The γ-Core Motif Peptides of AMPs from Grasses Display Inhibitory Activity against Human and Plant Pathogens

Antimicrobial peptides (AMPs) constitute an essential part of the plant immune system. They are regarded as alternatives to conventional antibiotics and pesticides. In this study, we have identified the γ-core motifs, which are associated with antimicrobial activity, in 18 AMPs from grasses and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. All the tested peptides displayed antimicrobial properties. We discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides designed from the 4-Cys-containing defensin-like peptides, whose role in plant immunity has remained unknown, as well as the knottin-like peptide and the C-terminal prodomain of the thionin, which points to the direct involvement of these peptides in defense mechanisms. Studies of the mode of action of the eight most active γ-core motif peptides on yeast cells using staining with propidium iodide showed that all of them induced membrane permeabilization leading to cell lysis. In addition to identification of the antimicrobial determinants in plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to control plant pathogens.     

Cationic Amphiphilic Peptides: Synthetic Antimicrobial Agents Inspired by Nature

Antimicrobial peptides are ubiquitous in multicellular organisms and have served as defense mechanisms for their successful evolution and throughout their life cycle. These peptides are short cationic amphiphilic polypeptides of fewer than 50 amino acids containing either a few disulfide-linked cysteine residues with a characteristic β-sheet-rich structure or linear α-helical conformations with hydrophilic side chains at one side of the helix and hydrophobic side chains on the other side. Antimicrobial peptides cause bacterial cell lysis either by direct cell-surface damage via electrostatic interactions between the cationic side chains of the peptide and the negatively charged cell surface, or by indirect modulation of the host defense systems. Electrostatic interactions lead to bacterial cell membrane disruption followed by leakage of cellular components and finally bacterial cell death. Because of their unusual mechanism of cell damage, antimicrobial peptides are effective against drug-resistant bacteria and may therefore prove more effective than classical antibiotics in certain cases. Currently, around 3000 natural antimicrobial peptides from six kingdoms (bacteria, archaea, protists, fungi, plants, and animals) have been isolated and sequenced. However, only a few of them are under clinical trials and/or in the commercial development stage for the treatment of bacterial infections caused by antibiotic-resistant bacteria. Moreover, high structural complexity, poor pharmacokinetic properties, and low antibacterial activity of natural antimicrobial peptides hinder their progress in drug development. To overcome these hurdles, researchers have become increasingly interested in modification and nature-inspired synthetic antimicrobial peptides. This review discusses some of the recent studies reported on antimicrobial peptides.     

Rational Design of Tryptophan‐Rich Antimicrobial Peptides with Enhanced Antimicrobial Activities and Specificities

Trp‐rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp‐rich peptides derived from the C‐terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A₂ (PLA₂), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK—designated PEM‐2—was found to display improved activity against both Gram‐positive and Gram‐negative bacteria. To improve the antimicrobial activity of PEM‐2 for potential clinical applications further, we determined the solution structure of PEM‐2 bound to membrane‐mimetic dodecylphosphocholine (DPC) micelles by two‐dimensional NMR methods. The DPC micelle‐bound structure of PEM‐2 adopts an α‐helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM‐2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM‐2 could be due to interactions with bacterial membranes. Potent analogues of PEM‐2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.     

Short Peptides Make a Big Difference: The Role of Botany-Derived AMPs in Disease Control and Protection of Human Health

Botany-derived antimicrobial peptides (BAMPs), a class of small, cysteine-rich peptides produced in plants, are an important component of the plant immune system. Both in vivo and in vitro experiments have demonstrated their powerful antimicrobial activity. Besides in plants, BAMPs have cross-kingdom applications in human health, with toxic and/or inhibitory effects against a variety of tumor cells and viruses. With their diverse molecular structures, broad-spectrum antimicrobial activity, multiple mechanisms of action, and low cytotoxicity, BAMPs provide ideal backbones for drug design, and are potential candidates for plant protection and disease treatment. Lots of original research has elucidated the properties and antimicrobial mechanisms of BAMPs, and characterized their surface receptors and in vivo targets in pathogens. In this paper, we review and introduce five kinds of representative BAMPs belonging to the pathogenesis-related protein family, dissect their antifungal, antiviral, and anticancer mechanisms, and forecast their prospects in agriculture and global human health. Through the deeper understanding of BAMPs, we provide novel insights for their applications in broad-spectrum and durable plant disease prevention and control, and an outlook on the use of BAMPs in anticancer and antiviral drug design.     

