Antibacterial and Antibiofilm Activity of Chemically and Biologically Synthesized Silver Nanoparticles.

Bacterial biofilms are a significant problem in the food industry, as they are difficult to eradicate and represent a threat to consumer health. Currently, nanoparticles as an alternative to traditional chemical disinfectants have garnered much attention due to their broad-spectrum antibacterial activity and low toxicity. In this study, silver nanoparticles (AgNPs) were synthesized by a biological method using a Jacaranda mimosifolia flower aqueous extract and by a chemical method, and the factors affecting both syntheses were optimized. The nanoparticles were characterized by Ultraviolet-visible (UV-Vis) spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS), X-ray diffraction (XRD), and Transmission electron microscopy (TEM) with a spherical and uniform shape. The antibacterial and antibiofilm formation activity was carried out on bacterial species of Pseudomonas aeruginosa and Staphylococcus aureus with the capacity to form biofilm. The minimum inhibitory concentration was 117.5 µg/mL for the chemical and 5.3 µg/mL for the biological nanoparticles. Both types of nanoparticles showed antibiofilm activity in the qualitative Congo red test and in the quantitative microplate test. Antibiofilm activity tests on fresh lettuce showed that biological nanoparticles decreased the population of S. aureus and P. aeruginosa by 0.63 and 2.38 logarithms, respectively, while chemical nanoparticles had little microbial reduction. In conclusion, the biologically synthesized nanoparticles showed greater antibiofilm activity. Therefore, these results suggest their potential application in the formulation of sanitizing products for the food and healthcare industries.

Thermal Treatment to Obtain 5-Hydroxymethyl Furfural (5-HMF), Furfural and Phenolic Compounds from Vinasse Waste from Agave.

Vinasses represent important final disposal problems due to their physical-chemical composition. This work analyzed the composition of tequila vinasses and increased 5-hydroxymethylfurfural, furfural, and phenolic compounds using thermal hydrolysis with hydrogen peroxide as a catalyst. A statistical Taguchi design was used, and a UPLC-MS (XEVO TQS Micro) analysis determined the presence and increase of the components. The treatment at 130 °C, 40 min, and 0.5% of catalyst presented the highest increase for 5-HMF (127 mg/L), furfural (3.07 mg/L), and phenol compounds as chlorogenic (0.36 mg/L), and vanillic acid (2.75 mg/L). Additionally, the highest removal of total sugars (57.3%), sucrose (99.3%), and COD (32.9%). For the treatment T130:30m:0P the syringic (0.74 mg/L) and coumaric (0.013 mg/L) acids obtained the highest increase, and the treatment T120:30m:1P increased 3-hydroxybenzoic (1.30 mg/L) and sinapic (0.06 mg/L) acid. The revaluation of vinasses through thermal treatments provides guidelines to reduce the impact generated on the environment.

Approximations to Diagnosis and Therapy of COVID-19 in Nervous Systems Using Extracellular Vesicles.

The SARS-CoV-2 virus was first identified at the end of December 2019, causing the disease known as COVID-19, which, due to the high degree of contagion, was declared a global pandemic as of 2020. The end of the isolation was in 2022, thanks to the global multidisciplinary work of the massive vaccination campaigns. Even with the current knowledge about this virus and the COVID-19 disease, there are many questions and challenges regarding diagnosis and therapy in the fight against this virus. One of the big problems is the so-called "long COVID", prolonged symptomatology characterized as a multiorgan disorder manifested as brain fog, fatigue, and shortness of breath, which persist chronically after the disease resolution. Therefore, this review proposes using extracellular vesicles (EVs) as a therapeutic or diagnostic option to confront the sequelae of the disease at the central nervous system level. Development: the review of updated knowledge about SARS-CoV-2 and COVID-19 is generally addressed as well as the current classification of extracellular vesicles and their proposed use in therapy and diagnosis. Through an analysis of examples, extracellular vesicles are highlighted to learn what happens in the central nervous system during and after COVID-19 and as a therapeutic option. Conclusions: even though there are limitations in the knowledge of the neurological manifestations of COVID-19, it is possible to observe the potential use of extracellular vesicles in therapy or as a diagnostic method and even the importance of their study for the knowledge of the pathophysiology of the disease.

Identifying Bixa orellana L. New Carotenoid Cleavage Dioxygenases 1 and 4 Potentially Involved in Bixin Biosynthesis.

