Intracellular Aminopeptidase Activity Determination from the Fungus Sporisorium reilianum: Purification and Biochemical Characterization of psrAPEi Enzyme.

The aims of this study were to, first, determine the intracellular aminopeptidase activity (APEi) and second, purify and biochemically characterize one intracellular aminopeptidase enzyme from the phytopathogen fungus Sporisorium reilianum (psrAPEi), the causal agent of head smut in corn. The fungus produced APEi activity in all media cultures evaluated. The psrAPEi was purified by a procedure that involved ammonium sulfate fractionation and four chromatographic steps using an FPLC system (Fast Protein Liquid Chromatography). Results showed an estimated molecular mass of 52.2 kDa. Enzymatic activity was optimal at pH 7.0 and 35 °C and was inhibited by EDTA-Na2, 1,10-phenanthroline, bestatin, and PMSF. This aminopeptidase showed a preference for leucine, arginine, and lysine at the N-position. The Km and Vmax values were 3.72 µM and 188.0 µmol/min, respectively, for L-lysyl-4-nitroanilide. This is the first study to report on intracellular aminopeptidase activity in S. reilianum and the purification and characterization of an intracellular metallo-serine-aminopeptidase (psrAPEi).

Evaluation in the performance of the biodegradation of herbicide diuron to high concentrations by Lysinibacillus fusiformis acclimatized by sequential batch culture.

We evaluated and characterized the biodegradation of the herbicide diuron in its commercial form above its saturation concentration by Lysinibacillus fusiformis acclimatized by sequential batch culturing. Acclimatization was carried out in eight cycles in liquid culture, improving the capacity of L. fusiformis to remove diuron from 55.13 ± 1.3% in the first batch to 87.2 ± 0.11% in the eighth batch. Diuron biosorption was characterized with Langmuir and Freundlich isotherms, obtaining a maximum biosorption (qmax) of 0.00885 mg mg-1. In diuron biodegradation assays, a consumption substrate biomass yield (YSD/X) of 6.266 mg mg-1 was obtained, showing that biodegradation was the main mechanism in diuron removal. Diuron biodegradation by L. fusiformis was characterized by the Monod model, with a maximum specific growth rate (µmax) of 0.0245 h-1 and an affinity constant (KSD) of 344.09 mg L-1. A low accumulation of 3,4-dichloroaniline with the production of chloride ions indicated dechlorination when diuron was present at high concentrations. A phytotoxic assay conducted with Lactuca sativa showed that the toxicity of an effluent with diuron at 250 mg L-1 decreased when it was pretreated with acclimatized L. fusiformis. Acclimatization by sequential batch culturing improved the ability of L. fusiformis to biodegrade diuron at high concentrations, showing potential in the bioremediation of diuron-contaminated sites.

Purification and characterization of the extracellular aspartyl protease APSm1 from the phytopathogen fungus Stenocarpella maydis.

The extracellular protease APSm1 was purified to homogeneity from Stenocarpella maydis that was grown in acidic minimal media with glucose and ammonium sulfate. The purification procedure consisted of ion exchange chromatography coupled to an FPLC (Fast Protein Liquid Chromatography) system, resulting in a 15.3% recovery and a 2.3-fold increase in specific activity. The molecular weight of the purified enzyme was estimated to be 56.8 kDa by SDS-PAGE. Enzymatic activity toward hemoglobin was optimal at pH 2.0 and at 25 °C. The effects of six protease inhibitors on APSm1 activity were tested. Pepstatin A inhibited APSm1 activity, as the protein is in fact an aspartyl protease. The pure enzyme degraded hemoglobin, albumin and proteins obtained from corn germ at pH 3 but did not have any milk-clotting activities. The Km and Vmax values obtained were 0.514 mg/mL and 0.222 µmol/min, respectively, using hemoglobin as the substrate. This work is the first to report the purification of a secreted aspartyl protease from S. maydis.

Biochemical study of the extracellular aspartyl protease Eap1 from the phytopathogen fungus Sporisorium reilianum.

