Green Extraction of Natural Colorants from Food Residues: Colorimetric Characterization and Nanostructuring for Enhanced Stability.

Food residues are a promising resource for obtaining natural pigments, which may replace artificial dyes in the industry. However, their use still presents challenges due to the lack of suitable sources and the low stability of these natural compounds when exposed to environmental variations. In this scenario, the present study aims to identify different food residues (such as peels, stalks, and leaves) as potential candidates for obtaining natural colorants through eco-friendly extractions, identify the colorimetric profile of natural pigments using the RGB color model, and develop alternatives using nanotechnology (e.g., liposomes, micelles, and polymeric nanoparticles) to increase their stability. The results showed that extractive solution and residue concentration influenced the RGB color profile of the pigments. Furthermore, the external leaves of Brassica oleracea L. var. capitata f. rubra, the peels of Cucurbita maxima, Cucurbita maxima x Cucurbita moschata, and Beta vulgaris L. proved to be excellent resources for obtaining natural pigments. Finally, the use of nanotechnology proved to be a viable alternative for increasing the stability of natural colorants over storage time.

Influence of biopolymer composition and crosslinking agent concentration on the micro- and nanomechanical properties of hydrogel-based filaments.

Hydrogel filaments were manufactured using wet spinning technique, incorporating variations in the concentrations of sodium alginate, gelatin, and calcium chloride (crosslinking agent). The combination of biopolymer concentrations was determined using design of experiments (DoE) approach. The resulting filaments were produced from the developed hydrogels. Tensile and vertical strength analyses of the filaments were conducted using an electromechanical extensor. Atomic force microscopy was employed to evaluate the roughness, viscoelasticity, retraction, and deflection of the hydrogels. By employing DoE, a total of seventeen different combinations of biopolymers and crosslinkers were generated to construct the hydrogels. The filaments exhibited variations in electromechanical traction (measured in kPa) and produced distinct stress peaks. Furthermore, diverse roughness values were observed among the tested materials, with the combinations featuring higher concentrations of sodium alginate displaying the highest Young's modulus. This study demonstrates that manipulating the concentrations of biopolymers and crosslinking agents can modulate the micro and nanomechanical properties of biopolymeric filaments.

Rapid and Versatile Biosensing of Liposome Encapsulation Efficiency Using Electrical Conductivity Sensor.

Liposomes are prominent nanosystems for drug delivery, with potential extending beyond isolated drugs. Ethanol-aqueous plant extracts can be encapsulated within liposomes to protect bioactive compounds (secondary metabolites) from rapid oxidation and enable sustained release. Determining which compound classes are present in each extract and the encapsulation efficiency (EE) of these extracts in liposomes is crucial for nanocarrier functionality. This involves assessing the ratio of bioactive substances within liposomes to the total content. However, quantifying EE for non-isolated compounds poses challenges due to the need for advanced analytical equipment and biosensing approaches. This study introduces an innovative method for EE quantification, using a conductivity electrode (k = 0.842/cm) to establish an EE biosensing technology. By correlating dynamic light scattering (DLS), zeta potential (ZP), and electrical conductivity (Cnd) data with the conductivity meter's calibration curve, a robust relationship between the free extract concentration and Cnd (r2 ≥ 0.950) was established. Lavender-loaded liposomes demonstrated an EE of 56.33%, while wormwood and oregano formulations exhibited high EEs of 94.33% and 91.70%, respectively. In contrast, sage-loaded liposomes exhibited an inadequate EE, encapsulating only approximately 0.57% of the extract. The straightforward quantification of the free extract within liposome formulations, compared to more complex approaches, could facilitate EE determination and support future characterizations.

Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine.

BACKGROUND: Influenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility. METHODS: This study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated. RESULTS: Only intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens. CONCLUSIONS: All findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

Unveiling the Remarkable Antioxidant Activity of Plant-Based Fish and Seafood Analogs through Electrochemical Sensor Analysis.