Plant Kunitz Inhibitors and Their Interaction with Proteases: Current and Potential Pharmacological Targets

The action of proteases can be controlled by several mechanisms, including regulation through gene expression; post-translational modifications, such as glycosylation; zymogen activation; targeting specific compartments, such as lysosomes and mitochondria; and blocking proteolysis using endogenous inhibitors. Protease inhibitors are important molecules to be explored for the control of proteolytic processes in organisms because of their ability to act on several proteases. In this context, plants synthesize numerous proteins that contribute to protection against attacks by microorganisms (fungi and bacteria) and/or invertebrates (insects and nematodes) through the inhibition of proteases in these organisms. These proteins are widely distributed in the plant kingdom, and are present in higher concentrations in legume seeds (compared to other organs and other botanical families), motivating studies on their inhibitory effects in various organisms, including humans. In most cases, the biological roles of these proteins have been assigned based mostly on their in vitro action, as is the case with enzyme inhibitors. This review highlights the structural evolution, function, and wide variety of effects of plant Kunitz protease inhibitors, and their potential for pharmaceutical application based on their interactions with different proteases.     

Mammalian Neuropeptides as Modulators of Microbial Infections: Their Dual Role in Defense versus Virulence and Pathogenesis

The regulation of infection and inflammation by a variety of host peptides may represent an evolutionary failsafe in terms of functional degeneracy and it emphasizes the significance of host defense in survival. Neuropeptides have been demonstrated to have similar antimicrobial activities to conventional antimicrobial peptides with broad-spectrum action against a variety of microorganisms. Neuropeptides display indirect anti-infective capacity via enhancement of the host’s innate and adaptive immune defense mechanisms. However, more recently concerns have been raised that some neuropeptides may have the potential to augment microbial virulence. In this review we discuss the dual role of neuropeptides, perceived as a double-edged sword, with antimicrobial activity against bacteria, fungi, and protozoa but also capable of enhancing virulence and pathogenicity. We review the different ways by which neuropeptides modulate crucial stages of microbial pathogenesis such as adhesion, biofilm formation, invasion, intracellular lifestyle, dissemination, etc., including their anti-infective properties but also detrimental effects. Finally, we provide an overview of the efficacy and therapeutic potential of neuropeptides in murine models of infectious diseases and outline the intrinsic host factors as well as factors related to pathogen adaptation that may influence efficacy.     

Synthesis and in Silico Modelling of the Potential Dual Mechanistic Activity of Small Cationic Peptides Potentiating the Antibiotic Novobiocin against Susceptible and Multi-Drug Resistant Escherichia coli

Cationic antimicrobial peptides have attracted interest, both as antimicrobial agents and for their ability to increase cell permeability to potentiate other antibiotics. However, toxicity to mammalian cells and complexity have hindered development for clinical use. We present the design and synthesis of very short cationic peptides (3–9 residues) with potential dual bacterial membrane permeation and efflux pump inhibition functionality. Peptides were designed based upon in silico similarity to known active peptides and efflux pump inhibitors. A number of these peptides potentiate the activity of the antibiotic novobiocin against susceptible Escherichia coli and restore antibiotic activity against a multi-drug resistant E. coli strain, despite having minimal or no intrinsic antimicrobial activity. Molecular modelling studies, via docking studies and short molecular dynamics simulations, indicate two potential mechanisms of potentiating activity; increasing antibiotic cell permeation via complexation with novobiocin to enable self-promoted uptake, and binding the E. coli RND efflux pump. These peptides demonstrate potential for restoring the activity of hydrophobic drugs.     

Prediction and Activity of a Cationic α-Helix Antimicrobial Peptide ZM-804 from Maize

Antimicrobial peptides (AMPs) are small molecules consisting of less than fifty residues of amino acids. Plant AMPs establish the first barrier of defense in the innate immune system in response to invading pathogens. The purpose of this study was to isolate new AMPs from the Zea mays L. inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria. In silico, the Collection of Anti-Microbial Peptides (CAMP_R3), a computational AMP prediction server, was used to screen a cDNA library for AMPs. A ZM-804 peptide, isolated from the Z. mays L. inbred line B73 cDNA library, was predicted as a new cationic AMP with high prediction values. ZM-804 was tested against eleven pathogens of Gram-negative and Gram-positive bacteria and exhibited high antimicrobial activities as determined by the minimal inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). A confocal laser scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes. SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato (Pst) DC3000 caused by ZM-804. In planta, ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by Pst DC3000. Moreover, four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response (HR) in tobacco leaves in response to low ZM-804 concentrations. ZM-804 exhibits low hemolytic activity against mouse red blood cells (RBCs) and is relatively safe for mammalian cells. In conclusion, the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control. Additionally, the ZM-804 peptide is considered a promising candidate for human and animal drug development.     