In this work, the extracellular protease Eap1 from Sporisorium reilianum was characterized in solid and liquid cultures using different culture media. The results showed that Eap1 was produced in all media and under all culture conditions, with the most activity in solid culture at an acidic pH of 3-5. Following purification, the 41 kDa protease demonstrated aspartyl protease activity. The enzyme was stable at a wide range of temperatures and pH values, but 45°C and pH 3 were optimal. The K(m) and V(max( values obtained were 0.69 mg/mL and 0.66 µmol/min, respectively, with albumin as the substrate. Eap1 degraded hemoglobin as well as proteins obtained from corn germ, roots, stems and slides at pH 3 and also had milk-clotting activity. Sequencing analysis showed that this protein has 100% similarity to the peptide sequence theoretically obtained from the sr11394 gene, which encodes an aspartyl protease secreted by S. reilianum.

Isolation and selection of autochthonous strains of Trichoderma spp. with inhibitory activity against Sporisorium reilianum.

Head smut is a worldwide disease caused by the fungus Sporisorium reilianum. In Mexico, this phytosanitary problem has been described in the central part of the country, specifically in the Mezquital Valley in the state of Hidalgo, where this basidiomycete causes significant economic losses. In this work, seven strains of Trichoderma spp. were isolated from corn rhizospheres collected from crops in the affected zone. The isolates were identified as Trichoderma asperellum MH1, T. asperellum T4H1, T. harzianum T1H1, T. harzianum T1H3, T. atrobrunneum T1H2, T. tomentosum T2H4, and T. brevicompactum T3H1. All strains showed the ability to grow on the phytopathogen but with distinct degrees of mycoparasitism. SEM observations demonstrated the ability of T. asperellum T4H1 to invade the S. reilianum yeast growth. All the strains produced volatile compounds with antifungal activity. With the exception of T. asperellum MH1, all strains inhibited the development of the pathogen by means of non-volatile compounds. Production of the extracellular enzymes (lipase, cellulase, chitinase, protease, and laccase) was evaluated, with most strains presenting high lipolytic activity and low proteolytic activity. The production of cellulase and chitinase was observed only in five strains. Laccase production was found in three isolates. Evaluations at the greenhouse of the sequential application of three mixtures of the isolates were conducted in a greenhouse; findings showed that the phytopathogen was not detected by specific PCR in the plants that received the treatment.

Effect of ß - hydroxy - γ -aminophosphonate (ß - HPC) on the hydrolytic activity of Nocardia brasiliensis as determined by FT-IR spectrometry.

The use of immunomodulatory and metabolic modulating drugs has been considered a better strategy to improve the efficacy of conventional treatments against pathogens and metabolic diseases. L-carnitine is relevant in fatty acid metabolism and energy production by ß-oxidation, but it also has a beneficial therapeutic immunomodulatory effect. The ß-hydroxy-γ-aminophosphonate (ß-HPC) was developed, synthesized and studied in different pathologies as a more soluble and stable analog than L-carnitine, which has been studied in bacterial physiology and metabolism; therefore, we set out to investigate the direct effect of ß-HPC on the metabolism of N. brasiliensis, which causes actinomycetoma in Mexico and is underdiagnosed. To analyze the effect of ß-HPC on the metabolic capacity of the bacterium for the hydrolysis of substrate casein, L-tyrosine, egg yolk, and tween 80, Fourier transform infrared spectroscopy (FT-IR) was employed. It was found that ß-HPC increases the metabolic activity of N. brasiliensis associated with increased growth and increased hydrolysis of the substrates tested. By the effect of ß-HPC, it was observed that, in the hydrolysis of L-tyrosine, the aromatic ring and functional groups were degraded. At 1515 cm-1, any distinctive signal or peak for this amino acid was missing, almost disappearing at 839, 720, 647, and 550 cm-1. In casein, hydrolysis is enhanced in the substrate, which is evident by the presence of NH, OH, amide, and CO. In casein, hydrolysis is enhanced in the substrate, which is evident by the presence of NH, OH, amide, COO, and P = O signals, characteristic of amino acids, in addition to the increase of the amide I and II bands. In Tween 80 the H-C = and C = C signals disappear and the ether signals are concentrated, it was distinguished by the intense band at 1100 cm-1. Egg yolk showed a large accumulation of phosphate groups at 1071 cm-1, where phosvitin is located. FT-IR has served to demonstrate that ß-HPC is a hydrolysis enhancer. Furthermore, by obtaining the spectrum of N. brasiliensis, we intend to use it as a quick comparison tool with other spectra related to actinobacteria. Eventually, FT-IR may serve as a species identification option.

