Europium Ion-Based Magnetic-Trapping and Fluorescence-Sensing Method for Detection of Pathogenic Bacteria.

Europium ions (Eu3+) have been utilized as a fluorescence-sensing probe for a variety of analytes, including tetracycline (TC). When Eu3+ is chelated with TC, its fluorescence can be greatly enhanced. Moreover, Eu3+ possesses 6 unpaired electrons in its f orbital, which makes it paramagnetic. Being a hard acid, Eu3+ can chelate with hard bases, such as oxygen-containing functional groups (e.g., phosphates and carboxylates), present on the cell surface of pathogenic bacteria. Due to these properties, in this study, Eu3+ was explored as a magnetic-trapping and sensing probe against pathogenic bacteria present in complex samples. Eu3+ was used as a magnetic probe to trap bacteria such as Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Bacillus cereus, and Pseudomonas aeruginosa. The addition of TC facilitated the easy detection of magnetic Eu3+-bacterium conjugates through fluorescence spectroscopy, with a detection limit of approximately ∼104 CFU mL-1. Additionally, matrix-assisted laser desorption/ionization mass spectrometry was employed to differentiate bacteria tapped by our magnetic probes.

Biofouling inhibition by Staphylococcus aureus extracts and their potential use for paints / Inhibición de la bioincrustación por extractos de Staphylococcus aureus y su uso potencial para pinturas

For the control of biofouling, some paints based on compounds that are toxic to marine organisms have been used. There is an intensive search for biodegradable solutions that are friendly to non-target organisms. Bacteria have been shown to be a source of compounds with antifouling potential. In this work, the antifouling activity of a strain of Staphylococcus aureus was evaluated. Extracts activity against biofilm-forming bacteria and the toxicity against Artemia franciscana were evaluated. The extracts were incorporated in a hard gel and a paint matrix, and they were exposed to the sea. In both the laboratory and field, we found that the compounds produced by S. aureus have antifouling activity. The non-toxicity of the tested extracts against Artemia franciscana nauplii suggests that the extracts obtained from S. aureus could have a low ecological impact over non-target organisms. Significant differences were found in the percentage of organisms cover in hard gels with extracts and control. After 90 days, important differences were also observed between the percentage of organisms cover of the paints that contained extracts and the control. Dichloromethane extract is the most effective for the inhibition or delay of the settlement of organisms For this reason, they could be used in matrices with different applications, such as in the shipping industry, aquaculture, or any other in which biofouling is a cause of inconvenience.(AU)

Rapid and ultra-sensitive lateral flow assay for pathogens based on multivalent aptamer and magnetic nanozyme.

Ultra-sensitive LFA methods for pathogen detection commonly depended on tedious and time-consuming nucleic acid amplification. Here, a high affinity multivalent aptamer (multi-Apt) for S. aureus was obtained through exquisite engineering design. The scaffold and conformation of the multi-Apt were found to be key factors in the detection signal of aptsensors. After optimization, the binding affinity of the multi-Apt to S. aureus was improved by more than 8-fold from 135.9 nM to 16.77 nM. By the joint use of the multi-Apt and a multifunctional nanozyme Fe3O4@MOF@PtPd, a fast and ultra-sensitive LFA for S. aureus was developed (termed MA-MN LFA). In this method, a Fe3O4@MOF@PtPd nanozyme was modified with vancomycin and could efficiently capture and separate S. aureus. Moreover, the multi-Apt worked together with the nanozyme to bind with S. aureus to form a ternary complex at the same time, which simply the fabrication of LFA strip. The developed MA-MN LFA could detect S. aureus as low as 2 CFU/mL within 30 min and a wide linear range of 10-1 × 108 CFU/mL was obtained. The detection is easily operated, fast (can be completed within 30 min) and versatile for Gram-positive pathogens, thus has great potential as a powerful tool in pathogen detection.

"Five birds one stone" tri-modal monitoring driven lab-on-magnetic aptasensor for accurate pathogen detection and enhanced germicidal application.

