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Quantification of ultraviolet-C irradiation induced cyclobutane pyrimidine dimers and their removal in Beauveria bassiana conidiospore DNA.

Ultraviolet (UV) radiation-induced DNA damage leading to entomopathogenic fungal inactivation is commonly measured by viability counts. Here we report the first quantification of UV-induced cyclobutane pyrimidine dimers (CPD) in DNA of the entomopathogenic fungus, Beauveria bassiana. Changes in the mobility of UV-C irradiated DNA were resolved with CPD specific bacteriophage T4 endonuclease V and alkaline agarose gel electrophoresis. The maximum number of CPD formed in B. bassiana DNA in vitro by UV-C irradiation was 28 CPD/ 10 kb after 720 J/m2 dose. The maximum number of CPDs formed in B. bassiana conidiospore DNA irradiated in vivo was 15 CPD/10 kb after 480 J/m2 dose and was quantified from conidiospores that were incubated to allow photoreactivation and nucleotide excision repair. The conidiospores incubated for photoreactivation and nucleotide excision repair showed decreased number of CPD/10 kb DNA and a higher percent survival of conidiospore populations than conidiospores not allowed to repair.

Colony Count, Microbial↗

Efficacy of Candida sake CPA-1 formulation for controlling Penicillium expansum decay on pome fruit from different Mediterranean regions.

The effectiveness of a formulated product of the yeast Candida sake CPA-1 for controlling postharvest diseases on pome fruits was demonstrated in laboratory, semicommercial, and commercial trials carried out in the major pome fruit producing region of the European Union. First, one wettable powder and seven liquid formulations were tested in laboratory trials that involved two varieties of apples and two varieties of pears. In all cases, an efficacy similar to that of fresh cells was demonstrated in the control of artificial Penicillium expansum infection. After these trials, the formulated product chosen for semicommercial and commercial trials was LF1, a liquid formulation that is particularly suitable for commercial applications. In semicommercial trials, LF1 showed a performance similar to fresh cells in most trials, and the population dynamics of both fresh and formulated cells were quite stable throughout the storage period. This indicates the high viability of C. sake CPA-1 in this formulation and the absence of adverse effects during the formulation of the product, which may significantly affect both its ability to grow on fruit and its antagonistic activity. We evaluated the control of natural infection after applying the formulated product in a commercial drencher in different packinghouses. A significant reduction in the incidence of diseases was observed with a recommended dose of around 10(7) CFU/ml when natural infections were greater than 1%. In general, large quantities of yeast were observed on the surface of unwounded fruits of different pome fruit cultivars. Moreover, populations of this biocontrol agent increased rapidly on fruit surfaces and remained quite stable for a long time under commercial storage conditions. Commercial practices used in packinghouses were therefore successfully applied for this formulated product.

Antibiosis↗

Phage therapy: past history and future prospects.

Bacterial viruses (bacteriophages, also called "phages") can be robust antibacterial agents in vitro. However, their use as therapeutic agents, during a number of trials from the 1920s to the 1950s, was greatly handicapped by a number of factors. In part, there were certain limitations inherent in phage physiology (e. g. narrow host range, and rapid clearance from the body); in part there were technological limitations in the era (e.g. lysogeny not yet discovered); but the greatest limitation was the highly inadequate scientific methodologies used by practitioners at the time (e.g., their failure to conduct placebo-controlled studies, to remove endotoxins from the preparations, and to re-confirm phage viability after adding sterilizing agents to the preparations). In recent years, well-controlled animal models have demonstrated that phages can rescue animals from a variety of fatal infections, while non-controlled clinical reports published in Eastern Europe have shown that phages can be effective in treating drug-resistant infections in humans. This encouraging data, combined with the fact that drug-resistant bacteria have become a global crisis, have created a window of opportunity for phage therapy to be tested anew, this time using modem technologies and placebo-controlled designs. If successful, it can be used as a stand-alone therapy when bacteria are fully resistant to antibiotics, and as a valuable adjunct to antibiotics when the bacteria are still susceptible.

Administration, Oral↗

Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture.

