New Type Of Antibacterial Agent Research Articles

A Highly Substituted Ring-Fused 2-Pyridone Compound Targeting PrfA and the Efflux Regulator BrtA in Listeria monocytogenes.

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

Bioinspired Green Synthesis of Silver Nanoparticles Using Three Plant Extracts and Their Antibacterial Activity against Rice Bacterial Leaf Blight Pathogen Xanthomonas oryzae pv. oryzae.

Rice bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is responsible for a significant reduction in rice production. Due to the small impact on the environment, biogenic nanomaterials are regarded as a new type of antibacterial agent. In this research, three colloids of silver nanoparticles (AgNPs) were synthesized with different biological materials such as Arctium lappa fruit, Solanum melongena leaves, and Taraxacum mongolicum leaves, and called Al-AgNPs, Sm-AgNPs and Tm-AgNPs, respectively. The appearance of brown colloids and the UV-Visible spectroscopy analysis proved the successful synthesis of the three colloids of AgNPs. Moreover, FTIR and XRD analysis revealed the formation of AgNPs structure. The SEM and TEM analysis indicated that the average diameters of the three synthesized spherical AgNPs were 20.18 nm, 21.00 nm, and 40.08 nm, respectively. The three botanical AgNPs had the strongest bacteriostatic against Xoo strain C2 at 20 μg/mL with the inhibition zone of 16.5 mm, 14.5 mm, and 12.4 mm, while bacterial numbers in a liquid broth (measured by OD600) decreased by 72.10%, 68.19%, and 65.60%, respectively. Results showed that the three AgNPs could inhibit biofilm formation and swarming motility of Xoo. The ultrastructural observation showed that Al-AgNPs adhered to the surface of bacteria and broke the bacteria. Overall, the three synthetic AgNPs could be used to inhibit the pathogen Xoo of rice bacterial leaf blight.

Toxicity and modulation of silver nanoparticles synthesized using abalone viscera hydrolysates on bacterial community in aquatic environment.

Polysaccharide decorated silver nanoparticles (AgNPs) are a new type of antibacterial agent in aquaculture, but their effects on the bacterial community structure in aquaculture water are still unknown. In this study, the primary hydrolysate from abalone (Haliotis discus hannai) viscera (AVH) was used to biosynthesize AVH-AgNPs by in situ reduction, and the crystallinity nature, size, morphology, and chemical composition were analyzed by high-resolution characterization techniques such as Ultraviolet–visible spectroscopy (UV–vis), X-rays diffraction (XRD), Transmission Electron Microscope (TEM), Dynamic light scattering (DLS), Zeta potential, inductively coupled plasma-optical emission spectrometry (ICP-OES) and Turbiscan stability index (TSI) values. Furthermore, the acute toxicity of AVH-AgNPs to zebrafish (Danio rerio) and their effects on bacterial community structure in fish culture water at low concentrations were studied. The results showed that the spherical AVH-AgNPs with an average diameter of 54.57 ± 12.96 nm had good stability, low toxicity, and good in vitro antibacterial activity. Within the experimental concentration range, all AVH-AgNPs treatments had decreased the bacterial diversity in zebrafish culture water to varying degrees. The bacteria with significantly decreased abundances were pathogenic or potential pathogenic, such as Aeromonas veronii, Flavobacterium columnare, and genera Flectobacillus and Bosea. The abundance of Haliscomenobacter sp. JS224, which might cause sludge swelling, also decreased significantly. On the other hand, the relative abundance of some bacterial taxa could remove xenobiotics (e.g., Runella defluvii and Phenylobacterium), control water eutrophication (Sediminibacterium), and reduce toxic algae proliferation (Candidatus Intestinusbacter nucleariae and Candidatus Finniella), increased significantly. Thus, the application of AVH-AgNPs in aquaculture water at low concentrations is relatively safe and has positive significance for improving the aquaculture environment. Also, AVH-AgNPs have good prospects in aquaculture.

Antimicrobial Properties of Metal Nanoparticles and Their Oxide Materials and Their Applications in Oral Biology

Some scholars have shown that metal nanoparticles have excellent antibacterial properties and can be used as a new type of antibacterial agent. In recent years, with the in-depth research on nanomaterials, its applications in the medical field have gradually increased. The oral cavity has a unique anatomical structure, and for oral infections, the current clinically commonly used treatment measures are oral or topical antibiotics. However, due to bacterial resistance and the special structure of dental plaque, the effect of antibiotics is not ideal. Metal and metal oxide nanoparticles have become the research hotspot of new antibacterial materials due to their small particles, large specific surface area, physical, mechanical, and chemical properties, and antibacterial properties. This article describes the antibacterial effect, antibacterial mechanism, biological toxicity, and application progress of metal nanomaterials in the oral cavity.