The effective combination of ultra-precise detection and on-demand sterilization stands out as one of the most valuable antifouling methods to combat pathogenic bacteria source and ensure the environment and food safety. Herein, an innovative "five birds one stone" aptasensor has been reported. It integrates magnetic separation, tri-modal precision detection, and efficient sterilization for monitoring of Staphylococcus aureus. Firstly, as a switch of the aptasensor, aptamer-modified potassium chloride-doped carbon dots (apt/KCl@CDs) could be adsorbed onto the surface of magnetic multi-walled carbon nanotube composites (M-MWCNTs) through π-π stacking, which could be replaced by the specific binding of the target bacteria to the aptamer. The mutual interference between the nanomaterials could be eliminated by this reverse magnetosorption strategy, enhancing the test sensitivity. In addition to the fluorescence properties, the peroxidase activity possessed by apt/KCl@CDs enables the conversion of the (3,3',5,5'-tetramethylbenzidine) TMB-H2O2 colorimetric system to a photothermal modal. Then, the ultra-precision detection in the assay was achieved by the fluorescence-colorimetric-photothermal tri-modal sensing from the formation of S. aureus-apt/KCl@CDs in the supernatant. Besides, the efficient sterilization could be ensured by adsorbing the apt/KCl@CDs on the surface of S. aureus, generating toxic •OH for direct attacking cells. This was the first report that was more beneficial for bacterial eradication. The detection limits of fluorescence, colorimetric and photothermal modals were 4.81 cfu/mL, 3.40 cfu/mL and 6.74 cfu/mL, respectively. The magnetic nanoplatform integrating tri-modal detection-sterilization meets the demand for highly sensitive and precise detection in different scenarios, providing immediate control for pathogens and broad application prospects.

Double phage displayed peptides co-targeting-based biosensor with signal enhancement activity for colorimetric detection of Staphylococcus aureus.

The development of simple, fast, sensitive, and specific strategies for the detection of foodborne pathogenic bacteria is crucial for ensuring food safety and promoting human health. Currently, detection methods for Staphylococcus aureus still suffer from issues such as low specificity and low sensitivity. To address this problem, we proposed a sensitivity enhancement strategy based on double phage-displayed peptides (PDPs) co-targeting. Firstly, we screened two PDPs and analyzed their binding mechanisms through fluorescent localization, pull-down assay, and molecular docking. The two PDPs target S. aureus by binding to specific proteins on its outer membrane. Based on this phenomenon, a convenient and sensitive double PDPs colorimetric biosensor was developed. Double thiol-modified phage-displayed peptides (PDP-SH) enhance the aggregation of gold nanoparticles (AuNPs), whereas the specific interaction between the double PDPs and bacteria inhibits the aggregation of AuNPs, resulting in an increased visible color change before and after the addition of bacteria. This one-step colorimetric approach displayed a high sensitivity of 2.35 CFU/mL and a wide detection range from 10-2 × 108 CFU/mL. The combination with smartphone-based image analysis improved the portability of this method. This strategy achieves the straightforward, highly sensitive and portable detection of pathogenic bacteria.

Characterization of an Autoinducing Peptide Signal Reveals Highly Efficacious Synthetic Inhibitors and Activators of Quorum Sensing and Biofilm Formation in Listeria monocytogenes.

Bacteria can use chemical signals to assess their local population density in a process called quorum sensing (QS). Many of these bacteria are common pathogens, including Gram-positive bacteria that utilize agr QS systems regulated by macrocyclic autoinducing peptide (AIP) signals. Listeria monocytogenes, an important foodborne pathogen, uses an agr system to regulate a variety of virulence factors and biofilm formation, yet little is known about the specific roles of agr in Listeria infection and its persistence in various environments. Herein, we report synthetic peptide tools that will enable the study of QS in Listeria. We identified a 6-mer AIP signal in L. monocytogenes supernatants and selected it as a scaffold around which a collection of non-native AIP mimics was designed and synthesized. These peptides were evaluated in cell-based agr reporter assays to generate structure-activity relationships for AIP-based agonism and antagonism in L. monocytogenes. We discovered synthetic agonists with increased potency relative to native AIP and a synthetic antagonist capable of reducing agr activity to basal levels. Notably, the latter peptide was able to reduce biofilm formation by over 90%, a first for a synthetic QS modulator in wild-type L. monocytogenes. The lead agr agonist and antagonist in L. monocytogenes were also capable of antagonizing agr signaling in the related pathogen Staphylococcus aureus, further extending their utility and suggesting different mechanisms of agr activation in these two pathogens. This study represents an important first step in the application of chemical methods to modulate QS and concomitant virulence outcomes in L. monocytogenes.