Dissecting the Binding Interactions of Teixobactin with the Bacterial Cell-Wall Precursor Lipid II

The prevalence of life-threatening, drug-resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered “resistance-proof” antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram-positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram-positive and Gram-negative bacteria. Furthermore, we show that when combined with known outer membrane-disrupting peptides, teixobactin is active against Gram-negative organisms.     

Isolation of Antimicrobial Genes from Oryza rufipogon Griff by Using a Bacillus subtilis Expression System with Potential Antimicrobial Activities

Antimicrobial genes are distributed in all forms of life and provide a primary defensive shield due to their unique broad-spectrum resistance activities. To better isolate these genes, we used the Bacillus subtilis expression system as the host cells to build Oryza rufipogon Griff cDNA libraries and screen potential candidate genes from the library at higher flux using built-in indicator bacteria. We observed that the antimicrobial peptides OrR214 and OrR935 have strong antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria, as well as several fungal pathogens. Owing to their high thermal and enzymatic stabilities, these two peptides can also be used as field biocontrol agents. Furthermore, we also found that the peptide OrR214 (MIC 7.7–10.7 μM) can strongly inhibit bacterial growth compared to polymyxin B (MIC 5–25 μM) and OrR935 (MIC 33–44 μM). The cell flow analysis, reactive oxygen burst, and electron microscopy (scanning and transmission electron microscopy) observations showed that the cell membranes were targeted by peptides OrR214 and OrR935, which revealed the mode of action of bacteriostasis. Moreover, the hemolytic activity, toxicity, and salt sensitivity experiments demonstrated that these two peptides might have the potential to be used for clinical applications. Overall, OrR214 and OrR935 antimicrobial peptides have a high-throughput bacteriostatic activity that acts as a new form of antimicrobial agent and can be used as a raw material in the field of drug development.     

New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.     

The Development of Antimicrobial α‐AApeptides that Suppress Proinflammatory Immune Responses

Herein we describe the development of a new class of antimicrobial and anti‐inflammatory peptidomimetics: cyclic lipo‐α‐AApeptides. They have potent and broad‐spectrum antibacterial activity against a range of clinically relevant pathogens, including both multidrug‐resistant Gram‐positive and Gram‐negative bacteria. Fluorescence microscopy suggests that cyclic lipo‐α‐AApeptides kill bacteria by disrupting bacterial membranes, possibly through a mechanism similar to that of cationic host‐defense peptides (HDPs). Furthermore, the cyclic lipo‐α‐AApeptide can mimic cationic host‐defense peptides by antagonizing Toll‐like receptor 4 (TLR4) signaling responses and suppressing proinflammatory cytokines such as tumor necrosis factor‐α (TNF‐α). Our results suggest that by mimicking HDPs, cyclic lipo‐α‐AApeptides could emerge as a new class of antibiotic agents that directly kill bacteria, as well as novel antiinflammatory agents that act through immunomodulation.     

Short Antimicrobial Lipo‐α/γ‐AA Hybrid Peptides

The last two decades have seen the rise of antimicrobial peptides (AMPs) to combat emerging antibiotic resistance. Herein we report the solid‐phase synthesis of short lipidated α/γ‐AA hybrid peptides. This family of lipo‐chimeric peptidomimetics displays potent and broad‐spectrum antimicrobial activity against a range of multi‐drug resistant Gram‐positive and Gram‐negative bacteria. These lipo‐α/γ‐AA hybrid peptides also demonstrate high biological specificity, with no hemolytic activity towards red blood cells. Fluorescence microscopy suggests that these lipo‐α/γ‐AA chimeric peptides can mimic the mode of action of AMPs and kill bacterial pathogens via membrane disintegration. As the composition of these chimeric peptides is simple, therapeutic development could be economically feasible and amenable for a variety of antimicrobial applications.     

