Modeling the growth of Aspergillus brasiliensis affected by a nonthermal plasma.

AIM: The main objective of the study was to develop and validate a model for the growth of Aspergillus brasiliensis on surfaces, specifically on agar culture medium. An additional aim was to determine conditions for complete growth inhibition of this micromycete using two different nonthermal plasma (NTP) sources. METHODS AND RESULTS: The developed model uses two key parameters, namely the growth rate and growth delay, which depend on the cultivation temperature and the amount of inoculum. These parameters well describe the growth of A. brasiliensis and the effect of NTP on it. For complete fungus inactivation, a single 10-minute exposure to a diffuse coplanar surface barrier discharge was sufficient, while a point-to-ring corona discharge required several repeated 10-minute exposures at 24-h intervals. CONCLUSIONS: The article presents a model for simulating the surface growth of A. brasiliensis and evaluates the effectiveness of two NTP sources in deactivating fungi on agar media.

Defensin-like peptides from Capsicum chinense induce increased ROS, loss of mitochondrial functionality, and reduced growth of the fungus Colletotrichum scovillei.

In the present study, we identified and characterized two defensin-like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed-phase in a high-performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse-phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1-Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 µg mL-1, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 µg mL-1 and 200 µg mL-1, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1-Fraction indicated the presence of two defensin-like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1-Fraction was also found to inhibit the activity of insect α-amylases. In summary, the F1-Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin-like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.

Impact of Bt corn expressing Bacillus thuringiensis Berliner insecticidal proteins on the growth and survival of Spodoptera frugiperda larvae in Colombia.

Bioassays were conducted under controlled conditions to determine the response of Spodoptera frugiperda (J. E. Smith) larvae fed with corn materials expressing Bacillus thuringiensis (Bt) insecticidal endotoxins: (1) VT Double Pro® (VT2P) expressing Cry1A.105-Cry2Ab2 proteins and (2) VT Triple Pro® (VT3P) expressing Cry1A.105-Cry2Ab2-Cry3Bb1 proteins. The parameters assessed were: (i) mortality rate, and (ii) growth inhibition (GI) with respect to the control. To conduct this study, larvae were collected from commercial non-Bt corn fields, in four agricultural sub-regions in Colombia, between 2018 and 2020. Fifty-two populations were assessed from the field and neonate larvae from each of the populations were used for the bioassays. The study found that mortality rates in the regions for larvae fed with VT2P corn ranged from 95.1 to 100.0%, with a growth inhibition (%GI) higher than 76.0%. Similarly, mortality rate for larvae fed with VT3P corn were between 91.4 and 100.0%, with a %GI above 74.0%. The population collected in Agua Blanca (Espinal, Tolima; Colombia) in 2020, showed the lowest mortality rate of 53.2% and a %GI of 73.5%, with respect to the control. The population that exhibited the lowest %GI was collected in 2018 in Agua Blanca (Espinal, Tolima, Colombia) with a 30.2%, growth inhibition, with respect to the control. In recent years, the use of plant tissue to monitor susceptibility to fall armyworm has proven to be useful in the resistance management program for corn in Colombia determining that the FAW populations are still susceptible to Bt proteins contained in VT2P and VT3P.

Potential of yeasts as biocontrol agents against Fusarium graminearum in vitro and on corn.

AIMS: The antifungal effect of the yeast species Kluyveromyces marxianus, Meyerozyma caribbica, and Wickerhamomyces anomalus was evaluated against two Fusarium graminearum strains (FRS 26 and FSP 27) in vitro and on corn seeds. METHODS AND RESULTS: The antifungal effect of the yeasts against F. graminearum was evaluated using scanning electron microscopy and extracellular chitinase and glucanase production to further elucidate the biocontrol mode of action. In addition, the germination percentage and vigor test were investigated after applying yeast on corn seeds. All the yeast strains inhibited fungal growth in vitro (57.4%-100.0%) and on corn seeds (18.9%-87.2%). In co-culture with antagonistic yeasts, F. graminearum showed collapsed hyphae and turgidity loss, which could be related to the ability of yeasts to produce chitinases and glucanases. The three yeasts did not affect the seed corn germination, and W. anomalus and M. caribbica increased corn seed growth parameters (germination percentage, shoot and root length, and shoot dry weight). CONCLUSION: Meyerozyma caribbica and W. anomalus showed satisfactory F. graminearum growth inhibition rates and did not affect seed growth parameters. Further studies are required to evaluate the application of these yeasts to the crop in the field.