Smart Copolymer Surface Derived from Geminized Cationic Amphiphilic Polymers for Reversibly Switchable Bactericidal and Self-Cleaning Abilities.

Bacterial adhesion and colonization on material surfaces pose a serious problem for healthcare-associated devices. Cationic amphiphilic polymer brushes are usually used as surface coatings in antibacterial materials to endow an interface with excellent bactericidal efficiency, but they are easily contaminated, which puts a great limitation on their application. Herein, novel antibacterial copolymer brush surfaces containing geminized cationic amphiphilic polymers (pAGC8) and thermoresponsive poly(N-isopropylacrylamide) polymers (pNIPAm) have been synthesized. Surface functionalization of polymer brushes was investigated by X-ray photoelectron spectroscopy, spectroscopic ellipsometry, atomic force microscopy, and water contact angle measurements. A proportion of AGC8 and NIPAm units in copolymer brushes has been adjusted to obtain a high-efficiency bactericidal surface with minimal interference to its self-cleaning property. The killing and releasing efficiency of the optimized surface simultaneously reached up to above 80% for both Staphylococcus aureus and Escherichia coli bacteria, and the bactericidal and self-cleaning abilities are still excellent even after three kill-release cycles. Such a novel copolymer brush system provides innovative guidance for the development of high-efficiency antibacterial materials in biomedical application.

1H, 13C, and 15N resonance assignments of SarA monomer from Staphylococcus aureus in complex with DNA.

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarAΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1H, 13C and 15N NMR assignment of SarAΔN19/DNA complex which is the first step towards further structure and function analysis.

Quantitative Determination of Staphylococcus aureus Using Aptamer-Based Recognition and DNA Amplification Machinery.

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that threatens human health and safety. It is significant to develop sensitive detection methods for the monitoring of S. aureus contamination in food and environment. Herein, a novel machinery based on aptamer recognition, DNA walker, and rolling circle amplification (RCA) was designed, which can form unique DNA nanoflower and subsequently detect low-level S. aureus contamination in samples. To this end, two rationally designed DNA duplexes were modified on the surface of the electrode to identify S. aureus through the high affinity between aptamers and S. aureus. Combined with the repeated movement of DNA walker machinery on the electrode surface and RCA technology, a unique DNA nanoflower structure was formed. This can effectively transform the biological information of aptamer recognition of S. aureus into a significantly amplified electrochemical signal. Through reasonable design and optimization of the parameters of each part, the linear response range of the S. aureus biosensor is from 60 to 6 × 107 CFU/mL and the detection limit is as low as 9 CFU/mL.

Structural insights into CodY activation and DNA recognition.

Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers.

Iron-regulated surface determinant protein A (IsdA) is a key surface protein found in the foodborne bacteria─Staphylococcus aureus (S. aureus)─which is known to be critical for bacterial survival and colonization. S. aureus is pathogenic and has been linked to foodborne diseases; thus, early detection is critical to prevent diseases caused by this bacterium. Despite IsdA being a specific marker for S. aureus and several detection methods have been developed for sensitive detection of this bacteria such as cell culture, nucleic acids amplification, and other colorimetric and electrochemical methods, the detection of S. aureus through IsdA is underdeveloped. Here, by combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we presented a widely applicable and robust detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch a FRET construct to a high-FRET state in the presence of protein was verified. The presented approach demonstrated the detection of IsdA down to picomolar levels (×10-12 M, equivalent to ∼1.1 femtomoles IsdA) with a dynamic range extending to ∼40 nM. The FRET-based single-molecule technique that we reported here is capable of detecting the foodborne pathogen protein IsdA with high sensitivity and specificity and has a broader application in the food industry and aptamer-based sensing field by enabling quantitative detection of a wide range of pathogen proteins.

Complete non-proline backbone resonance assignments of the S. aureus neutrophil serine protease inhibitor, EapH1.

The S. aureus extracellular adherence protein (Eap) and its homologs, EapH1 and EapH2, serve roles in evasion of the human innate immune system. EapH1 binds with high-affinity and inhibits the neutrophil azurophilic granule proteases neutrophil elastase, cathepsin-G and proteinase-3. Previous structural studies using X-ray crystallography have shown that EapH1 binds to neutrophil elastase and cathepsin-G using a globally similar binding mode. However, whether the same holds true in solution is unknown and whether the inhibitor experiences dynamic changes following binding remains uncertain. To facilitate solution-phase structural and biochemical studies of EapH1 and its complexes with neutrophil granule proteases, we have characterized EapH1 by multidimensional NMR spectroscopy. Here we report a total of 100% of the non-proline backbone resonance assignments of EapH1 with BMRB accession number 50,304.