Development of Bactericidal Peptides against Multidrug-Resistant Acinetobacter baumannii with Enhanced Stability and Low Toxicity

Pathogenic superbugs are the root cause of untreatable complex infections with limited or no treatment options. These infections are becoming more common as clinical antibiotics have lost their effectiveness over time. Therefore, the development of novel antibacterial agents is urgently needed to counter these microbes. Antimicrobial peptides (AMPs) are a viable treatment option due to their bactericidal potency against multiple microbial classes. AMPs are naturally selected physiological microbicidal agents that are found in all forms of organisms. In the present study, we developed two tilapia piscidin 2 (TP2)-based AMPs for antimicrobial application. Unlike the parent peptide, the redesigned peptides showed significant antimicrobial activity against multidrug-resistant bacterial species. These peptides also showed minimal cytotoxicity. In addition, they were significantly active in the presence of physiological salts, 50% human serum and elevated temperature. The designed peptides also showed synergistic activity when combined with clinical antibiotics. The current approach demonstrates a fruitful strategy for developing potential AMPs for antimicrobial application. Such AMPs have potential for progression to further trials and drug development investigations.     

Bio-Engineered Nisin with Increased Anti-Staphylococcus and Selectively Reduced Anti-Lactococcus Activity for Treatment of Bovine Mastitis

Bovine mastitis is a significant economic burden for dairy enterprises, responsible for premature culling, prophylactic and therapeutic antibiotic use, reduced milk production and the withholding (and thus wastage) of milk. There is a desire to identify novel antimicrobials that are expressly directed to veterinary applications, do not require a lengthy milk withholding period and that will not have a negative impact on the growth of lactic acid bacteria involved in downstream dairy fermentations. Nisin is the prototypical lantibiotic, a family of highly modified antimicrobial peptides that exhibit potent antimicrobial activity against many Gram-positive microbes, including human and animal pathogens including species of Staphylococcus and Streptococcus. Although not yet utilized in the area of human medicine, nisin is currently applied as the active agent in products designed to prevent bovine mastitis. Over the last decade, we have harnessed bioengineering strategies to boost the specific activity and target spectrum of nisin against several problematic microorganisms. Here, we screen a large bank of engineered nisin derivatives to identify novel derivatives that exhibit improved specific activity against a selection of staphylococci, including mastitis-associated strains, but have unchanged or reduced activity against dairy lactococci. Three such peptides were identified; nisin A M17Q, nisin A T2L and nisin A HTK.     

Structure–Activity Relationship of New Chimeric Analogs of Mastoparan from the Wasp Venom Paravespula lewisii

Mastoparan (MP) is an antimicrobial cationic tetradecapeptide with the primary structure INLKALAALAKKIL-NH₂. This amphiphilic α-helical peptide was originally isolated from the venom of the wasp Paravespula lewisii. MP shows a variety of biological activities, such as inhibition of the growth of Gram-positive and Gram-negative bacteria, as well as hemolytic activity and activation of mast cell degranulation. Although MP appears to be toxic, studies have shown that its analogs have a potential therapeutic application as antimicrobial, antiviral and antitumor agents. In the present study we have designed and synthesized several new chimeric mastoparan analogs composed of MP and other biologically active peptides such as galanin, RNA III inhibiting peptide (RIP) or carrying benzimidazole derivatives attached to the ε-amino side group of Lys residue. Next, we compared their antimicrobial activity against three reference bacterial strains and conformational changes induced by membrane-mimic environments using circular dichroism (CD) spectroscopy. A comparative analysis of the relationship between the activity of peptides and the structure, as well as the calculated physicochemical parameters was also carried out. As a result of our structure–activity study, we have found two analogs of MP, MP-RIP and RIP-MP, with interesting properties. These two analogs exhibited a relatively high antibacterial activity against S. aureus compared to the other MP analogs, making them a potentially attractive target for further studies. Moreover, a comparative analysis of the relationship between peptide activity and structure, as well as the calculated physicochemical parameters, may provide information that may be useful in the design of new MP analogs.     