Aspergillus ochraceus biocontrol by Hanseniaspora opuntiae in vitro and on coffee fruits.

Aspergillus ochraceus is an ochratoxin-producing fungus which contaminates coffee. In this study the antifungal effect of the yeast Hanseniaspora opuntiae on three Aspergillus ochraceus strains (IOC 4417, IOC 4462, Ao 14) was evaluated in vitro and on coffee fruits. H. opuntiae (106 and 107 cells mL-1) reduced in vitro fungal growth from 82% to 87%, when co-cultivated with A. ochraceus. The yeast cell free supernatant (CFS) inhibited conidial germination from 76.5% to 92.5%, and hyphal growth from 54% to 78%. The yeast (107 and 109 cells mL-1) applied on coffee fruits delayed fruit decay by A. ochraceus (IOC 4417 and Ao 14) until the 9th day, and was significantly different (p < 0.05) from the controls. Furthermore, the ultrastructure of the yeast-fungus interaction on the coffee fruit surface showed yeast attachment to A. ochraceus hyphae, and morphological alterations in fungal structures, with hyphal abnormalities, such as tortuous hyphae with irregular, non-uniform surface compared to the control without yeast. H. opuntiae showed efficacy as biocontrol agent and, to the best of our knowledge, this is the first study on the antifungal activity of H. opuntiae against A. ochraceus on coffee fruits Nevertheless, application of H. opuntiae to the crop in the field requires further studies.

Combined Effect of High Hydrostatic Pressure and Proteolytic Fraction P1G10 from Vasconcellea cundinamarcensis Latex against Botrytis cinerea in Grape Juice.

The effect of high hydrostatic pressure (HHP) and the proteolytic fraction P1G10 from papaya latex was studied to find out whether a synergy exists in the growth inhibition of Botrytis cinerea in grape juice, contributing to the improvement of conservation techniques and extending the shelf life and quality of food products. Grape juice (GJ) diluted to 16 °Brix with a water activity (aw) of 0.980 was prepared from a concentrated GJ and used in this study. Results indicated a 92% growth inhibition of B. cinerea when exposed to 1 mg/mL of P1G10 and 250 MPa/4 min of pressure treatment. The proximate composition and antioxidant compounds present in the GJ were not significantly affected after the treatments. Eight phenolic compounds and two flavonoids in GJ were identified and quantified, with values fluctuating between 12.77 ± 0.51 and 240.40 ± 20.9 mg/L in the control sample (0.1 MPa). The phenolic compounds showed a significant decrease after the applied treatments, with the HHP sample having a content of 65.4 ± 6.9 mg GAE/100 mL GJ. In conclusion, a synergistic effect at moderate HHP of 250 MPa/4 min with the addition of P1G10 was observed, and the successful development of a stable and acceptable GJ product was possible.

Evaluación antifúngica del ozono contra Candida albicans: estudio in vitro / Antifungal evaluation of ozone against Candida albicans: in vitro study

Introducción: la Candida albicans (C. albicans) es un patógeno fúngico que puede causar infecciones superficiales o potencialmente mortales. Los biofilms de C. albicans muestran rasgos fenotípicos únicos, el más destacado es su notable resistencia a una amplia variedad de agentes antimicóticos. Una de las alternativas para inhibir el crecimiento de este microorganismo es el ozono debido a sus propiedades bactericidas, fungicidas y virucidas; sin embargo, escasa información ha sido reportada en C. albicans. Objetivo: el objetivo de este estudio fue evaluar el efecto fungicida del ozono en C. albicans. Material y métodos: la metodología consistió en agregar ozono a tubos de ensayo con medios de caldo nutritivo en diversas concentraciones y tiempos de ozonización. El efecto fungicida fue determinado con la determinación del número de colonias de C. albicans en agar nutritivo a través de procedimiento microbiológicos estandarizados por triplicado. Resultados: todas las muestras con ozono mostraron adecuados niveles de inhibición de crecimiento del microorganismo. Además, el efecto fungicida del ozono se encontró para ser significativamente dependiente del tiempo de ozonización y de la concentración. Conclusión: el uso de terapia con ozono podría tener potencial en el control de infecciones micóticas causadas por la presencia de C. albicans (AU)