Hispánico cheese, a semi-hard Spanish variety, was manufactured from a mixture of pasteurized cows' and ewes' milks (4:1) using a commercial mesophilic LD-type starter comprising Lactococcus lactis subsp. cremoris, Lc. lactis subsp. lactis, Lc. lactis subsp. lactis var diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Varying amounts (0-1.0 g/kg) of an Enterococcus faecalis INIA 4 culture in milk were added as a bacteriocin-producing adjunct. Differences in pH between cheeses manufactured with and without the bacteriocin producer did not exceed 0.11 pH units. Starter lactococci lost viability more rapidly in cheeses made with the bacteriocin producer, which reached counts of up to 6 x 10(7) cfu/g during ripening. Aminopeptidase activity in 1-d-old cheese made from milk inoculated with 1.0 g bacteriocin-producing culture/kg was twice that in control cheese. Degrees of overall proteolysis and levels of total free amino acids in 45-d-old cheese made with 1.0 g bacteriocin-producing culture/kg were 1.80-fold and 2.17-fold those in control cheese of the same age. Inoculating milk with 1.0 g/kg bacteriocin-producing culture reduced the level of hydrophobic peptides in the resultant cheese, increased the concentrations of 3-methyl-1-butanal, diacetyl and acetoin, and resulted in the highest scores for flavour quality and flavour intensity throughout ripening.

Aminopeptidases↗

Inactivation of Escherichia coli by a combination of nisin, pulsed electric fields, and water activity reduction by sodium chloride.

The effect of nisin combined with pulsed electric fields (PEF) and water activity reduction by sodium chloride (NaCl) on the inactivation of E. coli in simulated milk ultrafiltrate media was studied with a Doehlert design and a response surface method. The reduction of water activity from 0.99 to 0.95 by the addition of NaCl (without any other hurdle) did not affect E. coli viability of approximately 10(8) CFU/ml. A reduction in PEF effectiveness occurred when the NaCl concentration was increased because of an increase in conductance, which reduced the pulse decay time. In cells submitted to PEF nisin activity was decreased, probably as a consequence of the nonspecific binding of nisin to cellular debris or the emergence of new binding sites in or from cells. However, the lethal effect due to nisin was reestablished and further improved when water activity was reduced to 0.95. A synergistic effect was evidenced when low-intensity PEF were applied. Decreasing water activity to 0.95 and applying PEF at 5 kV/cm (a nonlethal intensity when no other hurdle is used) with the further addition of nisin (1,200 IU/ml) resulted in a 5-log cycle reduction of the bacterial population.

Animals↗

Acute ethanol treatment modulates Toll-like receptor-4 association with lipid rafts.

BACKGROUND: Alcohol, a substance that is most frequently abused, suppresses innate immune responses to microbial pathogens. The host senses pathogens via Toll-like receptors (TLRs). Recent studies indicate that alcohol affects TLR signaling. METHODS: Here, we hypothesized that acute alcohol treatment may interfere with early steps of membrane-associated TLR2 and TLR4 signaling at the level of lipid rafts. Human monocytes and Chinese hamster ovary (CHO) cells, transfected with human TLR2, TLR4, or CD14, were stimulated with peptidoglycan (PGN, TLR2 ligand) or lipopolysaccharide (LPS, TLR4 ligand) with or without alcohol (50 mM) and analyzed for cytokine production (enzyme-linked immunosorbent assay), nuclear factor-kappaB (NF-kappaB) activation (electrophoretic mobility shift assay), membrane fluidity (fluorescent pyrene eximer formation), and partition of cellular membrane into cholesterol-rich, detergent-resistant domains (DRMs; Western blot). RESULTS: We determined that both TLR2 and TLR4 were located outside the rafts; flotillin, a DRM marker, was resident in the rafts, while CD14 was equally distributed in and outside the rafts in a steady-state condition. PGN forced TLR2 to migrate into DRMs. Engagement of TLR4 and CD14 with LPS induced their migration into the rafts. Alcohol prevented TLR4 partitioning; however, it did not affect TLR2 migration into the rafts. Furthermore, alcohol downregulated TLR4-induced, but not TLR2-induced, NF-kappaB activation and cytokine production in monocytes. We found that alcohol increased membrane fluidity and depleted cellular cholesterol in CHO cells without affecting cell viability. CONCLUSIONS: These data demonstrate for the first time that alcohol disturbs TLR4 and CD14 association with lipid rafts. We propose that alcohol-induced effects on lipid rafts may contribute to modulation of TLR4-CD14-triggered early cellular responses.