The global consumption of vegan foods is experiencing an expressive upward trend, underscoring the critical need for quality control measures based on nutritional and functional considerations. This study aimed to evaluate the functional quality of caviar and salmon analog food inks based on pulses combined with nano ingredients and produced in our laboratory (LNANO). The primary objective of this work was to determine the total antioxidant compounds contained in these samples using a voltammetric technique with a glassy carbon electrode. The samples underwent ethanolic extraction (70%) with 1 h of stirring. The voltammograms were acquired in a phosphate buffer electrolyte, pH 3.0 with Ag/AgCl (KCl 3 mol L-1) as the reference electrode and platinum wire as the auxiliary electrode. The voltammograms revealed prominent anodic current peaks at 0.76-0.78 V, which are attributed to isoflavones. Isoflavones, known secondary metabolites with substantial antioxidant potential commonly found in pulses, were identified. The total isoflavone concentrations obtained ranged from 31.5 to 64.3 mg Eq genistein 100 g-1. The results not only validated the efficacy of the electrochemical sensor for quantifying total antioxidant compounds in the samples but also demonstrated that the concentration of total isoflavones in caviar and salmon analogs fell within the expected limits.

Nucleotides Entrapped in Liposome Nanovesicles as Tools for Therapeutic and Diagnostic Use in Biomedical Applications.

The use of nucleotides for biomedical applications is an old desire in the scientific community. As we will present here, there are references published over the past 40 years with this intended use. The main problem is that, as unstable molecules, nucleotides require some additional protection to extend their shelf life in the biological environment. Among the different nucleotide carriers, the nano-sized liposomes proved to be an effective strategic tool to overcome all these drawbacks related to the nucleotide high instability. Moreover, due to their low immunogenicity and easy preparation, the liposomes were selected as the main strategy for delivery of the mRNA developed for COVID-19 immunization. For sure this is the most important and relevant example of nucleotide application for human biomedical conditions. In addition, the use of mRNA vaccines for COVID-19 has increased interest in the application of this type of technology to other health conditions. For this review article, we will present some of these examples, especially focused on the use of liposomes to protect and deliver nucleotides for cancer therapy, immunostimulatory activities, enzymatic diagnostic applications, some examples for veterinarian use, and the treatment of neglected tropical disease.

Eco-Friendly Silver Nanoparticles Synthesized from a Soybean By-Product with Nematicidal Efficacy against Pratylenchus brachyurus.

This study explores an eco-friendly approach to synthesizing silver nanoparticles (AgNPs) using soybean leaf extracts, employing a reaction with silver nitrate at 65 °C for 2.5 h. Optimal results were achieved at extract concentrations of 3.12 and 6.25 mg of the leaf mL-1, termed 3.12AgNP and 6.25AgNP, respectively. UV-Vis spectrophotometric analysis between 350 and 550 nm exhibited a peak at 410-430 nm, along with a color transition in the suspensions from pale yellow to brown, indicating successful synthesis. Dynamic light scattering (DLS) further delineated the favorable properties of these AgNPs, including nanometric dimensions (73-104 nm), negative charge, and moderate polydispersity, portraying stable and reproducible synthesis reactions. The bioreduction mechanism, possibly expedited by leaf extract constituents such as amino acids, phenolic acids, and polysaccharides, remains to be fully elucidated. Notably, this study underscored the potent nematicidal effectiveness of biosynthesized AgNPs, especially 6.25AgNP, against Pratylenchus brachyurus, which is a common plant-parasitic nematode in tropical soybean cultivation regions. In vitro tests illustrated significant nematicidal activity at concentrations above 25 µmol L-1, while in vivo experiments displayed a pronounced nematode population diminishment in plant roots, particularly with a 6.25AgNP rhizosphere application at concentrations of 500 µmol L-1 or twice at 250 µmol L-1, attaining a reproduction factor below 1 without any morphological nematode alterations. This research highlights the potential of 6.25AgNPs derived from soybean leaf extracts in forging sustainable nematicidal solutions, marking a significant stride toward eco-friendly phytonematode management in soybean cultivation. This novel methodology signals a promising avenue in harnessing botanical resources for nematode control and propelling a greener agricultural horizon.

Priming of defense-related genes in Brassica oleracea var. capitata using concentrated metabolites produced by Rhizobium tropici CIAT 899.