Carotene cleavage dioxygenases (CCDs) are a large family of Fe2+ dependent enzymes responsible for the production of a wide variety of apocarotenoids, such as bixin. Among the natural apocarotenoids, bixin is second in economic importance. It has a red-orange color and is produced mainly in the seeds of B. orellana. The biosynthesis of bixin aldehyde from the oxidative cleavage of lycopene at 5,6/5',6' bonds by a CCD is considered the first step of bixin biosynthesis. Eight BoCCD (BoCCD1-1, BoCCD1-3, BoCCD1-4, CCD4-1, BoCCD4-2, BoCCD4-3 and BoCCD4-4) genes potentially involved in the first step of B. orellana bixin biosynthesis have been identified. However, the cleavage activity upon lycopene to produce bixin aldehyde has only been demonstrated for BoCCD1-1 and BoCCD4-3. Using in vivo (Escherichia coli) and in vitro approaches, we determined that the other identified BoCCDs enzymes (BoCCD1-3, BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4) also participate in the biosynthesis of bixin aldehyde from lycopene. The LC-ESI-QTOF-MS/MS analysis showed a peak corresponding to bixin aldehyde (m/z 349.1) in pACCRT-EIB E. coli cells that express the BoCCD1 and BoCCD4 proteins, which was confirmed by in vitro enzymatic assay. Interestingly, in the in vivo assay of BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4, bixin aldehyde was oxidized to norbixin (m/z 380.2), the second product of the bixin biosynthesis pathway. In silico analysis also showed that BoCCD1 and BoCCD4 proteins encode functional dioxygenases that can use lycopene as substrate. The production of bixin aldehyde and norbixin was corroborated based on their ion fragmentation pattern, as well as by Fourier transform infrared (FTIR) spectroscopy. This work made it possible to clarify at the same time the first and second steps of the bixin biosynthesis pathway that had not been evaluated for a long time.

Cocoa Nanoparticles to Improve the Physicochemical and Functional Properties of Whey Protein-Based Films to Extend the Shelf Life of Muffins.

A novel nanocomposite whey protein-based film with nanoemulsified cocoa liquor (CL) was prepared using one-stage microfluidization to evaluate the emulsion properties and the effect of CL on the film properties by response surface methodology (RSM). The results indicated that the number of cycles by microfluidization had a significant effect (p < 0.05) on the particle size and polydispersity of the nanoemulsion, with a polyphenol retention of approximately 83%. CL decreased the solubility (<21.87%) and water vapor permeability (WVP) (<1.57 g mm h-1 m-2 kPa-1) of the film. FTIR analysis indicated that CL modified the secondary protein structure of the whey protein and decreased the mechanical properties of the film. These results demonstrate that applying the film as a coating is feasible and effective to improve the shelf life of bakery products with a high moisture content. This nanocomposite film is easy to produce and has potential applications in the food industry.

Proteomic characterization of a second-generation version of the BCGΔBCG1419c vaccine candidate by means of electrospray-ionization quadrupole time-of-flight mass spectrometry.

Tuberculosis (TB) is the most important infectious disease worldwide, based on the number of new cases and deaths reported by the World Health Organization. Several vaccine candidates against TB have been characterized at preclinical and clinical levels. The BCGΔBCG1419c vaccine candidate, which lacks the BCG1419c gene that encodes for a c-di-GMP phosphodiesterase, provides improved efficacy against chronic TB, reactivation from latent-like infection and against chronic TB in the presence of type 2 diabetes in murine models. We previously reported that compared with wild type BCG, BCGΔBCG1419c changed levels of several proteins. Here, using a label-free proteomic approach, we confirmed that a novel, second-generation version of BCGΔBCG1419c maintains changes in antigenic proteins already reported, and here we further found differences in secreted proteins, as well as that this new BCGΔBCG1419c version modifies its production of proteins involved in redox and nitrogen/protein metabolism compared with wild type BCG. This work contributes to the proteomic characterization of a novel vaccine candidate that is more effective against TB than parental BCG in diverse murine models.

Transcriptional and Mycolic Acid Profiling in Mycobacterium bovis BCG In Vitro Show an Effect for c-di-GMP and Overlap between Dormancy and Biofilms.

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA, kasB, and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR, therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.

Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto (Bixa orellana L.).

Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world's food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5',6'.

Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence.

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1ß, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4+ and T CD8+ activation. This correlated with protection versus lethality in mice infected with the highly virulent M. tuberculosis 5186 afforded by vaccination with all the tested BCG strains, and controlled the growth of the mildly virulent M. tuberculosis H37Rv in lungs by vaccination with BCGΔBCG1419c during chronic late infection from 4 to 6 months after challenge. Furthermore, when grown as surface pellicles, a condition used to manufacture BCG vaccine, in comparison to BCG wild type, both rBCGs changed expression of antigenic proteins such as DnaK, HbhA, PstS2, 35KDa antigen, GroEL2, as well as AcpM, a protein involved in synthesis of mycolic acids, molecules relevant to modulate inflammatory responses.

Demulsification of crude oil-in-water emulsions by means of fungal spores.

The present feature describes for the first time the application of spores from Aspergillus sp. IMPMS7 to break out crude oil-in-water emulsions (O/W). The fungal spores were isolated from marine sediments polluted with petroleum hydrocarbons. The spores exhibited the ability to destabilize different O/W emulsions prepared with medium, heavy or extra-heavy Mexican crude oils with specific gravities between 10.1 and 21.2°API. The isolated fungal spores showed a high hydrophobic power of 89.3 ± 1.9% and with 2 g of spores per liter of emulsion, the half-life for emulsion destabilization was roughly 3.5 and 0.7 h for extra-heavy and medium crude oil, respectively. Then, the kinetics of water separation and the breaking of the O/W emulsion prepared with heavy oil through a spectrofluorometric technique were studied. A decrease in the fluorescence ratio at 339 and 326 nm (I339/I326) was observed in emulsions treated with spores, which is similar to previously reported results using chemical demulsifiers.

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. CONCLUSIONS: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-µm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.