Study on the Performance of Titanium Materials Based on Nano Silver Particles in Orthodontic Healing.

Nanomaterials are used as a new type of antibacterial agent in clinical medical research projects due to their unique physical and chemical properties. In this study, silane coupling method was used to coat nano-silver particles on titanium sheets to make the smooth titanium surface have antibacterial properties. Scanning electron microscope to observe the surface morphology of silane coupling modified titanium plate, the shape and size of nano-silver particles, and the adhesion on titanium plate; X-ray energy spectroscopy and line scan analysis of each element on the titanium wafer; X-ray photoelectron spectroscopy quantitative analysis of surface elements on the titanium wafer. The experimental group was nano-silver modified titanium sheet, and the control group was smooth titanium sheet. The experimental results show that the titanium material loaded with nano-silver particles has better antibacterial performance in the orthodontic process than ordinary materials. This material is highly safe and can be promoted for use.

Multivalent Bacteria Binding by Flexible Polycationic Microsheets Matching Their Surface Charge Density

AbstractAiming at the overall negative surface charge of bacteria, a new strategy of antibacterial agents based on large polymer‐modified graphene oxide (GO) sheets is assessed. The presented flexible, polycationic sheets match the size and charge density of the Escherichia coli surface charge density (2 × 1014 cm−2). These matching parameters create an unspecific but very strong bacteria adsorber by multivalent, electrostatic attraction. Their interaction with bacteria is visualized via atomic force and confocal microscopy and shows that they effectively bind and wrap around E. coli cells, and thereby immobilize them. The incubation of Gram‐negative and ‐positive bacteria (E. coli and methicillin‐resistant Staphylococcus aureus, MRSA) with these polycationic sheets leads to the inhibition of proliferation and a reduction of the colony forming bacteria over time. This new type of antibacterial agent acts in a different mode of action than classical biocides and could potentially be employed in medicinal, technical, or agriculture applications. The presented microsheets and their unspecific binding of cell interfaces could further be employed as adsorber material for bacterial filtration or immobilization for imaging, analysis, or sensor technologies.

Nanosilver induces the formation of neutrophil extracellular traps in mouse neutrophil granulocytes

As a new type of antibacterial agent, nanosilver has attracted great attention in biomedical applications. However, the safety of nanosilver to humans and the environment has not been well elucidated. The objective of this study was to investigate the influence of nanosilver on novel effector mechanism of neutrophil extracellular traps (NETs), and its possible molecular mechanisms. In this study, nanosilver (10, 20 and 40 μg/mL) was incubated with neutrophils for 90 min. Then, nanosilver-induced the release of NETs was observed by laser confocal microscopy. Nanosilver-induced NETs release was also quantitatively detected by pico Green®. In addition, the role of NADPH oxidase, extracellular signal-regulated kinase (ERK) and p38 signaling pathways in nanosilver-induced NETs release were detected by the inhibitors and pico Green®. The results indicated that nanosilver significantly activated polymorphonuclear neutrophils (PMN) to release NETs, which was a DNA-based network structure modified with histones (H3) and neutrophil elastase (NE). The inhibitors of NADPH oxidase, ERK and p38 signaling pathways significantly inhibited the formation of nanosilver-induced NETs. Furthermore, nanosilver did not alter the extracellular lactate dehydrogenase (LDH) level of PMN cells. All these results showed that nanosilver significantly induced NETs release, and the potential molecular mechanisms were correlated with reactive oxygen species (ROS) production-dependent on NADPH oxidase, ERK and p38 signaling pathways, which might provide a new perspective on nanosilver-induced excess NETs release related to the host immune damage.

Remarkably Improvement in Antibacterial Activity of Carbon Nanotubes by Hybridizing with Silver Nanodots.

In view of their super capacity of adsorbing microbial, the carbon nanotubes (CNTs) were used as the carriers for in situ synthesizing well-dispersed and small-sized silver nanodots (AgND), to prepare a new type of antibacterial agent with remarkably improved activity. Polyethyleneimine (PEI) was introduced as a linkage for guaranteeing the as-generated AgND to be anchored onto the CNTs and to prevent them from agglomeration. The obtained hybridizing materials were characterized by transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy, and the results showed that the AgND with an average size of 2.6 nm were uniformly loaded on surfaces of CNTs. There existed special interactions between silver atoms and CNTs. The antibacterial activities of the as-prepared hybrids against Escherichia coli were evaluated by disk diffusion assay method and minimal inhibitory concentration measurements. The results showed that the as-prepared hybrids displayed a remarkable improvement in antibacterial activity as compared to CNTs, acidified-CNTs and even the identical silver amount of AgNO3 solution, which was mainly attributed to the small size of AgND and the hybridizing effect between AgND and CNTs.