Adolescent↗

Antimicrobial peptides: a potential role in ocular therapy.

Bacterial pathogens are often involved in contact lens-related adverse responses. This study aimed to find antimicrobial peptides and proteins that effectively eradicate or inhibit ocular bacteria. The antimicrobials were screened against gram-negative and gram-positive bacteria originating from ocular sources. The viability of these ocular bacteria was measured after exposure to the peptides and proteins. Two conditions were used to grow bacteria, low nutrient phosphate-buffered saline and high nutrient tryptone soya broth. Samples were taken at different times up to 48 h. In low nutrient conditions, protamine was found to be the most effective against all strains. Melittin was very effectve against all strains except Serratia and one Pseudomonas isolate which were partially affected. In high nutrient condition, only melittin was effective in killing Staphylococcus aureus. Protamine and the combination of protamine and melittin had the greatest effect in eradicating the bacteria tested in low nutrient condition. Protamine alone and its combination with melittin may have potential therapeutic agents for ocular infections in an era of emerging antibiotic resistance.

Anti-Bacterial Agents↗

Application of cereals and cereal components in functional foods: a review.

The food industry is directing new product development towards the area of functional foods and functional food ingredients due to consumers' demand for healthier foods. In this respect, probiotic dairy foods containing human-derived Lactobacillus and Bifidobacterium species and prebiotic food formulations containing ingredients that cannot be digested by the human host in the upper gastrointestinal tract and can selectively stimulate the growth of one or a limited number of colonic bacteria have been recently introduced into the market. The aim of these products is to affect beneficially the gut microbial composition and activities. Cereals offer another alternative for the production of functional foods. The multiple beneficial effects of cereals can be exploited in different ways leading to the design of novel cereal foods or cereal ingredients that can target specific populations. Cereals can be used as fermentable substrates for the growth of probiotic microorganisms. The main parameters that have to be considered are the composition and processing of the cereal grains, the substrate formulation, the growth capability and productivity of the starter culture, the stability of the probiotic strain during storage, the organoleptic properties and the nutritional value of the final product. Additionally, cereals can be used as sources of nondigestible carbohydrates that besides promoting several beneficial physiological effects can also selectively stimulate the growth of lactobacilli and bifidobacteria present in the colon and act as prebiotics. Cereals contain water-soluble fibre, such as beta-glucan and arabinoxylan, oilgosaccharides, such as galacto- and fructo-oligosaccharides and resistant starch, which have been suggested to fulfil the prebiotic concept. Separation of specific fractions of fibre from different cereal varieties or cereal by-products, according to the knowledge of fibre distribution in cereal grains, could be achieved through processing technologies, such as milling, sieving, and debranning or pearling. Finally, cereal constituents, such as starch, can be used as encapsulation materials for probiotics in order to improve their stability during storage and enhance their viability during their passage through the adverse conditions of the gastrointestinal tract. It could be concluded that functional foods based on cereals is a challenging perspective, however, the development of new technologies of cereal processing that enhance their health potential and the acceptability of the food product are of primary importance.

Bacteria↗

Viability of bacteria in glycerin and ethanol preserved sclera.

BACKGROUND: Sclera is commonly preserved in glycerin or ethanol before being used for ophthalmic surgery. The purpose of this study was to determine the ability of bacteria to survive in sclera preserved in glycerin or ethanol. METHODS: Fresh sclera was inoculated with Staphylococcus aureus, Streptococcus pneumoniae, or Pseudomonas aeruginosa and transferred to preservative vials containing glycerin, 95% ethanol, or trypticase soy broth (control) and stored at room temperature. Pieces of sclera were removed from preservative at designated intervals over a 14-day period. The sclera was then homogenized, plated on blood agar, and incubated at 37 degrees C. Colonies were counted at 24, 48, and 72 hours. RESULTS: S. pneumoniae, P. aeruginosa, and S. aureus were recovered from glycerin preserved sclera for up to 12 hours, 1.5 days, and 8 days, respectively. No bacteria was recovered from the ethanol preserved sclera. CONCLUSIONS: Bacteria cannot be recovered from ethanol preserved sclera but can survive in glycerin preserved sclera for at least 8 days. Ethanol may offer advantages over glycerin as a scleral preservative due to its greater antibacterial activity.