To verify the potential of metabolites extracted from Rhizobium tropici to trigger the priming of defense responses in cruciferous plants, we analyzed the expression of defense-related genes by qRT-PCR. Brassica oleracea var. capitata, susceptible to Xanthomonas campestris pv. campestris, were grown in greenhouse conditions. At 18 days after sowing, plants were inoculated with 1 mL of 1% concentrated metabolites produced by R. tropici (CM-RT) in the root. In a second experiment, leaves were sprayed with 1 mL of a solution containing 1% CM-RT. Aerial and root tissue were collected separately at 0 (non-treated control condition), 24, and 48 h after application, submitted to RNA extraction and gene expression analysis by qRT-PCR. The results showed that, after root treatment with CM-RT, most evaluated genes were upregulated at 24 h after application and downregulated at 48 h after application in roots, while in leaves, genes were downregulated both at 24 and 48 h after application. On the other hand, leaf treatment with CM-RT showed that most evaluated genes in leaves and roots were upregulated at 24 and 48 h after application. These results indicate that the effect of CM-RT applied in roots seems restricted to the applied region and is not sustained, while the application in leaves results in a more systemic response and maintenance of the effect of CM-RT for a longer period. The results obtained in this study emphasize the biotechnological potential of using metabolites of R. tropici as an elicitor of active defense responses in plants.

Initial steps on mapping differentially expressed proteins in bovine preantral follicles and ovarian tissue: An approach using single-follicle MALDI-MS and mass spectrometry imaging (MSI) analysis.

The molecular mechanisms regulating follicular development and ensuring primordial follicle activation remain undefined. To help elucidate these mechanisms, this proteomic study of bovine ovarian tissue identified the differential molecular profiles of preantral follicles together with the spatial distribution of the most abundant molecular components in the tissue. Isolated primordial, primary and secondary follicles were individually placed on a MALDI target plate for mass spectral acquisitions, with detection of different m/z ranges. Ovarian tissue was sectioned and analysed in the m/z 400-2,000 range. Results of the first analysis indicated a similarity pattern in the molecular protein profile among different follicular classes in the m/z ranges of 100-1000 and 25,000-200,000, but in the m/z ranges of 800-4000, 4000-20,000 and 15,000-70,000, primary and secondary follicles shared similar clustering profiles which were different from primordial follicles (p < .05). In the second analysis, it was possible to correlate some intense molecular components in the tissue from global mass spectrum with the ions detected in the first analysis. Molecular components at m/z 11,325 (±230) were also detected in primary and secondary follicles in the experiment with isolated follicles, in addition to ions at m/z 4,029 (±120), 13,799 (±70), 5,547 (±9), 15,313 (±200), 7,018 (±40) and 7,663 (±90) which were also intensely detected in primary and secondary follicles. The present proteomic approaches evaluated different mass ranges of preantral follicles in bovine ovarian tissue and also indicated the spatial distribution of the most abundant molecular components. This study hopes to pave the way for future research identifying and characterizing specific proteins involved in follicle activation in bovine follicles, in order to better understand folliculogenesis and potentially improve mammalian follicle culture systems.

Development of a Graphene-Based Biosensor for Detecting Recombinant Cyanovirin-N.

We present a graphene-based biosensor selective to recombinant cyanovirin-N (rCV-N), an antiviral protein that has proven to be an effective microbicide to inhibit HIV replication. We modified the graphene monolayer devices with 1-pyrenebutanoic acid succinimidyl ester, which interacts with both graphene and the primary and secondary amines of antibodies. By monitoring the change in the electrical resistance of the device, we were able to detect rCV-N in solutions in the range of 0.01 to 10 ng/mL, and found that the detection limit was 0.45 pg/mL, which is much smaller than that obtained with currently available techniques. This is important for applications of this microbicide against HIV, since it may be produced at a large scale from soya bean seeds processed using the available industrial processing technologies. The sensor showed high sensitivity, selectivity, and reproducibility.

Liposomes Composed by Membrane Lipid Extracts from Macrophage Cell Line as a Delivery of the Trypanocidal N,N'-Squaramide 17 towards Trypanosoma cruzi.

