Tizoxanide Antiviral Activity on Dengue Virus Replication.

Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections.

Chemical composition and anti-Mayaro virus activity of Schinus terebinthifolius fruits.

Brazilian traditional medicine has explored the antiviral properties of many plant extracts, including those from the Brazilian pepper tree, Schinus terebinthifolius. In the present study, we investigated the chemical composition and anti-mayaro virus (MAYV) activity of S. terebinthifolius fruit. Extensive virucidal activity (more than 95%) was detected for the ethyl acetate extract and the isolated biflavonoids. From the ethyl acetate extract of Schinus terebinthifolius fruits, two bioflavonoids were isolated ((2S, 2″S)-2,3,2″,3″-tetrahydroamentoflavone and agathisflavone), which showed strong virucidal activity against Mayaro virus. Furthermore, several other compounds like terpenes and phenolics were identified by hyphenated techniques (GC-MS, LC-MS and HPLC-UV), as well as by mass spectrometry. Immunofluorescence assay confirmed antiviral activity and transmission electron microscopy revealed damage in viral particles treated with biflavonoids. The data suggest the direct action of the extract and the biflavonoids on the virus particles. The biflavonoids tetrahydroamentoflavone and agathisflavone had strong virucidal activity and reduced MAYV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00698-z.

The influence of polycyclic aromatic hydrocarbons in protein profile of Medicago sativa L.

Medicago sativa L. (alfalfa) are studied as potential phytoremediation agents of priority pollutants like polycyclic aromatic hydrocarbons (PAH). However, elucidation of the biochemical mechanisms involved in phytoremediation is a topic to be explored with knowledge gaps. This study aims to identify and classify proteins expressed in the aerial parts of laboratory-cultivated alfalfa in the presence and absence of pyrene, anthracene, and phenanthrene. Soil samples were amended with 100 mg.kg-1 of each PAH (total concentration of 300 ppm) and cultivated with alfalfa plants for 20 days. After this, aerial parts of cultivated plants from each condition were collected for qualitative proteomic analysis (ESI-Q/TOF). The results showed a significant increase (Student's t-test p < 0.05) of 41.7% in the concentration of proteins from plants grown in PAH-amended substrates, changes in the protein profile, with intense protein bands observed at 40-55, 34, 28, and 15 kDa when compared to the control. A total of 504 proteins were identified and classified into 12 functional categories, highlighting the identification of 11 phytoremediation-related proteins candidates in plants grown in the presence of PAH, with biological functions related to diverse metabolisms involved in the xenobiotics biodegradation (included PAH), glutathione and response to stress.

Phytoremediation of polycyclic aromatic hydrocarbons (PAH) by cv. Crioula: A Brazilian alfalfa cultivar.

This work aimed to evaluate the phytoremediation capacity of the alfalfa cultivar Crioula in soils contaminated with polycyclic aromatic hydrocarbons (PAHs), primary pollutants with mutagenic and carcinogenic potential. Alfalfa was grown from seed for 40 days on soil amended with anthracene, pyrene, and phenanthrene. Soil and plant tissue was collected for biometric assay, dry mass analysis, and PAH analysis by liquid chromatography. Increased total PAH concentration was associated with decreases in plant biomass, height, and internode length. The Crioula cultivar had a satisfactory phytoremediation effect, reducing total PAH concentration (300 ppm) in the experimental soil by 85% in 20 days, and by more than 95% in 40 days. The PAH showed a tendency to be removed in the temporal order: phenanthrene before pyrene before anthracene, and the removal ratio was influenced by the initial soil concentration of each PAH.

Proteomics-based identification of differentially abundant proteins reveals adaptation mechanisms of Xanthomonas citri subsp. citri during Citrus sinensis infection.