Bacteria↗

The role of RelMtb-mediated adaptation to stationary phase in long-term persistence of Mycobacterium tuberculosis in mice.

Long-term survival of nonreplicating Mycobacterium tuberculosis (Mtb) is ensured by the coordinated shutdown of active metabolism through a broad transcriptional program called the stringent response. In Mtb, this response is initiated by the enzymatic action of RelMtb and deletion of relMtb produces a strain (H37RvDeltarelMtb) severely compromised in the maintenance of long-term viability. Although aerosol inoculation of mice with H37RvDeltarelMtb results in normal initial bacterial growth and containment, the ability of this strain to sustain chronic infection is severely impaired. Significant histopathologic differences were noted in lungs and spleens of mice infected with H37RvDeltarelMtb compared with controls throughout the course of the infection. Microarray analysis revealed that H37RvDeltarelMtb suffers from a generalized alteration of the transcriptional apparatus, as well as specific changes in the expression of virulence factors, cell-wall biosynthetic enzymes, heat shock proteins, and secreted antigens that may alter immune recognition of the recombinant organism. Thus, RelMtb is critical for the successful establishment of persistent infection in mice by altering the expression of antigenic and enzymatic factors that may contribute to successful latent infection.

Adaptation, Physiological↗

Adherence of viable and nonviable bacteria to soft contact lenses.

Bacterial viability in bacterial adherence to new soft contact lenses (SCLs) was studied by exposing polymacon and lidofilcon A lenses to Pseudomonas aeruginosa: L, live, untreated bacteria; H, bacteria heated for 1 h at 100 degrees C; and P, bacteria treated with 3% hydrogen peroxide for 1 h. Cultures of groups L, H, and P showed heavy, no, and minimal bacterial growth, respectively. Scanning electron microscopy (SEM) showed significantly less bacteria on new SCLs in groups H and P. Transmission electron microscopy (TEM) showed that bacterial shape was mostly preserved in all groups, with marked intracellular and cell membrane changes and breaks (groups H and P), margination of intracellular electron-dense material (group H), and central and peripheral vacuolation (group P). Adherence to new SCLs appears to be an active process, as killed or altered bacteria adhere less.

Bacterial Adhesion↗

Inhibition of nitric oxide with aminoguanidine reduces bacterial translocation after endotoxin challenge in vivo.

BACKGROUND: Administration of lipopolysaccharide (LPS) has been shown to increase bacterial translocation (BT) in vivo and in vitro. In addition, LPS upregulates inducible nitric oxide synthase expression in the intestinal epithelium-a phenomenon that can either enhance microbial killing, or alternatively, promote BT by impairing the gut barrier. OBJECTIVE: To determine the effect, if any, of an inhibitor of nitric oxide synthase, namely, aminoguanidine (AG), on BT after LPS challenge. DESIGN: Sprague-Dawley rats were randomized to receive either AG or normal saline solution via subcutaneously placed osmotic pumps (Alzet), followed 18 hours later by LPS injection (5 mg/kg or 20 mg/kg intraperitoneally). Quantitative cultures of the cecum, mesenteric lymph nodes, liver, and spleen were obtained, and plasma nitrite and nitrate levels were measured at 24 hours. Transmembrane potential difference and mucosal permeability to fluorescein isothiocyanate-labeled dextran and fluorescein isothiocyanate-labeled Escherichia coli C25 were measured in the Using chamber. The intestinal membrane was examined by light, transmission electron, and confocal laser microscopy. RESULTS: Rats that were given high-dose LPS had elevated levels of nitrite and nitrate and a 100% incidence of BT. In contrast, AG infusion significantly reduced both BT (22%) and nitrite and nitrate levels. Animals that received LPS and normal saline solution had a significantly lower transmembrane potential difference than those that received LPS and AG. High-dose LPS resulted in sloughing of the apical enterocytes at the villus tips where bacterial entry seemed to occur, as seen with confocal laser microscopy. CONCLUSIONS: Inhibition of nitric oxide production with AG decreases BT after high-dose LPS challenge. The mechanism may involve increased cellular viability and decreased damage to the gut mucosal barrier in rats that receive AG.