Chagas is a neglected tropical disease caused by Trypanosoma cruzi, and affects about 25 million people worldwide. N, N'-Squaramide 17 (S) is a trypanocidal compound with relevant in vivo effectiveness. Here, we produced, characterized, and evaluated cytotoxic and trypanocidal effects of macrophage-mimetic liposomes from lipids extracted of RAW 264.7 cells to release S. As results, the average hydrodynamic diameter and Zeta potential of mimetic lipid membranes containing S (MLS) was 196.5 ± 11 nm and -61.43 ± 2.3 mV, respectively. Drug entrapment efficiency was 73.35% ± 2.05%. After a 72 h treatment, MLS was observed to be active against epimastigotes in vitro (IC50 = 15.85 ± 4.82 µM) and intracellular amastigotes (IC50 = 24.92 ± 4.80 µM). Also, it induced low cytotoxicity with CC50 of 1199.50 ± 1.22 µM towards VERO cells and of 1973.97 ± 5.98 µM in RAW 264.7. MLS also induced fissures in parasite membrane with a diameter of approximately 200 nm in epimastigotes. MLS showed low cytotoxicity in mammalian cells and high trypanocidal activity revealing this nanostructure a promising candidate for the development of Chagas disease treatment.

Evaluation of behavior, growth, and swarming formation of Escherichia coli and Staphylococcus aureus in culture medium modified with silver nanoparticles.

Silver nanoparticles (AgNPs), as well as silver ions, are described as toxic to a broad spectrum of microorganisms, especially bacteria. In contrast to this, a current trend is to develop and carry out the in vitro cultivation of microorganisms, facilitating the study of interactions between populations of cells and species. Thus, the goal of this study was to evaluate the behavior, growth, and swarming formation of bacteria under conditions of co-culture in solid medium modified with AgNPs. The aqueous extract from the leaves of Handroanthus serratifolius was used to chemically reduce silver nitrate (AgNO3) solution, forming AgNPs. This synthesis route was performed in an aqueous medium at 50 °C for 3 h. The hydrodynamic diameter (HD) and polydispersity index (PdI) were obtained by dynamic light scattering (DLS), and Zeta potential (ZP) of the AgNPs were measured by electrophoretic mobility. Atomic force microscopy (AFM) was used to evaluate the shape of the AgNPs. Luria Bertani (LB) medium was used for the liquid culture steps and for the solid medium, bacterial agar was added. Solutions containing AgNPs or AgNO3 were added at final concentrations of 256, 128, or 64 µM. Subsequently, microorganism Escherichia coli ATCC® 8739 and Staphylococcus aureus ATCC® 25923 were plated with AgNPs, AgNO3, and control media. Analyses of the AgNPs showed an average HD of 76.02 ± 3.08 nm, PdI of 0.461 ± 0.012, and ZP of -21.5 ± 2.2 mV; in addition, AgNPs were nearly spherical. The solid culture medium elaborated and modified with AgNPs at the concentrations of 256 and 128 µM inhibited the growth of the tested microorganisms and decreased the swarming formation. However, those media modified at a concentration of 64 µM did not induce any alteration in the growth and proliferation of the microorganisms. Furthermore, it was observed that plates containing modified culture media with 128 µM, increased proximity between both co-cultured bacteria occurred. Thus, the application of AgNPs in solid culture media becomes a promising and potentially reproducible strategy for evaluating the behavior, swarming formation, and toxicity of AgNPs, making the understanding of possible bactericidal or bacteriostatic effects, and also colonizing strategies.

Fraternine, a Novel Wasp Peptide, Protects against Motor Impairments in 6-OHDA Model of Parkinsonism.