Introduction: Candida albicans (C. albicans) is a fungal pathogen that can cause superficial or life-threatening infections. Biofilms of C. albicans display unique phenotypic traits, the most prominent being their remarkable resistance to a wide variety of antifungal agents. One of the alternatives to inhibit the growth of this microorganism is ozone due to its bactericidal, fungicidal and virucidal properties; however, little information has been reported on C. albicans. Objective: the objective of this study was to evaluate the fungicidal effect of ozone on C. albicans. Material and methods: the methodology consisted in adding ozone to test tubes with nutrient broth media in various concentrations and ozonation times. The fungicidal effect was determined by determining the number of colonies of C. albicans in nutrient agar through standardized microbiological procedures in triplicate. Results: all the ozone samples showed adequate levels of growth inhibition of the microorganism. Furthermore, the fungicidal effect of ozone was found to be significantly dependent on ozonation time and concentration. Conclusion: the use of ozone therapy could have potential in the control of fungal infections caused by the presence of C. albicans (AU)

Assessing the Larvicidal Properties of Endemic Campeche, Mexico Plant Piper cordoncillo var. apazoteanum (Piperaceae) against Aedes aegypti (Diptera: Culicidae) Mosquitoes.

The research aims to investigate the mortality effect of essential oil from Piper cordoncillo var. apazoteanum, an endemic plant from Campeche, Mexico, on early second-instar Aedes aegypti larvae; it also aims to identify the volatile compounds present in the fresh leaves of the plant. To test the effectiveness of the essential oil, we followed World Health Organization Standard Procedures. Larvae were observed for 17 consecutive days after treatment to determine the mortality and growth-inhibitory effect exerted by the essential oil. The results showed that the essential oil was effective in controlling mosquito populations. At a concentration of 800 ppm, the oil achieved an effectiveness rate of 70.00 ± 8.16% after 24 h, increasing to 100.00 ± 0.01% mortality after 72 h. With a concentration of 400 ppm, the effectiveness was 98.33 ± 0.17% by the end of the experiment. Furthermore, the obtained results demonstrated that the LC50 value was 61.84 ± 6.79 ppm, while the LC90 value was 167.20 ± 11.49 ppm. Essential oil concentrations inhibited the growth of immature insect stages, with concentrations between 800-100 ppm demonstrating very high inhibitory activity, and the lowest concentration of 50 ppm showing high inhibitory activity. The study also identified 24 chemical compounds representing 86.71% of the volatile compound composition of the fresh leaves of P. cordoncillo; the most abundant compounds were Safrole, Caryophyllene oxide, E-Nerolidol, and Calarene epoxide. The method used to extract the volatile compounds, solvent-free microwave extraction (SFME), is a promising alternative to traditional methods that avoids the use of potentially harmful solvents, making it more ecologically friendly and potentially safer for professionals handling the extracted compounds. Overall, the study demonstrates the potential of P. cordoncillo essential oil as an effective means of controlling mosquito populations, and provides valuable information on the chemical composition of the plant.Moreover, our study is the first to report on the biological activity and chemical composition of P. cordoncillo worldwide.

Phytotoxicity of 2,4-D and fipronil mixtures to three green manure species.

Sugarcane expansion has been associated with soil contamination by agrochemicals. Pesticides can affect plant growth, and their mixture might have potentiated effects on exposed species. This research aimed to evaluate the influence of fipronil on the phytotoxicity of 2,4-D on three green manure plant species: Canavalia ensiformis, Dolichos lablab, and Lupinus albus. Plants were exposed (for 21 days, at 25 °C) to a control soil and five concentrations of each pesticide and their combinations (36 treatments, considering a full-factorial approach). Effect concentrations of 50% growth inhibition (EC50) were estimated. No phytotoxicity effects were identified when plants were exposed to different fipronil concentrations (up to 0.12 mg kg-1). All species exposed to 2,4-D showed a decrease in shoot and root length and fresh/dry biomass. L. albus and D. lablab roots showed the highest sensitivity when exposed to 2,4-D among the endpoints (EC50 = 0.02 and 0.05 mg kg-1, respectively), while C. ensiformis roots were the most tolerant (EC50 = 0.98 mg kg-1). However, the interference of fipronil on the toxicity of 2,4-D was not detected in different mixture proportions, indicating no interaction between pesticides. Residues of 2,4-D might also impair other crops' growth, compromise productivity, and limit phytotechnologies for soil recovery.