Antibiotic-enzyme-inorganic nanoflowers based immunoassay for the ultrasensitive detection of Staphylococcus aureus.

In this work, we constructed a moderate and convenient approach for the determination of staphylococcus aureus (S. aureus) by using organic-inorganic flower-like hybrid nanoflowers and Pig IgG together in an enzyme-linked immunosorbent assay (ELISA) system. To ensure efficient capture, the hybrid nanoflowers were prepared by encapsulating horseradish peroxidase (HRP) and vancomycin (VAN) in the inorganic nanocrystal composites (calcium ion solution), just like the mimic biomineralization process. Owing to the self-assembly technique, the synthesized VAN-HRP-CaHPO4 nanoflowers (NFs) can not only retain the ability to particularly capture the gram-positive bacteria but also enhance the stability and enzymatic activity to achieve the signal output amplification. Then, taking advantage of the integration of signal amplification elements (HRP) and biorecognition unit (VAN), the VAN-HRP-CaHPO4 NFs were utilized as a new kind of capture & signal regent in the procedure of S. aureus detection. Based on this ELISA system, S. aureus could be clearly detected within the concentration ranging from 1.0 × 102 to 1.0 × 107 CFU mL-1. The detection limit was defined as 4.3 CFU mL-1, which performance is superior to some commercial ELISA kits. Additionally, this system detected the S. aureus in food samples and showed an acceptable recovery. As a cost-effective and sensitive platform, this proposed assay was enable to fulfill the requirement of a quick and effective detection of S. aureus.

Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

Identification of potential Staphylococcus aureus dihydrofolate reductase inhibitors using QSAR, molecular docking, dynamics simulations and free energy calculation.

Herein we describe the use of molecular docking simulations, quantitative structure-activity relationships studies and ADMETox predictions to analyse the molecular recognition of a series of 7-aryl-2,4-diaminoquinazoline derivatives on the inhibition of Staphylococcus aureus dihydrofolate reductase and conducted a virtual screening to discover new potential inhibitors. A quantitative structure-activity relationship model was developed using 40 compounds and two selected descriptors. These descriptors indicated the importance of pKa and molar refractivity for the inhibitory activity against SaDHFR. The values of R2train, CVLOO and R2test generated by the model were 0.808, 0.766, and 0.785, respectively. The integration between QSAR, molecular docking, ADMETox analysis and molecular dynamics simulations with binding free energies calculation, yielded the compounds PC-124127620, PC-124127795 and PC-124127805 as promising candidates to SaDHFR inhibitors. These compounds presented high potency, good pharmacokinetics and toxicological profile. Thus, these molecules are good potential antimicrobial agent to treatment of infect disease caused by S. aureus.Communicated by Ramaswamy H. Sarma.

Differentiating interactions of antimicrobials with Gram-negative and Gram-positive bacterial cell walls using molecular dynamics simulations.

Developing molecular models to capture the complex physicochemical architecture of the bacterial cell wall and to study the interaction with antibacterial molecules is an important aspect of assessing and developing novel antimicrobial molecules. We carried out molecular dynamics simulations using an atomistic model of peptidoglycan to represent the architecture for Gram-positive S. aureus. The model is developed to capture various structural features of the Staphylococcal cell wall, such as the peptide orientation, area per disaccharide, glycan length distribution, cross-linking, and pore size. A comparison of the cell wall density and electrostatic potentials is made with a previously developed cell wall model of Gram-negative bacteria, E. coli, and properties for both single and multilayered structures of the Staphylococcal cell wall are studied. We investigated the interactions of the antimicrobial peptide melittin with peptidoglycan structures. The depth of melittin binding to peptidoglycan is more pronounced in E. coli than in S. aureus, and consequently, melittin has greater contacts with glycan units of E. coli. Contacts of melittin with the amino acids of peptidoglycan are comparable across both the strains, and the D-Ala residues, which are sites for transpeptidation, show enhanced interactions with melittin. A low energetic barrier is observed for translocation of a naturally occurring antimicrobial thymol with the four-layered peptidoglycan model. The molecular model developed for Gram-positive peptidoglycan allows us to compare and contrast the cell wall penetrating properties with Gram-negative strains and assess for the first time binding and translocation of antimicrobial molecules for Gram-positive cell walls.