Synthesis and Bioactivity Evaluation of N-Arylsulfonylindole Analogs Bearing a Rhodanine Moiety as Antibacterial Agents.

Due to the rapidly growing bacterial resistance to antibiotics and the scarcity of novel agents under development, bacterial infections are still a pressing global problem, making new types of antibacterial agents, which are effective both alone and in combination with traditional antibiotics, urgently needed. In this paper, seven series of N-arylsulfonylindole analogs 5–11 bearing rhodanine moieties were synthesized, characterized, and evaluated for antibacterial activity. According to the in vitro antimicrobial results, half of the synthesized compounds showed potent inhibition against four Gram-positive bacteria, with MIC values in the range of 0.5–8 µg/mL. For multidrug-resistant strains, compounds 6a and 6c were the most potent, with MIC values of 0.5 µg/mL, having comparable activity to gatifloxacin, moxiflocaxin and norfloxacin and being 128-fold more potent than oxacillin (MIC = 64 µg/mL) and 64-fold more active than penicillin (MIC = 32 µg/mL) against Staphylococcus aureus ATCC 43300.

Synthesis and Antibacterial Evaluation of Novel 3-Substituted Ocotillol-Type Derivatives as Leads.

Due to the rapidly growing bacterial antibiotic-resistance and the scarcity of novel agents in development, bacterial infection is still a global problem. Therefore, new types of antibacterial agents, which are effective both alone and in combination with traditional antibiotics, are urgently needed. In this paper, a series of antibacterial ocotillol-type C-24 epimers modified from natural 20(S)-protopanaxadiol were synthesized and evaluated for their antibacterial activity. According to the screening results of Gram-positive bacteria (B. subtilis 168 and MRSA USA300) and Gram-negative bacteria (P. aer PAO1 and A. baum ATCC19606) in vitro, the derivatives exhibited good antibacterial activity, particularly against Gram-positive bacteria with an minimum inhibitory concentrations (MIC) value of 2–16 µg/mL. The subsequent synergistic antibacterial assay showed that derivatives 5c and 6c enhanced the susceptibility of B. subtilis 168 and MRSA USA300 to chloramphenicol (CHL) and kanamycin (KAN) (FICI < 0.5). Our data showed that ocotillol-type derivatives with long-chain amino acid substituents at C-3 were good leads against antibiotic-resistant pathogens MRSA USA300, which could improve the ability of KAN and CHL to exhibit antibacterial activity at much lower concentrations with reduced toxicity.

Batch synthesis of polyoxometalate-based phosphonium compounds by one-step room-temperature mechanochemical process, and their morphology-dependent antibacterial activities

Based on the Keggin-type polyoxometalate and quaternary phosphonium salt, the (MePh3P)n-POMKeggin compounds were synthesized via one-step mechanochemical process at room temperature. It showed the advantages of convenient operation, lower cost, less pollution, and mass production. Wonderfully, the morphology of compounds presented a strong dependence on the number of crystal water in the source heteropoly acids. A hypothesis of ‘semi-solid nonlocalized waters’ was brought to discuss the formation mechanism of polyhedrons. Antibacterial experiments indicate that the samples have good antibacterial activities, which resulted from the increase of the positive electrical charge of the phosphonium cation caused by the polarization by POM groups. Additionally, a formula of inhibitory zone per unit surface area was designed to more precisely evaluate the antibacterial activity of the materials. The calculation result indicated that the polyhedral particles give a higher surface antibacterial activity than the grain powder. This work developed an alternative synthesis method for composites from a new perspective, and will promote the research of new type of antibacterial agent.

Synthesis and characterization of new α,α′-diaminoalkane-bridged dicarbonyl(η5-cyclopentadienyl)ruthenium(II) complex salts: Antibacterial activity tests of η5-cyclopentadienyl dicarbonyl ruthenium(II) amine complexes