Peptide-pulsed dendritic cells' immunomodulating effect regarding Mycobacterium tuberculosis growth in macrophages.

Mycobacterium tuberculosis is one of the most successful pathogens affecting humans, being the main cause of tuberculosis. It accounts for most infectious agent-related deaths worldwide; it has been estimated that a third of the world's population are bacillus carriers. This pathogen's evolutionary adaptation is mainly due to its ability to block a host's immune system by preventing it using an effective immune response in cases of active tuberculosis. Peptide-based synthetic vaccines represent an alternative for counteracting tuberculosis; however, although peptide antigens can be identified, they are not recognised by a host's immune system. An approach using dendritic cells as immunomodulating agents for increasing synthetic peptides' antigenic capacity has thus been advanced. Dendritic cells obtained from IL to 4- and GM-CSF-treated peripheral blood mononuclear cells were pulsed with synthetic Mtb protein peptides which have been reported as participating in mycobacteria-host interactions; their amino acid sequences were modified to improve MHC-II coupling and thus increase their recognition by a host's immune system. pMHC-II/TCR interaction triggered a lymphocyte response which controlled Mtb intracellular growth in infected macrophages. This work has been aimed at contributing to understanding dendritic cells' role in Mycobacterium tuberculosis protein peptide antigen presentation, thereby increasing individuals' immune response as a means of controlling the disease.

Inhibition of Serine Protease, α-Amylase and Growth of Phytopathogenic Fungi by Antimicrobial Peptides from Capsicum chinense Fruits.

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

Inhibition of adenosine deaminase activity reverses resistance to the cytotoxic effect of high adenosine levels in cervical cancer cells.

Adenosine (ADO) generation in the tumor microenvironment (TME) plays important roles in the promotion of tumor growth, invasion, and metastasis and in suppression of the antitumor immune response. Recently, adenosine deaminase (ADA) activity in the TME has been proposed to be a compensatory mechanism against toxic accumulation of ADO in cancerous tissues. In the present study, the expression and functional activity of ADA in cervical cancer (CeCa) tumor cells were analyzed: C33A (HPV-), CaSki (HPV + ), and HeLa (HPV + ) cells. CeCa tumor cells, as well as activated T lymphocytes (ATLs), which were used as a positive control, showed different ADA contents in the membrane and intracellularly and a strong ability to convert ADO into inosine (INO). Treatment of tumor cells with EHNA, a specific ADA inhibitor, decreased the viability of CeCa tumor cells in a dose-dependent manner. In C33A (EHNA half maximal inhibitory concentration (IC50) = 374 µM), CaSki (EHNA IC50 = 273.6 µM), and HeLa (EHNA IC50 = 252.2 µM) cells, EHNA strongly reversed the resistance of tumor cells to the cytotoxic effect of high concentrations of ADO; 38.82 ± 3.1%, 47.18 ± 4.7%, and 71.63 ± 6.9% of the cells were apoptotic, and 40 ± 4.8%, 52 ± 5.3% and 70 ± 6.8% of the cells had mitochondrial membrane damage, respectively. In ATLs (EHNA IC50 = 391.8 µM) treated with EHNA, 32.4 ± 4.4% were apoptotic, and 32 ± 4.3% had mitochondrial membrane damage. These results suggest that the presence and activity of ADA in CeCa tumor cells can provide protection against the cytotoxic effect of high ADO contents in the TME. Therefore, the inhibition of ADA could be a strategy for the treatment of CeCa.

Diatraea saccharalis harbors microorganisms that can affect growth of sugarcane stalk-dwelling fungi.

