a genuine unmet need for a new generation of innovative energy-harvesting materials that are soft, flexible, bio-compatible, and bio-degradable. Piezoelectric gels or PiezoGels are a smart crystalline form of gels with polar ordered structures that belongs to the broader family of piezoelectric material, which generate electricity in response to mechanical stress or deformation. Given that PiezoGels are structurally similar to hydrogels, they offer several advantages including intrinsic chirality, crystallinity, degree of ordered structures, mechanical flexibility, biocompatibility, and biodegradability, emphasizing their potential applications ranging from power generation to bio-medical applications. Herein, we describe recent examples of new functional PiezoGel materials employed for energy harvesting, sensing, and wound dressing applications. First, this review focuses on the principles of piezoelectric generators (PEGs) and the advantages of using hydrogels as PiezoGels in energy and biomedical applications. Next, we provide a detailed discussion on the preparation, functionalization, and fabrication of PiezoGel-PEGs (P-PEGs) for the applications of energy harvesting, sensing and wound healing/dressing. Finally, this review concludes with a discussion of the current challenges and future directions of P-PEGs.

Manipulating the Piezoelectric Response of Amino Acid-Based Assemblies by Supramolecular Engineering.

Variation in the molecular architecture significantly affects the electronic and supramolecular structure of biomolecular assemblies, leading to dramatically altered piezoelectric response. However, relationship between molecular building block chemistry, crystal packing and quantitative electromechanical response is still not fully understood. Herein, we systematically explored the possibility to amplify the piezoelectricity of amino acid-based assemblies by supramolecular engineering. We show that a simple change of side-chain in acetylated amino acids leads to increased polarization of the supramolecular arrangements, resulting in significant enhancement of their piezoelectric response. Moreover, compared to most of the natural amino acid assemblies, chemical modification of acetylation increased the maximum piezoelectric tensors. The predicted maximal piezoelectric strain tensor and voltage constant of acetylated tryptophan (L-AcW) assemblies reach 47 pm V-1 and 1719 mV m/N, respectively, comparable to commonly used inorganic materials such as bismuth triborate crystals. We further fabricated an L-AcW crystal-based piezoelectric power nanogenerator that produces a high and stable open-circuit voltage of over 1.4 V under mechanical pressure. For the first time, the illumination of a light-emitting diode (LED) is demonstrated by the power output of an amino acid-based piezoelectric nanogenerator. This work presents the supramolecular engineering toward the systematic modulation of piezoelectric response in amino acid-based assemblies, facilitating the development of high-performance functional biomaterials from simple, readily available, and easily tailored building blocks.

The Metabolostasis Network and the Cellular Depository of Aggregation-Prone Metabolites.

The vital role of metabolites across all branches of life and their involvement in various disorders have been investigated for decades. Many metabolites are poorly soluble in water or in physiological buffers and tend to form supramolecular aggregates. On the other hand, in the cell, they should be preserved in a pool and be readily available for the execution of biochemical functions. We thus propose that a quality-control network, termed "metabolostasis", has evolved to regulate the storage and retrieval of aggregation-prone metabolites. Such a system should control metabolite concentration, subcellular localization, supramolecular arrangement, and interaction in dynamic environments, thus enabling normal cellular physiology, healthy development, and preventing disease onset. The paradigm-shifting concept of metabolostasis calls for a reevaluation of the traditional view of metabolite storage and dynamics in physiology and pathology and proposes unprecedented directions for therapeutic targets under conditions where metabolostasis is imbalanced.

Peptide- and Metabolite-Based Hydrogels: Minimalistic Approach for the Identification and Characterization of Gelating Building Blocks.

Minimalistic peptide- and metabolite-based supramolecular hydrogels have great potential relative to traditional polymeric hydrogels in various biomedical and technological applications. Advantages such as remarkable biodegradability, high water content, favorable mechanical properties, biocompatibility, self-healing, synthetic feasibility, low cost, easy design, biological function, remarkable injectability, and multi-responsiveness to external stimuli make supramolecular hydrogels promising candidates for drug delivery, tissue engineering, tissue regeneration, and wound healing. Non-covalent interactions such as hydrogen bonding, hydrophobic interactions, electrostatic interactions, and π-π stacking interactions play key roles in the formation of peptide- and metabolite-containing low-molecular-weight hydrogels. Peptide- and metabolite-based hydrogels display shear-thinning and immediate recovery behavior due to the involvement of weak non-covalent interactions, making them supreme models for the delivery of drug molecules. In the areas of regenerative medicine, tissue engineering, pre-clinical evaluation, and numerous other biomedical applications, peptide- and metabolite-based hydrogelators with rationally designed architectures have intriguing uses. In this review, we summarize the recent advancements in the field of peptide- and metabolite-based hydrogels, including their modifications using a minimalistic building-blocks approach for various applications.

Self-Assembly of Cysteine into Nanofibrils Precedes Cystine Crystal Formation: Implications for Aggregation Inhibition.

The self-association of metabolites into well-ordered assemblies at the nanoscale has significant biological and medical implications. The thiol-containing amino acid cysteine (CYS) can assemble into amyloid-like nanofibrils, and its oxidized form, the disulfide-bonded cystine (CTE), forms hexagonal crystals as those found in cystinuria due to metabolic disorder. Yet, there have been no attempts to connect these two phenomena, especially the fibril-to-crystal transition. Here, we reveal that these are not separated events, and the CYS-forming amyloid fibrils are mechanistically linked to hexagonal CTE crystals. For the first time, we demonstrated that cysteine fibrils are a prerequisite for forming cystine crystals, as observed experimentally. To further understand this mechanism, we studied the effects of thiol-containing cystinuria drugs (tiopronin, TIO; and d-penicillamine, PEN) and the canonical epigallocatechin gallate (EGCG) amyloid inhibitor on fibril formation by CYS. The thiol-containing drugs do not solely interact with monomeric CYS via disulfide bond formation but can disrupt amyloid formation by targeting CYS oligomers. On the other hand, EGCG forms inhibitor-dominant complexes (more than one EGCG molecule per cysteine unit) to prevent CYS fibril formation. Interestingly, while CYS can be oxidized into CTE, the thiol drugs can reduce CTE back to CYS. We thus suggest that the formation of crystals in cystinuria could be halted at the initial stage by targeting CYS fibril formation as an alternative to solubilizing the water-insoluble hexagonal CTE crystals at a later stage. Taken together, we depicted a complex hierarchical organization in a simple amino acid assembly with implications for therapeutic intervention.

Rational Design of Biological Crystals with Enhanced Physical Properties by Hydrogen Bonding Interactions.

Hydrogen bonds are non-covalent interactions and essential for assembling supermolecules into ordered structures in biological systems, endowing crystals with fascinating physical properties, and inspiring the construction of eco-friendly electromechanical devices. However, the interplay between hydrogen bonding and the physical properties is not fully understood at the molecular level. Herein, we demonstrate that the physical property of biological crystals with double-layer structures could be enhanced by rationally controlling hydrogen bonding interactions between amino and carboxyl groups. Different hydrogen bonding interactions result in various thermal, mechanical, electronic, and piezoelectric properties. In particular, the weak interaction between O and H atoms contributes to low mechanical strength that permits important ion displacement under stress, giving rise to a strong piezoelectric response. This study not only reveals the correlation between the hydrogen bonding and physical properties in double-layer structures of biological crystals but also demonstrates the potential of these crystals as functional biomaterials for high-performance energy-harvesting devices. Theoretical calculations and experimental verifications in this work provide new insights into the rational design of biomaterials with desirable physical properties for bioelectrical devices by modulating intermolecular interactions.