ß-Xylosidase SRBX1 Activity from Sporisorium reilianum and Its Synergism with Xylanase SRXL1 in Xylose Release from Corn Hemicellulose.

Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-ß-1,4-xylanases and one with α-L-arabinofuranosidase activity. In this study, the native protein extracellular with ß-xylosidase activity, called SRBX1, produced by this basidiomycete was analyzed by performing production kinetics and its subsequent purification by gel filtration. The enzyme was characterized biochemically and sequenced. Finally, its synergism with Xylanase SRXL1 was determined. Its activity was higher in a medium with corn hemicellulose and glucose as carbon sources. The purified protein was a monomer associated with the sr16700 gene, with a molecular weight of 117 kDa and optimal activity at 60 °C in a pH range of 4-7, which had the ability to hydrolyze the ρ-nitrophenyl ß-D-xylanopyranoside and ρ-Nitrophenyl α-L-arabinofuranoside substrates. Its activity was strongly inhibited by silver ions and presented Km and Vmax values of 2.5 mM and 0.2 µmol/min/mg, respectively, using ρ-nitrophenyl ß-D-xylanopyranoside as a substrate. The enzyme degrades corn hemicellulose and birch xylan in combination and in sequential synergism with the xylanase SRXL1.

Pressure Drives Rapid Burst-Like Coordinated Cellular Motion from 3D Cancer Aggregates.

A key behavior observed during morphogenesis, wound healing, and cancer invasion is that of collective and coordinated cellular motion. Hence, understanding the different aspects of such coordinated migration is fundamental for describing and treating cancer and other pathological defects. In general, individual cells exert forces on their environment in order to move, and collective motion is coordinated by cell-cell adhesion-based forces. However, this notion ignores other mechanisms that encourage cellular movement, such as pressure differences. Here, using model tumors, it is found that increased pressure drove coordinated cellular motion independent of cell-cell adhesion by triggering cell swelling in a soft extracellular matrix (ECM). In the resulting phenotype, a rapid burst-like stream of cervical cancer cells emerged from 3D aggregates embedded in soft collagen matrices (0.5 mg mL-1 ). This fluid-like pushing mechanism, recorded within 8 h after embedding, shows high cell velocities and super-diffusive motion. Because the swelling in this model system critically depends on integrin-mediated cell-ECM adhesions and cellular contractility, the swelling is likely triggered by unsustained mechanotransduction, providing new evidence that pressure-driven effects must be considered to more completely understand the mechanical forces involved in cell and tissue movement as well as invasion.

Isolation of bacteria with antifungal activity against the phytopathogenic fungi Stenocarpella maydis and Stenocarpella macrospora.

Stenocarpella maydis and Stenocarpella macrospora are the causal agents of ear rot in corn, which is one of the most destructive diseases in this crop worldwide. These fungi are important mycotoxin producers that cause different pathologies in farmed animals and represent an important risk for humans. In this work, 160 strains were isolated from soil of corn crops of which 10 showed antifungal activity against these phytopathogens, which, were identified as: Bacillus subtilis, Pseudomonas spp., Pseudomonas fluorescens, and Pantoea agglomerans by sequencing of 16S rRNA gene and the phylogenetic analysis. From cultures of each strain, extracellular filtrates were obtained and assayed to determine antifungal activity. The best filtrates were obtained in the stationary phase of B. subtilis cultures that were stable to the temperature and extreme pH values; in addition they did not show a cytotoxicity effect against brine shrimp and inhibited germination of conidia. The bacteria described in this work have the potential to be used in the control of white ear rot disease.

Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis.

Tension and mechanical properties of muscle tissue are tightly related to proper skeletal muscle function, which makes experimental access to the biomechanics of muscle tissue formation a key requirement to advance our understanding of muscle function and development. Recently developed elastic in vitro culture chambers allow for raising 3D muscle tissue under controlled conditions and to measure global tissue force generation. However, these chambers are inherently incompatible with high-resolution microscopy limiting their usability to global force measurements, and preventing the exploitation of modern fluorescence based investigation methods for live and dynamic measurements. Here, we present a new chamber design pairing global force measurements, quantified from post-deflection, with local tension measurements obtained from elastic hydrogel beads embedded in muscle tissue. High-resolution 3D video microscopy of engineered muscle formation, enabled by the new chamber, shows an early mechanical tissue homeostasis that remains stable in spite of continued myotube maturation.

Detection of SARS-CoV-2 in Wastewater Northeast of Mexico City: Strategy for Monitoring and Prevalence of COVID-19.

A month-long wastewater sampling project was conducted along the northeast periphery of Mexico City, specifically in the state of Hidalgo, to assess the presence of SARS-CoV-2. To determine the prevalence of infection and obtain a range of COVID-19 cases in the main metropolitan zones. Viral RNA residues (0-197,655 copies/L) were measured in wastewater from the five central municipalities in the state. By recording the number of RNA viral copies per liter, micro-basins delimitation, demographic and physiological data, an interval of infected people and virus prevalence was estimated using a Monte Carlo model (with 90% confidence) in the micro-basin of five municipalities with metropolitan influence or industrial activity. Our procedure determined that the percentage of the infected population ranges from 1.4% to 41.7%, while the official data reports 0.1-0.3%. This model is proposed as a helpful method of regional epidemiological monitoring through the analysis of viral prevalence.

Physicochemical characterization of an arabinoxylan-rich fraction from brewers' spent grain and its application as a release matrix for caffeine.

The brewers' spent grain is a by-product generated during brewery process and is a potential source for arabinoxylans (AX) extraction. In the present work, the extraction and characterization of an arabinoxylan-rich fraction from brewers' spent grain (BSG-AX) were performed, and BSG-AX was evaluated as release matrix for caffeine. The BSG-AX showed an AX content of 72% (w/w), a ferulic acid content of 3.52 µg/mg BSG-AX, an Ara/Xyl ratio of 0.89, an intrinsic viscosity of 41.18 mL g-1, and a molecular weight of 43.80 kDa. The studied BSG-AX showed a good antioxidant capacity compared with other polysaccharide gums and was estimated by DPPH (114.41 µM Trolox equivalent/g BSG-AX) and FRAP (49.01 µmol Fe2+/g BSG-AX) assays. The partial specific volume (0.63 cm3 g-1), loss on drying (10.68%), swelling (10.87%), solubility (80.93%) and electrostatic interactions (by zeta potential, -3.44 to -9.17 mV) were determined and used to evaluate the application of the BSG-AX as release matrix. A film containing the BSG-AX, glycerol (as plasticizer) and caffeine (target drug) was prepared as release matrix. Glycerol promoted an increase in the extensibility and the surface smoothness of the BSG-AX-caffeine film. The drug was released (≈98%) in about 7 h. These results are promising to concern the design and use of BSG-AX based biofilms for the controlled release of bioactive compounds.

Use of water hyacinth as a substrate for the production of filamentous fungal hydrolytic enzymes in solid-state fermentation.