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), the sugarcane borer, spends most of its life cycle inside the galleries it burrows into sugarcane stalk, where two rot-causing fungi Colletotrichum falcatum (Went, 1893) and Fusarium verticillioides (Nirenberg, 1976) are commonly found. Results have shown that microbiota harbored by D. saccharalis inhibits the growth of F. verticillioides and C. falcatum. D. saccharalis larvae were collected from chemical-free field plants, and yeast and bacteria from third and fourth-instar D. saccharalis regurgitate were isolated onto appropriate media. The percentage of F. verticillioides and C. falcatum mycelial growth inhibition was recorded. Out of 32 yeast isolates, 9 exerted 30 to 40% growth inhibition of C. falcatum or F. verticillioides. When 24 bacterial isolates were confronted with rot-causing fungi, six inhibited C. falcatum growth by 30 to 60%, and 24 isolates inhibited 30 to 60% of F. verticillioides growth. Bacteria and yeast isolates were identified through DNA sequencing of part of 16S rDNA and part of ITS1-5.8S-ITS2, respectively, revealing an abundance of isolates with sequence similarity to Klebsiella and Bacillus and Meyerozyma, which have been used as biological control agents and their ability to promote plant growth has been demonstrated. We have shown that microorganisms from borer regurgitate inhibit phytopathogen growth in vitro. Still, further investigation of the possible functions of D. saccharalis-associated microorganisms may help understand their ecological role in plant-insect-phytopathogen interaction.

Biological Control of Charcoal Rot in Peanut Crop through Strains of Trichoderma spp., in Puebla, Mexico.

Charcoal rot is an emerging disease for peanut crops caused by the fungus Macrophomina phaseolina. In Mexico, peanut crop represents an important productive activity for various rural areas; however, charcoal rot affects producers economically. The objectives of this research were: (a) to identify and morphologically characterize the strain "PUE 4.0" associated with charcoal rot of peanut crops from Buenavista de Benito Juárez, belonging to the municipality of Chietla in Puebla, Mexico; (b) determine the in vitro and in vivo antagonist activity of five Trichoderma species on M. phaseolina, and (c) determine the effect of the incidence of the disease on peanut production in the field. Vegetable tissue samples were collected from peanut crops in Puebla, Mexico with the presence of symptoms of charcoal rot at the stem and root level. The "PUE 4.0" strain presented 100% identity with M. phaseolina, the cause of charcoal rot in peanut crops from Buenavista de Benito Juárez. T. koningiopsis (T-K11) showed the highest development rate, the best growth speed, and the highest percentage of radial growth inhibition (PIRG) over M. phaseolina (71.11%) under in vitro conditions, in addition, T. koningiopsis (T-K11) showed higher production (1.60 ± 0.01 t/ha-1) and lower incidence of charcoal rot under field conditions. The lowest production with the highest incidence of the disease occurred in plants inoculated only with M. phaseolina (0.67 ± 0.01 t/ha-1) where elongated reddish-brown lesions were observed that covered 40% of the total surface of the main root.

Exogenous ethylene reduces growth via alterations in central metabolism and cell wall composition in tomato (Solanum lycopersicum).

Ethylene is a gaseous hormone with a well-established role in the regulation of plant growth and development. However, its role in the modulation of carbon assimilation and central metabolism remains unclear. Here, we investigated the morphophysiological and biochemical responses of tomato plants (Solanum lycopersicum) following the application of ethylene in the form of ethephon (CEPA - 2-chloroethylphosphonic acid), forcing the classical triple response phenotype. CEPA-treated plants were characterized by growth inhibition, as revealed by significant reductions in both shoot and root dry weights, coupled with a reduced number of leaves and lower specific leaf area. Growth inhibition was associated with a reduction in carbon assimilation due to both lower photosynthesis rates and stomatal conductance, coupled with impairments in carbohydrate turnover. Furthermore, exogenous ethylene led to the accumulation of cell wall compounds (i.e., cellulose and lignin) and phenolics, indicating that exposure to exogenous ethylene also led to changes in specialized metabolism. Collectively, our findings demonstrate that exogenous ethylene disrupts plant growth and leaf structure by affecting both central and specialized metabolism, especially that involved in carbohydrate turnover and cell wall biosynthesis, ultimately leading to metabolic responses that mimic stress situations.

Comparative Growth Inhibition of Bread Spoilage Fungi by Different Preservative Concentrations Using a Rapid Turbidimetric Assay System.