Bacterial resistance to antimicrobial drugs is a significant threat to humans and requires urgent intervention. There is, therefore, a clear need for the development of new types of antibacterial agents. We have thus synthesized the α,α′-diaminoalkane-bridged diruthenium complex salts [Rp2NH2(CH2)nNH2]Y2 (Rp = CpRu(CO)2 where Cp = η5–C5H5; n = 2, 3, 4 and 6; Y=BF4 (1) or SO3CF3(2)) by the reaction of [RpNCCH3]Y and NH2(CH2)nNH2 (n = 2 (DAE) (3); n = 3 (DAP) (4); n = 4 (DAB) (5) and n = 6 (DAH) (6 (BF)4, 7 (SO3CF3)) at ambient temperature and pressure. These compounds are reported for the first time and have been fully characterized by infrared spectroscopy, 1H and 13C NMR spectroscopy, elemental analyses and melting points. The crystal structures of compounds 1 and 3 were obtained by single crystal X-ray crystallography. The two compounds crystallized in the monoclinic crystal system in the P21/n space group. Antimicrobial susceptibility tests were done for the new dinuclear complexes 3–7 as well as for mononuclear ruthenium complexes, [RpBA]BF4 (8), [RpMBA]BF4 (9), [RpMeOBA]BF4 (10) and [RpAMBN]BF4 (11) (BA=NH2CH2C6H5, MBA = NH2CH(CH3)C6H5, MeOBA = NH2CH2C6H4OCH3 and AMBN = NH2CH2C6H5) against selected drug-resistant and drug-susceptible Gram-positive and Gram-negative bacteria. Some of the dinuclear and mononuclear ruthenium complexes demonstrated good potential abilities to inhibit growth of the bacteria tested, with some showing better growth inhibition ability than well-known antibiotics such as ampicillin (AMP10). The ruthenium moiety CpRu(CO)2 triggered or enhanced the ability of the coordinated ligands to inhibit bacterial growth. In vitro cytotoxicity of the complexes 8 and 10 was investigated through cell viability evaluation using human colorectal cancer (Caco-2) cells and the non-cancerous human embryonic kidney (HEK293) cells.

N-Containing Ag(I) and Hg(II) Complexes: A New Class of Antibiotics

Several classes of antimicrobial compounds are presently available; microorganism's resistance to these drugs constantly emerges. In order to prevent this serious medical problem, the elaboration of new types of antibacterial agents or the expansion of bioactivity of the naturally known biosensitive compounds is a very interesting research problem. The synthesis and characterization of metal complexes with organic bioactive ligands is one of the promising fields for the search. The biological activities of the metal complexes differ from those of either the ligand or the metal ion. The results obtained thus far have led to the conclusion that structural factors, which govern antimicrobial activities, are strongly dependent on the central metal ion. A review of papers dealing with the Ag(I) and Hg(II) complexes of N donor ligands is presented. These metal complexes of N-chelating ligands have attracted considerable attention because of their interesting physicochemical properties and pronounced biological activities. This review will mainly focus on the preparation procedures and antibacterial properties of free organic ligands and the corresponding complexes. Finally, a research about antimicrobial properties of new Hg(II) complexes with 5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione (L) and various halogen ions, HgL2X2 (X = Cl¯ (49), Br¯ (50), and I¯ (51)), is reported. Noteworthy antimicrobial activities, evaluated by minimum inhibitory concentration, for these complexes were observed.

Update of Peptides with Antibacterial Activity

For many years a battle has been going on between bacteria and humans, with bacteria trying to survive against the antibiotics used by humans. Bacteria are found to be dominant in this battle since they can develop resistance. In fact, in the last decade multi-, extended- and pan-drug resistant bacteria have been isolated. On the other hand, the number of new antibiotics approved by the FDA has dramatically decreased during the last 20 years. Therefore, there is a desperate need for developing new types of antibacterial agents, where antimicrobial peptides may play an important role. This review provides an update of the recently identified antimicrobial peptides. Three valid approaches for developing a future antibacterial agent, as are the mechanisms of action as well as the in vitro and in vivo assays have been described in depth. In comparison to the antibacterial agents available at present, the targets for most of the antimicrobial peptides are not well known. However several proposals having been introduced for many antimicrobial peptides of different mechanisms of action, there still lies some uncertainty about their utility. Hundreds of antimicrobial peptides have been tested in vitro against all types of bacteria, but in this review we will highlight only those which have been tested against the most important Gram-positive and Gram-negative bacteria. The last step to get a potential antibiotic includes studies with an in vivo model. Therefore only antimicrobial peptides with good activity are tested that have been described in this review.