The objective of the present work was to evaluate the water hyacinth (WH) as a substrate for the production of hydrolytic enzymes (cellulases and hemicellulases) of 100 strains of filamentous fungi under conditions of solid growth. Five fungal strains, identified as Trichoderma harzianum, Trichoderma atroviride, Penicillium griseofulvum, Penicillium commune and Aspergillus versicolor, were selected and studied for their ability to grow on water hyacinth as a substrate and carbon source only, evaluating hydrolytic enzymatic activities (α-l-arabinofuranosidase, cellulase, xylanase and ß-d-xylopyranosidase) and extracellular protein per g of water hyacinth dry matter (gdm). The five strains selected were able to produce the four enzymes studied; however, T. harzianum strain PBCA produces the highest xylanase (149.3 ± 14.3 IU/gdm at 108 h), cellulase (16.4 ± 0.6 IU/gdm at 84 h) and ß-d-xylopyranosidase (127.7 ± 14.8 IU/gdm at 48 h). In contrast, the fungus with the highest α-l-arabinofuranosidase activity was A. versicolor, with 129.8 ± 13.3 IU/gdm after 108 h. In conclusion, T. harzianum showed the best production of the hydrolytic enzymes studied, using as a matrix and carbon source, water hyacinth. In addition, catalytic activities of arabinofuranosidase and xylopyranosidase were reported for the first time in T. versicolor and T. harzianum.

Nutritional Characterization of Prosopis laevigata Legume Tree (Mesquite) Seed Flour and the Effect of Extrusion Cooking on its Bioactive Components.

Mesquite (Prosopis laevigata) is a legume tree widely distributed in Aridoamerica. The mature fruit of this legume is a pod, which is currently underutilized and has high nutritional potential. In the present work, mesquite seed flour is described in terms of its nutritional value, as well as the effect of extrusion cooking on its bioactive components. Mesquite seed flour is rich in fiber (7.73 g/100 g) and protein (36.51 g/100 g), with valine as the only limiting amino acid. Total phenolic compound contents in raw and extruded seed flour were 6.68 and 6.46 mg of gallic acid equivalents/g (mg GAE/g), respectively. 2-2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity values in raw and extruded seed flour were 9.11 and 9.32 mg of ascorbic acid equivalent/g (mg AAE/g), respectively. The absorbance at 290 nm, as an indicator of generation of Maillard reaction product (MRP), was the same for raw and extruded samples. Apigenin was the only flavonoid found in mesquite seed flour (41.6 mg/kg) and was stable in the extrusion process. The water absorption index (WAI) and water solubility index (WSI) were changed significantly during extrusion. The expansion of mesquite seed flour extrudates was null due to the high protein and fiber content in the sample. Extrusion cooking of mesquite seed flour is a useful form of technology for the industrialization of this underutilized and nutritionally valuable legume.

Presence of Multidrug-Resistant Shiga Toxin-Producing Escherichia coli, Enteropathogenic Escherichia coli, and Enterotoxigenic Escherichia coli on Fresh Cheeses from Local Retail Markets in Mexico.

Cheesemaking is one of the most important industries in Mexico. Among all the Mexican cheeses, fresh cheeses are the most popular and most consumed cheese in Mexico and Latin America. However, in Mexico fresh cheese is frequently made with unpasteurized milk and sold in public markets. This may increase the risk for contamination of dairy products with pathogenic bacteria. The presence of multidrug-resistant pathogenic bacteria in food is an important public health concern. Diarrheagenic Escherichia coli pathotypes (DEPs) are foodborne bacteria. This study investigated the presence of indicator bacteria and multidrug-resistant DEPs in fresh cheeses. A total of 120 fresh cheese samples were collected from public markets in the city of Pachuca, Mexico. The samples were analyzed for presence of fecal coliforms (FC), E. coli, and antibiotic resistant DEPs. FC and E. coli were analyzed using the most-probable-number technique. DEPs were identified using two multiplex PCR methods. Susceptibility to 16 antibiotics was tested for the isolated DEPs strains by the standard assay. The frequency of FC, E. coli, and DEPs in the cheese samples was 50, 40, and 19%, respectively. The identified DEPs included Shiga toxin-producing E. coli (STEC; 8%), enteropathogenic E. coli (EPEC; 6%), and enterotoxigenic E. coli (ETEC; 5%). All isolated strains exhibited resistance to at least five antibiotics. One, one, two, and three STEC strains were resistant to 14, 12, 11, and 10 antibiotics, respectively. One strain of EPEC was resistant to 11 antibiotics, three EPEC strains to 9, and one strain to 7. One, one, and two strains of ETEC were resistant to 10, 8, and 7 antibiotics, respectively. The results of the present study indicate that fresh cheeses made with unpasteurized milk could be a risk for consumers, both for native people and visitors to Mexico.