Bread and intermediate moisture bakery products are mainly spoiled by yeasts and filamentous fungi. The inoculum load and preservation system used determines their shelf life. To extend the shelf life of such commodities, the use of chemical preservatives is the most common way to try and control the initiation of mold spoilage of bread. This study has utilized a rapid turbidimetric assay system (Bioscreen C) to examine the temporal efficacy of calcium propionate (CP) and potassium sorbate (PS) for controlling the growth of important bread spoilage fungi. The objectives were to compare the temporal growth of strains of three important spoilage fungi Hyphopichia burtonii (HB17), Paecilomyces variotii (PV11), and Penicillium roqueforti (PR06) isolated from visibly molded bread to (a) different concentrations of CP and PS (0-128 mM), (b) temperatures (25°C, 30°C), (c) water activity (aw; 0.95, 0.97), and (d) pH (5.0, 5.5). All three abiotic factors, pH, aw, and temperature, and preservative concentrations influenced the relative growth of the species examined. In general, PS was more effective than CP in inhibiting the growth of the strains of these three species. In addition, the Time to Detection (TTD) for the efficacy of the preservatives under the interacting abiotic factors was compared. The strain of Paecilomyces variotii (PV10) was the most tolerant to the preservatives, with the shortest TTD values for both preservatives. P. roqueforti was the most sensitive with the longest TTD values under all conditions examined. These results are discussed in the context of the evolution of resistance to food-grade preservatives by such spoilage fungi in bakery products.

Synthesis and Nano-Sized Characterization of Bioactive Oregano Essential Oil Molecule-Loaded Small Unilamellar Nanoliposomes with Antifungal Potentialities.

The development of greener nano-constructs with noteworthy biological activity is of supreme interest, as a robust choice to minimize the extensive use of synthetic drugs. Essential oils (EOs) and their constituents offer medicinal potentialities because of their extensive biological activity, including the inhibition of fungi species. However, their application as natural antifungal agents are limited due to their volatility, low stability, and restricted administration routes. Nanotechnology is receiving particular attention to overcome the drawbacks of EOs such as volatility, degradation, and high sensitivity to environmental/external factors. For the aforementioned reasons, nanoencapsulation of bioactive compounds, for instance, EOs, facilitates protection and controlled-release attributes. Nanoliposomes are bilayer vesicles, at nanoscale, composed of phospholipids, and can encapsulate hydrophilic and hydrophobic compounds. Considering the above critiques, herein, we report the in-house fabrication and nano-size characterization of bioactive oregano essential oil (Origanum vulgare L.) (OEO) molecules loaded with small unilamellar vesicles (SUV) nanoliposomes. The study was focused on three main points: (1) multi-compositional fabrication nanoliposomes using a thin film hydration-sonication method; (2) nano-size characterization using various analytical and imaging techniques; and (3) antifungal efficacy of as-developed OEO nanoliposomes against Trichophyton rubrum (T. rubrum) by performing the mycelial growth inhibition test (MGI). The mean size of the nanoliposomes was around 77.46 ± 0.66 nm and 110.4 ± 0.98 nm, polydispersity index (PdI) of 0.413 ± 0.015, zeta potential values up to -36.94 ± 0.36 mV were obtained by dynamic light scattering (DLS). and spherical morphology was confirmed by scanning electron microscopy (SEM). The presence of OEO into nanoliposomes was displayed by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. Entrapment efficiency values of 79.55 ± 6.9% were achieved for OEO nanoliposomes. In vitro antifungal activity of nanoliposomes tested against T. rubrum strains revealed that OEO nanoliposomes exhibited the highest MGI, 81.66 ± 0.86%, at a concentration of 1.5 µL/mL compared to the rest of the formulations. In summary, this work showed that bioactive OEO molecules with loaded nanoliposomes could be used as natural antifungal agents for therapeutical purposes against T. rubrum.

Mycobacterial Growth Inhibition Assay (MGIA) as a Host Directed Diagnostic Tool for the Evaluation of the Immune Response in Subjects Living With Type 2 Diabetes Mellitus.