Update of Peptides with Antibacterial Activity

For many years a battle has been going on between bacteria and humans, with bacteria trying to survive against the antibiotics used by humans. Bacteria are found to be dominant in this battle since they can develop resistance. In fact, in the last decade multi-, extended- and pan-drug resistant bacteria have been isolated. On the other hand, the number of new antibiotics approved by the FDA has dramatically decreased during the last 20 years. Therefore, there is a desperate need for developing new types of antibacterial agents, where antimicrobial peptides may play an important role. This review provides an update of the recently identified antimicrobial peptides. Three valid approaches for developing a future antibacterial agent, as are the mechanisms of action as well as the in vitro and in vivo assays have been described in depth. In comparison to the antibacterial agents available at present, the targets for most of the antimicrobial peptides are not well known. However several proposals having been introduced for many antimicrobial peptides of different mechanisms of action, there still lies some uncertainty about their utility. Hundreds of antimicrobial peptides have been tested in vitro against all types of bacteria, but in this review we will highlight only those which have been tested against the most important Grampositive and Gram-negative bacteria. The last step to get a potential antibiotic includes studies with an in vivo model. Therefore only antimicrobial peptides with good activity are tested that have been described in this review. Keywords: Antimicrobial peptides, mechanism of action, barrel model, toroidal model, aggregate model, carpet model, “in vitro” activity, “in vivo” activity, melittin, indolicidin, cecropin A, magainin II, pexiganan, buforin

StCT2, a new antibacterial peptide characterized from the venom of the scorpion Scorpiops tibetanus

Bacterial infection poses an increasing threat to global public health and new types of antibacterial agents are urgently needed to respond to the threat. Scorpion venom contains series of bioactive peptides, among which antibacterial peptide is an important part. Herein, a new antimicrobial peptide StCT2 was characterized from the venomous gland cDNA library of the Scorpiops tibetanus. The full-length cDNA of StCT2 is 369 nucleotides encoding the precursor that contains a putative 24 residues signal peptide, a presumed 14 residues mature peptide, and a putative 37 residues acidic propeptide at the C-terminus. The minimal inhibition concentrations (MICs) of StCT2 for Staphylococcus aureus were 6.25–25μg/ml, including antibiotic-resistant strains such as methicillin resistant S. aureus (MRSA). StCT2 was further found to show high in vivo antimicrobial activity by an S. aureus infection mouse model. StCT2 exerted its antimicrobial activity via a rapid bactericidal mechanism. Taken together, these results demonstrate the efficacy and general mechanism of StCT2 antimicrobial action and the therapeutic potential of StCT2 as a new antimicrobial peptide.

One-Pot Enantioselective Synthesis of Functionalized Pyranocoumarins and 2-Amino-4H-chromenes: Discovery of a Type of Potent Antibacterial Agent

Function-oriented design and synthesis of chiral small molecules with novel activity is a key goal in modern organic chemistry. As multiple antibiotic-resistant pathogens are emerging and causing serious diseases, the need for practical routes for the development of new types of antibacterial agents is very urgent. Herein, we present a highly efficient process for the synthesis of optically active pyranocoumarins and 2-amino-4H-chromenes through an organocatalytic Knoevenagel/Michael/cyclization sequence, and the preliminary biological studies of these new heterocyclic compounds revealed potent antibacterial activity. This study provides a novel strategy for further research and development of new types of antibacterial agents effective against human pathogens.

Standardization of Antibacterial Bone China

The developments of antibacterial ceramics technology were introduced. A new type of antibacterial agent used in bone china, multifuntional healthy ceramic materials antibacterial mechanism, was discussed. With the quick development of antibacterial bone china technology and market, it was necessary to estabish the antibacterial bone china standard.

Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790.

Dodecylglycerol has a minimum inhibitory concentration of 4 micrograms/ml compared to 9 micrograms/ml for monolaurin (dodecanoylglycerol) with Streptococcus faecium ATCC 9790 as the test organism. The greater potency of dodecylglycerol can be correlated to its greater retention by the cell. Gram-positive bacteria were more susceptible than Gram-negative bacteria to dodecylglycerol. The antibacterial action of dodecylglycerol is not through the physical dissolution of cell walls, but rather as an enzymatic effector. The autolysin activity of whole cells of S. faecium was greatly stimulated by dodecylglycerol. The stimulation of autolytic activity and inhibition of growth respond in parallel to different concentrations of dodecylglycerol, to dodecylglycerol versus some poorer effector such as monolaurin or a glycerol alkyl ether with a longer or shorter fatty alkyl side chain than dodecanol, and to the antagonistic effects of diphosphatidlyglycerol. This close relationship implies that the stimulation of autolysin activity could be a primary, but not necessarily the only, mechanism by which dodecylglycerol and related compounds exert their antibacterial activity. However, the autolysin activity is not stimulated by a direct interaction between the enzyme and dodecylglycerol. A non-wall entity, such as a proteinase, has been implicated as an intermediary (Ved, H. S., Gustow, E., and Pieringer, R. A. (1984) J. Biol. Chem. 259, 8122-8124).