Animals↗

Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans.

Prostaglandins are now known to be produced by Candida albicans and may play an important role in fungal colonization. Their synthesis in mammalian cells is decreased by inhibitors of the cyclooxygenase isoenzymes required for prostaglandin formation. In the present study, a catheter disk model system was used to investigate the effects of nonsteroidal anti-inflammatory drugs (all cyclooxygenase inhibitors) on biofilm formation by three strains of C. albicans. Seven of nine drugs tested at a concentration of 1 mM inhibited biofilm formation. Aspirin, etodolac, and diclofenac produced the greatest effects, with aspirin causing up to 95% inhibition. Celecoxib, nimesulide, ibuprofen, and meloxicam also inhibited biofilm formation, but to a lesser extent. Aspirin was active against growing and fully mature (48-h) biofilms; its effect was dose related, and it produced significant inhibition (20 to 80%) at pharmacological concentrations. Simultaneous addition of prostaglandin E(2) abolished the inhibitory effect of 25 or 50 micro M aspirin. At 1 mM, aspirin reduced the viability of biofilm organisms to 1.9% of that of controls. Surviving cells had a wrinkled appearance, as judged by scanning electron microscopy, and consisted of both yeasts and hyphae. Treatment with other cyclooxygenase inhibitors, such as etodolac, resulted in biofilms that consisted almost entirely of yeast cells. In conventional assays for germ tube formation, these drugs produced significant inhibition, whereas aspirin had little effect. Our findings suggest that cyclooxygenase-dependent synthesis of fungal prostaglandin(s) is important for both biofilm development and morphogenesis in C. albicans and may act as a regulator in these physiological processes. Our results also demonstrate that aspirin possesses potent antibiofilm activity in vitro and could be useful in combined therapy with conventional antifungal agents in the management of some biofilm-associated Candida infections.

Anti-Inflammatory Agents, Non-Steroidal↗

An antimicrobial effect associated with rabbit primary aqueous humour.

Viability curves were recorded for a variety of bacteria suspended in rabbit primary aqueous humour. Controls included neat rabbit serum, dilute rabbit serum (1/50), dilute heat treated fetal bovine serum (1/50), and balanced salt solution. Rabbit aqueous humour was bactericidal in vitro for Staphylococcus aureus and Pseudomonas aeruginosa. Micrococcus spp, Streptococcus pneumoniae and Escherichia coli were not affected. The factor mediating the bactericidal effect survives filtration (0.2 micron porosity) and freezing in liquid nitrogen (-196 degrees C) but is labile when stored in air at temperatures of 4 degrees C, 25 degrees C, and 37 degrees C for 1 hour. The bactericidal effect was stable when aqueous humour was stored in an atmosphere of 5% CO2 at 37 degrees C for 3 hours. The antibacterial component in rabbit aqueous humour has yet to be identified.

Animals↗

Effects of multiple deletions of murein hydrolases on viability, septum cleavage, and sensitivity to large toxic molecules in Escherichia coli.

The multiplicity of murein hydrolases found in most bacteria presents an obstacle to demonstrating the necessity of these potentially autolytic enzymes. Therefore, Escherichia coli mutants with deletions in multiple murein hydrolases, including lytic transglycosylases, amidases, and DD-endopeptidases, were constructed. Even a mutant from which seven different hydrolases were deleted was viable and grew at a normal rate. However, penicillin-induced lysis was retarded. Most of the mutants were affected in septum cleavage, which resulted in the formation of chains of cells. All three enzymes were shown to be capable of splitting the septum. Failure to cleave the septum resulted in an increase in outer membrane permeability, and thus the murein hydrolase mutants did not grow on MacConkey agar plates. In addition, the hydrolase mutants not only could be lysed by lysozyme in the absence of EDTA but also were sensitive to high-molecular-weight antibiotics, such as vancomycin and bacitracin, which are normally ineffective against E. coli.

Anti-Bacterial Agents↗

Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus.