Behavior of 11 Foodborne Bacteria on Whole and Cut Mangoes var. Ataulfo and Kent and Antibacterial Activities of Hibiscus sabdariffa Extracts and Chemical Sanitizers Directly onto Mangoes Contaminated with Foodborne Bacteria.

The behavior of foodborne bacteria on whole and cut mangoes and the antibacterial effect of Hibiscus sabdariffa calyx extracts and chemical sanitizers against foodborne bacteria on contaminated mangoes were investigated. Mangoes var. Ataulfo and Kent were used in the study. Mangoes were inoculated with Listeria monocytogenes, Shigella flexneri, Salmonella Typhimurium, Salmonella Typhi, Salmonella Montevideo, Escherichia coli strains (O157:H7, non-O157:H7 Shiga toxin-producing, enteropathogenic, enterotoxigenic, enteroinvasive, and enteroaggregative). The antibacterial effect of five roselle calyx extracts (water, ethanol, methanol, acetone, and ethyl acetate), sodium hypochlorite, colloidal silver, and acetic acid against foodborne bacteria were evaluated on contaminated mangoes. The dry extracts obtained with ethanol, methanol, acetone, and ethyl acetate were analyzed by nuclear magnetic resonance spectroscopy to determine solvent residues. Separately, contaminated whole mangoes were immersed in five hibiscus extracts and in sanitizers for 5 min. All foodborne bacteria attached to mangoes. After 20 days at 25 ± 2°C, all foodborne bacterial strains on whole Ataulfo mangoes had decreased by approximately 2.5 log, and on Kent mangoes by approximately 2 log; at 3 ± 2°C, they had decreased to approximately 1.9 and 1.5 log, respectively, on Ataulfo and Kent. All foodborne bacterial strains grew on cut mangoes at 25 ± 2°C; however, at 3 ± 2°C, bacterial growth was inhibited. Residual solvents were not detected in any of the dry extracts by nuclear magnetic resonance. Acetonic, ethanolic, and methanolic roselle calyx extracts caused a greater reduction in concentration (2 to 2.6 log CFU/g) of all foodborne bacteria on contaminated whole mangoes than the sodium hypochlorite, colloidal silver, and acetic acid. Dry roselle calyx extracts may be a potentially useful addition to disinfection procedures of mangoes.

Physiological, morphological, and mannanase production studies on Aspergillus niger uam-gs1 mutants

Mutant strains from Aspergillus niger UAM-GS1 were produced by UV radiation to increase their hemicellulolytic and cellulolytic activity production. The mutant strains showing more enzymatic activity were those labelled GS1-S059 and GS1-S067. These strains also showed the largest relationship between diameter of hydrolysis zone and colony diameter. The mutant GS1-S067 showed a colony radial extension rate and a biomass growth rate g biomass/(cm² h), 1.17 times higher than that achieved by strain UAM-GS1. The high invasive capacity makes this mutant strain a promising alternative for its use in solid substrate fermentation (SSF). The morphological properties of the two mutant strains were evaluated by using scanning electron microscopy. The diameter of the sporangium of the mutant strains GS1-S059 and GS1-S067 was significantly larger (P < 0.05) than that found for the parental strain. The hypha length and diameter of the mutant strains significantly changed (P < 0.05) compared to the parental strain. A Pearson correlation analysis on hypha length, sporangium diameter, and cellulase and xylanase activities indicated that there was a strong relationship among these variables in relation to mannanase activity. Mutant strains GS1-S059 and GS1-S067 significantly increased their level of mannanase, xylanase and cellulase production, compared to the parental strain, improving their potential industrial applications.