Parkinson's disease (PD) is a progressive neurodegenerative condition that affects the Central Nervous System (CNS). Insect venoms show high molecular variability and selectivity in the CNS of mammals and present potential for the development of new drugs for the treatment of PD. In this study, we isolated and identified a component of the venom of the social wasp Parachartergus fraternus and evaluated its neuroprotective activity in the murine model of PD. For this purpose, the venom was filtered and separated through HPLC; fractions were analyzed through mass spectrometry and the active fraction was identified as a novel peptide, called Fraternine. We performed two behavioral tests to evaluate motor discoordination, as well as an apomorphine-induced rotation test. We also conducted an immunohistochemical assay to assess protection in TH+ neurons in the Substantia Nigra (SN) region. Group treated with 10 µg/animal of Fraternine remained longer in the rotarod compared to the lesioned group. In the apomorphine test, Fraternine decreased the number of rotations between treatments. This dose also inhibited dopaminergic neuronal loss, as indicated by immunohistochemical analysis. This study identified a novel peptide able to prevent the death of dopaminergic neurons of the SN and recover motor deficit in a 6-OHDA-induced murine model of PD.

Magnetic nanoparticles coated with carbohydrates for 3D culture of bacteria.

Magnetic nanoparticles (MNPs) are a specific type of nanomaterial whose applications are widespread into several fields including biomedicine as a smart drug targeter and environmental engineering due to their interactions with contaminants. Lately, the use of MNPs has also been demonstrated in structuring three-dimensional (3D) cultures of mammalian cells. However, MNPs application to other cell types is still limited. In this sense, some planktonic microorganisms when adhered to surfaces perform the swarming phenomenon to guarantee the expansion of the colony and to guarantee more niches. Therefore, the aim of this study was to produce MNPs coated with four carbohydrates (galactose - gal, glucose - glu, sucrose - suc, and maltose - mal) aiming microorganism culture applications and also for possible 3D arrays. The results showed that carbohydrate-coated MNPs showed hydrodynamic diameters ranging from 100 to 200 nm and that their coatings influenced the chemical behavior in different ways. Indeed, when subjected to biological tests to determine their potential level of cytotoxicity, it was found that in concentrations of 1 mM, 800, 600, and 400 µM (iron equivalent), there was not any alteration on growth of model microorganisms when visually evaluated. Besides, magnetization of bacteria was promoted in different ways as well as the modulation of swarming formation in Escherichia coli when exposed to MNP-Glu. In sum, MNPs coated with carbohydrates and even uncoated were atoxic to bacteria and one of them was able to modulate E. coli swarming formation showing the potential for applications in 3D cultures of bacteria.

Shape and size of epididymal sperm from Gir bulls using atomic force microscopy: A nanoscale characterization of epididymal sperm.

As epididymal sperm (EP) are not exposed to seminal plasma, they are physiologically different from ejaculated spermatozoa (EJ). Therefore, the aim of this study was to morphologically characterize the head of EP recovered from the epididymis tail, and to evaluate if the physiological differences between EP and EJ were also expressed in the head's shape and size. EP and EJ were recovered from seven Gir bulls and were individually assessed. Sperm cells were washed, fixed, and 20 cells from each animal were analyzed by atomic force microscopy (AFM). The images were acquired through contact mode. Then, an off-line processing software was used and the images acquired were manually segmented using digital zoom of the original images. Twenty-four structural features were assessed including one, two, and three dimensional parameters, and also shape descriptors which were calculated based on the one and two dimensional parameters. Data were compared by t-test, then, a collective analysis was performed using principal component analysis (PCA). The EP group presented higher roughness and elongation (P ≤ 0.05), and smaller form factor and circularity rate than that of the EJ group (P ≤ 0.05). For the other parameters no differences (P ≥ 0.05) were observed. In addition, in the PCA analysis no differences among EP and EJ were observed either (P ≤ 0.05). This study showed that EP and EJ collected from the same sire presented similar characteristics in nineteen of the twenty-four parameters evaluated, indicating that absence of seminal plasma does not affect the morphology of EP.

MALDI TOF MS-profiling: Applications for bacterial and plant sample differentiation and biological variability assessment.