AIMS: The development of a rapid method for the selective detection and enumeration of the total and viable vegetative cell and spore content of thermophilic bacilli in milk powder by PCR. METHODS AND RESULTS: Quantitative PCR and microscopy indicate the presence of up to 2.9 log units more cells in milk powder than accounted for by plate counting due to the majority of cells being killed during milk processing. Two approaches for viable and dead cell differentiation of thermophilic bacilli by quantitative PCR were evaluated, these being the nucleic binding dye ethidium monoazide (EMA) and DNase I digestion. The former agent exposed to a viable culture of Anoxybacillus flavithermus caused considerable cell inactivation. In contrast, DNase I treatment had no effect on cell viability and was utilized to develop DNA extraction methods for the differential enumeration of total, viable vegetative cells and spores in milk powder. Moreover, the methods were further applied and evaluated to 41 factory powder samples taken throughout eight process runs to assess changes in numbers of vegetative cells and spores with time. DNase I treatment reduced vegetative cell numbers enumerated with PCR by up to 2.6 log units. The quantification of spores in the factory milk powders investigated indicates on average the presence of 1.2 log units more spores than determined by plate counting. CONCLUSIONS: The method presented in this study provides the ability to selectively enumerate the total and viable cell and spore content of reconstituted milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides a tool to monitor the extent of thermophilic contamination during milk powder manufacturing 60-90 min after sampling.

Azides↗

Separation of high and low metastatic subpopulations from solid tumors by centrifugal elutriation.

We have isolated from murine solid tumors (B16a) subpopulations of cells possessing high and low metastatic potential. Tumors were dispersed by collagenase treatment. The resulting heterogeneous population of cells (i.e., viable and non-viable tumor cells and host cells) were separated by centrifugal elutriation. Four of the fractions (100, 180, 260, 340) contained tumor cells of high viability (greater than 95%) and high purity (less than 1% host cell contamination). The four fractions were characterized by flow cytometry and found to differ in distribution of cells in G1, S and G2. The cell populations were also found to differ in metastatic potential as determined by their ability to form lung colonies following intravenous injection. The 340 fraction was approximately 5-fold more metastatic than the 100 fraction. We also observed that cells from the 100 fraction failed to induce platelet aggregation whereas cells from the 340 fraction induced significant platelet aggregation. These observations demonstrate that cells of B16a tumors are heterogeneous for phenotypic characteristics (i.e., metastatic potential; platelet aggregation, etc.) and that their ability to induce platelet aggregation is positively correlated with metastatic potential.

Animals↗

A novel colorimetric broth microdilution method to determine the minimum inhibitory concentration (MIC) of antibiotics and essential oils against Helicobacter pylori.

Helicobacter pylori infections have been associated with the pathogenesis of a number of stomach and gastroduodenal diseases. In order to find alternative drugs for their treatment the search is increasingly focused on new antimicrobial products. However, no standardized methods are available to test the anti-Helicobacter pylori activity in particular of natural substances. Therefore we developed a broth microdilution assay to investigate the susceptibility of this fastidious slow growing bacterium against 15 essential oils widely used to treat disorders of the gastrointestinal tract. The MIC values were determined colorimetrically using p-iodonitrophenyltetrazolium violet (INT) as an indicator for bacterial cell viability. The test sytem was evaluated with three common antibiotics: amoxicillin, ampicillin and levofloxacin. The antibiotic MICs were controlled by Etest. The Helicobacter reference strain was remarkably susceptible to both the antibiotics (amoxicillin MIC: 0.02 microg/ml, ampicillin MIC: 0.064 microg/ml, levofloxacin MIC: 0.39 microg/ml) and the essential oils. Most of their MICs ranged from 0.015 to 0.064% (v/v) and about 140.0 to 280.0 microg/ml, respectively. Interestingly, chamomile oil, orange flower oil and ginger oil inhibited the bacterial growth in extraordinarily low concentrations of 0.0075% (v/v) and about 65 microg/ml, respectively. The bactericidal concentrations were generally one to two dilution steps higher. In conclusion, we could develop an innovative assay for the MIC determination of essential oils and antibiotics against Helicobacter pylori, which is simple to handle, accurate, reproducible and not as time- and material-consuming as traditional agar dilution techniques.

Anti-Bacterial Agents↗