A Novel Antimicrobial Peptide Spampcin56–86 from Scylla paramamosain Exerting Rapid Bactericidal and Anti-Biofilm Activity In Vitro and Anti-Infection In Vivo

The abuse of antibiotics leads to the increase of bacterial resistance, which seriously threatens human health. Therefore, there is an urgent need to find effective alternatives to antibiotics, and antimicrobial peptides (AMPs) are the most promising antibacterial agents and have received extensive attention. In this study, a novel potential AMP was identified from the marine invertebrate Scylla paramamosain and named Spampcin. After bioinformatics analysis and AMP database prediction, four truncated peptides (Spa31, Spa22, Spa20 and Spa14) derived from Spampcin were screened, all of which showed potent antimicrobial activity with different antibacterial spectrum. Among them, Spampcin_56–86 (Spa31 for short) exhibited strong bactericidal activity against a variety of clinical pathogens and could rapidly kill the tested bacteria within minutes. Further analysis of the antibacterial mechanism revealed that Spa31 disrupted the integrity of the bacterial membrane (as confirmed by scanning electron microscopy observation, NPN, and PI staining assays), leading to bacterial rupture, leakage of cellular contents (such as elevated extracellular ATP), increased ROS production, and ultimately cell death. Furthermore, Spa31 was found to interact with LPS and effectively inhibit bacterial biofilms. The antibacterial activity of Spa31 had good thermal stability, certain ion tolerance, and no obvious cytotoxicity. It is worth noting that Spa31 could significantly improve the survival rate of zebrafish Danio rerio infected with Pseudomonas aeruginosa, indicating that Spa31 played an important role in anti-infection in vivo. This study will enrich the database of marine animal AMPs and provide theoretical reference and scientific basis for the application of marine AMPs in medical fields.     

Carnivore Attractant or Plant Elicitor? Multifunctional Roles of Methyl Salicylate Lures in Tomato Defense

Synthetic plant volatile lures attract natural enemies, but may have non-target effects due to the multifunctional nature of volatile signals. For example, methyl salicylate (MeSA) is used to attract predators, yet also serves as a signaling hormone involved in plant pathogen defense. We investigated the consequences of deploying MeSA lures to attract predators for tomato (Solanum lycopersicum) defense against herbivores. To understand the spatial distribution of the lure’s effect, we exposed tomatoes in the field to MeSA along a linear distance gradient and induced defenses by simulating feeding by hornworm caterpillars in a fully crossed factorial design (+/? MeSA, +/? herbivory). Subsequently, we analyzed activity of several defensive proteins (protease inhibitors, polyphenol oxidase, peroxidase), development of hornworm larvae (Manduca sexta), growth of fungal pathogens (Cladosporium and Alternaria), and attractiveness to herbivores and predators. Overall, MeSA-exposed plants were more resistant to both insects and pathogens. Secondary pathogen infection was reduced by 25% in MeSA exposed plants, possibly due to elevated polyphenol oxidase activity. Interestingly, we found that lures affected plant pathogen defenses equivalently across all distances (up to 4 m away) indicating that horizontal diffusion of a synthetic volatile may be greater than previously assumed. While thrips avoided colonizing hornworm– damaged tomato plants, this induced resistance was not observed upon pre-exposure to MeSA, suggesting that MeSA suppresses the repellant effect induced by herbivory. Thus, using MeSA lures in biological control may inadvertently protect crops from pathogens, but has mixed effects on plant resistance to insect herbivores.     

Comparative Antimicrobial Activity of Hp404 Peptide and Its Analogs against Acinetobacter baumannii

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion Heterometrus petersii. Hp1404 exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus but is cytotoxic. In this study, we designed antimicrobial peptides by substituting amino acids at the 14 C-terminal residues of Hp1404 to reduce toxicity and improve antibacterial activity. The analog peptides, which had an amphipathic α-helical structure, were active against gram-positive and gram-negative bacteria, particularly multidrug-resistant Acinetobacter baumannii, and showed lower cytotoxicity than Hp1404. N-phenyl-1-naphthylamine uptake and DisC₃-5 assays demonstrated that the peptides kill bacteria by effectively permeating the outer and cytoplasmic membranes. Additionally, the analog peptides inhibited biofilm formation largely than Hp1404 at low concentrations. These results suggest that the analog peptides of Hp1404 can be used as therapeutic agents against A. baumannii infection.     

Activity and Synergy of Cu-ATCUN Antimicrobial Peptides

Antibiotic resistance demands innovative strategies and therapies. The pairs of antimicrobial peptides tested in this work show broad-spectrum synergy and are capable of interacting with diverse bacterial membranes. In most cases, the ATCUN motif enhanced the activity of peptides tested in combination. Our studies also show CP10A to be a multifaceted peptide, displaying both cell membrane and intracellular activity and acting as a chameleon, improving the activity of other peptides as needed. The results of the synergy experiments demonstrate the importance of varied modes of action and how these changes can affect the ability to combat pathogens, while also illustrating the value of the metal-binding domain in enhancing the activity of antimicrobial peptides in combination.