The lack of efficient and cost-effective diagnostic tools contributes to poor control of tuberculosis in endemic countries. Moreover, host biological processes influence susceptibility, and infection resolution. It is well known that comorbidities such as type 2 diabetes mellitus (DM2) affect the host immune response, making individuals more susceptible to Mycobacterium tuberculosis infection. Currently, there are no laboratory tools that can identify those subjects who have a higher risk of developing the disease. In this study, we used a whole blood mycobacterial growth inhibition assay to assess the immune response capacity to inhibit mycobacterial growth between healthy subjects and those living with DM2 with optimal and poor glycemic control. We also measured cytokine levels in the culture supernatant by cytokine bead arrays. We included 89 patients with DM2: 54 patients with optimal control (mean age 56.2 ± 11.75 years) and 35 patients with poor control (mean age 52.05 ± 9.94 years). We also included 44 healthy subjects as controls (mean age 42.12 ± 11.75 years). We compared the Δlog UFC (a value that represents the difference between mycobacterial growth in the control tube versus the subject's blood) between each group. Our results demonstrate that patients with DM2 had a lower capacity to inhibit M. tuberculosis growth (Δlog UFC DM2 subjects 0.9581 (-0.3897 to 2.495) vs Δlog UFC healthy subjects 0.7190 (-0.2678 to 2.098); p=0.013). Comparing subjects living with DM2 (optimal and poor glycemic control) vs healthy subjects, we found only significant differences between healthy subjects and patients poorly controlled (Δlog UFC optimal control group 0.876 (-0.3897 to 2.495); Δlog UFC poor control group 1.078 (0.068 to 2.33); Δlog UFC healthy subjects 0.7190 (-0.2678 to 2.098); p= 0.022). Therefore, glycemic control assessed by glycosylated hemoglobin values influences the capacity of the host to control the infection. Our results confirm that the whole blood mycobacterial growth inhibition assay has potential utility as an in vitro marker of M. tuberculosis immunological control in vivo in subjects living with DM2. This assay can be used to evaluate the immune response of each individual against M. tuberculosis, allowing clinicians to choose a more specific host-directed therapy.

Effect of eight common Brazilian drugs on Lemna minor and Salvinia auriculata growth.

The growth of two species of macrophytes (Lemna minor and Salvinia auriculata) under the effect of a mixture of amoxicillin, caffeine, carbamazepine, dipyrone, ibuprofen, losartan, omeprazole, and tenivastatin was investigated by bioassay. Three concentration levels were utilized in this study (10, 200, and 500 µg L-1) using a growth inhibition test based on the OECD 221/2006 guidelines. The frond number, total area, and chlorophyll a level were selected as suitable end points. For L. minor, at all concentrations, a significant difference in the total frond number was observed and the growth inhibition varied from 30 to 70% at the low and high concentrations, respectively. No significant growth change was observed to S. auriculata exposed to the mixture of drugs. Thus, individual drug tests were performed for L. minor which demonstrated stimulation in growth, when exposed to most drugs individually, except tenivastatin which was identified as the drug responsible for the significant growth inhibition seen in the mixture. The L. minor enhanced growth was probably caused by N molecule transformation to ammonium and nitrate, essential nutrients for plants.

Chemical Genetics to Uncover Mechanisms Underlying Lipid-Mediated Signaling Events in Plants.

Like animals, plants use various lipids as signaling molecules to guide their growth and development. The focus of our work is on the N-acylethanolamine (NAE) group of lipid mediators, which have been shown to play important physiological roles in plants. However, mechanisms by which NAEs modulate plant function remain elusive. Chemical genetics has emerged as a potent tool to elucidate signaling pathways in plants, particularly those orchestrated by plant hormones. Like plant hormones, exogenous application of NAEs elicits distinct plant growth phenotypes that can serve as biological readouts for chemical genetic screens. For example, N-lauroylethanolamide (NAE 12:0) inhibits seedling development in the model plant Arabidopsis thaliana. Thus, a library of small synthetic chemical compounds can be rapidly screened for their ability to reverse the inhibitory effect of NAE 12:0 on seedling development. Chemicals identified through such screens could be potential agonists/antagonists of NAE receptors or signaling pathways and therefore serve as additional tools for understanding NAE function in plants. In this chapter, we describe general protocols for NAE 12:0-based chemical genetic screens in Arabidopsis. Although such screens were designed primarily for NAE 12:0, they could potentially be applied for similar work with other NAE species or plant lipid mediators.