In this study, we evaluated the potential use of MALDI-TOF MS Profiling for the differentiation of biological samples submitted to different treatments. We compared the bacterium Xanthomonas campestris pv. campestris (Xcc), grown in culture medium and in vivo (recovered from the plant). Plant samples were also analyzed and included explants at different somatic embryogenesis (SE) stages, as well as leaves from Brassica oleracea and Arabidopsis thaliana inoculated with Xcc, at different time points. The results showed that bacteria and highly divergent plant samples, such as those from embryogenic stages, can be unequivocally differentiated and the clustering was in accordance with proteomic analysis performed by 2-DE. These results show an important application of MALDI-TOF MS Profiling to select and prioritize samples to be analyzed prior to more complex approaches including transcriptomics and proteomics. We also show that in plant-pathogen interactions, when more subtle differences are obtained, the main contribution of MALDI-TOF MS Profiling is in the assessment of experimental variability. This is relevant since reproducibility is a challenging issue when dealing with complex experimental conditions such as plant-pathogen interactions. We propose the use of MALDI-TOF MS Profiling to aid researchers in minimizing experimental variability unrelated to the condition being analyzed. SIGNIFICANCE: MALDI-Profiling offers an inexpensive, rapid and reliable approach for investigating the protein profile to assess sample differentiation and experimental variability in microorganisms and plants and can be highly useful to analyze samples prior to more complex and expensive techniques such as proteomics and transcriptomics.

Correction to: Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry.

The original version of this article unfortunately contained a mistake. The presentation of Fig. 1was incorrect. The correct version is given below.

The dynamics of gene expression, lipid composition and DNA methylation reprogramming are different during in vitro maturation of pig oocytes obtained from prepubertal gilts and cycling sows.

This study aimed to characterize the gene expression, lipid composition and DNA methylation reprogramming during in vitro maturation (IVM) of pig oocytes with different developmental competencies. We used prepubertal gilts and cycling sows as a model to obtain oocytes with different levels of competency. We found that genes involved in lipid metabolism, SLC27A4, CPT2 and PLIN2, and DNA methylation, DNMT3A, TET1 and TET3, possessed altered transcript expression levels during IVM. Specifically, SLC27A4 mRNA (p = 0.05) increased in oocytes from cycling females, whereas CPT2 (p = 0.05), PLIN2 (p = 0.02) and DNMT3A (p = 0.02) increased in oocytes from prepubertal females during IVM. Additionally, TET3 mRNA increased during IVM in oocytes from prepubertal (p = 0.0005) and cycling females (p = 0.02). The TET1 transcript decreased (p = 0.05) during IVM in oocytes from cycling sows. Regarding lipid composition, mass spectrometry revealed a cluster of ions, with molecular masses higher than m/z 700, which comprises a group of complex phospholipids, was identified in all groups of oocytes, except in those from prepubertal gilts. With respect to DNA methylation reprogramming, it was noted that the less competent oocytes were not able to reprogramme the XIST gene during IVM. We conclude that the maternal mRNA store, lipid composition and epigenetic reprogramming are still being established during maturation and are related to oocyte competence. In addition, we propose that the methylation pattern of the XIST may be used as molecular marker for oocyte competence in pigs.

Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry.

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

Chloroplast Proteome of Nicotiana benthamiana Infected by Tomato Blistering Mosaic Virus.

Tymovirus is a genus of plant pathogenic viruses that infects several dicotyledonous plants worldwide, causing serious diseases in economically important crops. The known cytopathic effect on the host cell organelles involves chloroplast membrane deformation and the induction of vesicles in its periphery. These vesicles are known to be the location where tymoviral genomic RNA replication occurs. Tomato blistering mosaic virus (ToBMV) is a tymovirus recently identified in tomato plants in Brazil, which is able to infect several other plants, including tobacco. In this work, we investigated the chloroplast proteomic profile of ToBMV-infected N. benthamiana using bidimensional electrophoresis (2-DE) and mass spectrometry, aiming to study the virus-host interaction related to the virus replication and infection. A total of approximately 200 spots were resolved, out of which 36 were differentially abundant. Differential spots were identified by mass spectrometry including photosynthesis-related and defense proteins. We identified proteins that may be targets of a direct interaction with viral proteins, such as ATP synthase ß subunit, RNA polymerase beta-subunit, 50S ribosomal protein L6 and Trigger factor-like protein. The identification of these candidate proteins gives support for future protein-protein interaction studies to confirm their roles in virus replication and disease development.