Combination of DNA walker and Pb2+-specific DNAzyme-based signal amplification with a signal-off electrochemical DNA sensor for Staphylococcus aureus detection.

the accurate, reliable and specific analysis of foodborne pathogenic bacteria is vital for human health and safety. Staphylococcus aureus (S. aureus), as a common bacterium, is regularly found in food, water, and other biological samples. Herein, a signal-off electrochemical DNA sensor (E-DNA sensor) was designed for the sensitive detection ofS. aureusamplified withthecombination of a dna walker and pb2+-specific dnazyme. In this work, vancomycin functionalized gold nanoclusters (Van@Au NCs) and an aptamer strand as identification units were modified at the termini of two proximity probes. upon the addition of targetS. aureus, a dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pb2+-dependent dnazyme, achieving the conversion of oneS. aureus to many intermediate dna (t) strands. then, the released t strands hybridized with methylene blue-tagged hairpin dna (h-mb) on the electrode. consequently, the conformational alteration of t strands reduced the electron transfer efficiency of mb to the electrodeinterface (signal-off). therefore, sensitive analysis of S. aureus was readily acquired within a range of 10-107 CFU/mL and a low detection limit at 1 CFU/mL. Undoubtedly, dual recognition by aptamer and vancomycin in an integrated scheme brought about a good recognition performance of S. aureus in complex samples, as well as an efficient annihilation of harmful pathogenic bacteria during the experiment.

Identification of responsible amino acid residues in staphylococcal superantigen-like 12 for the activation of mast cells.

Staphylococcal superantigen-like 12 (SSL12) is reported to evoke the degranulation in murine mast cells. The allelic variant of SSL12 in the genome of reference strain NCTC8325 induced the degranulation of murine mast cells, that of MRSA252 strain did not, nevertheless relatively high sequence similarity (82%). To identify responsible amino acid residues of SSL12 for mast cell activation, we created a series of domain swap mutants and amino acid substitution mutants between the active and inactive variants. The mutants that harbored oligonucleotide/oligosaccharide binding (OB)-fold domain of the active variant activated mast cells. The replacement at position 56 (L56F) in the OB-fold domain diminished the mast cell stimulatory activity, and the combinatorial substitutions L56F/K92E, L56F/D95S, and L56F/S100V abolished the stimulatory activities of the mutant that harbored OB-fold domain of the active variant and the intact active variant. These indicate that the responsive elements of SSL12 for mast cell activation are in the OB-fold of SSL12, and L56 would be an essential amino acid residue for the activation of mast cells. The findings would contribute to the understanding of the molecular mechanism of SSL12 for mast cell activation and the development of toxoids preventing allergic inflammations associated with Staphylococcus aureus.

Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection.

CRISPR-Cas systems have been employed to detect a large variety of pathogenic microorganisms by simply changing the guide RNA sequence. However, these platforms usually rely on nucleic acid extraction and amplification to achieve good sensitivity. Herein, we developed a new platform for the highly specific and sensitive detection of live staphylococcus aureus (S. aureus) based on an Aptamer-based Cas14a1 Biosensor (ACasB), without the need for nucleic acid extraction or amplification. First, the S. aureus specific aptamer was hybrid with a blocker DNA. After the live S. aureus was added, the blocker can be released upon bacteria-aptamer binding. Finally, the released blocker can activate Cas14a1 protein by binding with the sgRNA to generate a change of fluorescent intensity. The ACasB indicates high specificity and sensitivity: it can directly distinguish 400 CFU/ml live S. aureus cells. Comparable to qPCR, the Cas14a1-aptamer biosensor can detect S. aureus with 100% accuracy in complex samples. Therefore, this ACasB for the on-site detection of live S. aureus can broaden its applications in food safety and environmental monitoring.

A detailed protocol for expression, purification, and activity determination of recombinant SaCas9.

Recombinant SaCas9 is useful for a broad range of applications in the context of genome editing, especially when the specific protospacer adjacent motifs of other Cas9 derivatives are missing. Here, we describe a detailed protocol for the expression and purification of recombinant SaCas9. We detail the main steps for immobilized metal affinity chromatography and size exclusion chromatography. In addition, we present an assay for activity determination of SaCas9. Active SaCas9 can be purified in a week by using this protocol.