BACKGROUND: Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. A proteomic analysis under in planta infectious and non-infectious conditions was conducted in order to increase our knowledge about the adaptive process of Xac during infection. RESULTS: For that, a 2D-based proteomic analysis of Xac at 1, 3 and 5 days after inoculation, in comparison to Xac growth in NB media was carried out and followed by MALDI-TOF-TOF identification of 124 unique differentially abundant proteins. Among them, 79 correspond to up-regulated proteins in at least one of the three stages of infection. Our results indicate an important role of proteins related to biofilm synthesis, lipopolysaccharides biosynthesis, and iron uptake and metabolism as possible modulators of plant innate immunity, and revealed an intricate network of proteins involved in reactive oxygen species adaptation during Plants` Oxidative Burst response. We also identified proteins previously unknown to be involved in Xac-Citrus interaction, including the hypothetical protein XAC3981. A mutant strain for this gene has proved to be non-pathogenic in respect to classical symptoms of citrus canker induced in compatible plants. CONCLUSIONS: This is the first time that a protein repertoire is shown to be active and working in an integrated manner during the infection process in a compatible host, pointing to an elaborate mechanism for adaptation of Xac once inside the plant.

Proteomic analysis of the kissing bug Rhodnius prolixus antenna.

Reception of odorants is essential in insects' life since the chemical signals in the environment (=semiochemicals) convey information about availability of hosts for a blood meal, mates for reproduction, sites for oviposition and other relevant information for fitness in the environment. Once they reach the antennae, these semiochemicals bind to odorant-binding proteins and are transported through the sensillar lymph until reach the odorant receptors. Such perireceptor events, particularly the interactions with transport proteins, are the liaison between the external environment and the entire neuroethological system and, therefore, a potential target to disrupt insect chemical communication. In this study, a proteomic profile of female and male antennae of Rhodnius prolixus, a vector of Chagas disease, was obtained in an attempt to unravel the entire repertoire of olfactory proteins involved in perireceptor events. Using shotgun proteomics and two-dimensional gel electrophoresis approaches followed by nano liquid chromatography coupled with tandem LTQ Velos Orbitrap mass spectrometry, we have identified 581 unique proteins. Putative olfactory proteins, including 17 odorant binding proteins, 6 chemosensory proteins, 2 odorant receptors, 3 transient receptor channels and 1 gustatory receptor were identified. Proteins involved in general cellular functions such as generation of precursor metabolites, energy generation and catabolism were expressed at high levels. Additionally, proteins that take part in signal transduction, ion binding, and stress response, kinase and oxidoreductase activity were frequent in antennae from both sexes. This proteome strategy unraveled for the first time the complex nature of perireceptor and other olfactory events that occur in R. prolixus antennae, including evidence for phosphorylation of odorant-binding and chemosensory proteins. These findings not only increase our understanding of the olfactory process in triatomine species, but also identify potential molecular targets to be explored for population control of such insect vectors.

Detection of polycyclic aromatic hydrocarbons (PAHs) in Medicago sativa L. by fluorescence microscopy.

Green technologies, such as phytoremediation, are effective for removing organic pollutants derived from oil and oil products, including polycyclic aromatic hydrocarbons (PAHs). Given the increasing popularity of these sustainable remediation techniques, methods based on fluorescence microscopy and multiphoton microscopy for the environmental monitoring of such pollutants have emerged in recent decades as effective tools for phytoremediation studies aimed at understanding the fate of these contaminants in plants. However, little is known about the cellular and molecular mechanisms involved in PAH uptake, responses and degradation by plants. Thus, the present study aimed to detect the location of pyrene, anthracene and phenanthrene using fluorescence microscopy techniques in shoots and roots of Medicago sativa L. (alfalfa) plants grown in artificially contaminated soil (150ppm PAHs) for 40days. Leaflet and root samples were then collected and observed under a fluorescence microscope to detect the presence of PAHs in various tissues. One important finding of the present study was intense fluorescence in the glandular secreting trichomes (GSTs) of plants grown in contaminated soil. These trichomes, with a previously unknown function, may be sites of PAH conjugation and degradation.

Comparative proteome analysis reveals that blood and sugar meals induce differential protein expression in Aedes aegypti female heads.

Aedes aegypti females ingest sugar or blood to obtain the nutrients needed to maintain cellular homeostasis. During human blood ingestion, female mosquitoes may transmit different viruses such as dengue, yellow fever and, more recently, zika and chikungunya. Here, we report changes in protein expression in the heads of adult female Ae. aegypti mosquitoes in response to the ingestion of blood or sugar. Proteins extracted from the heads of Ae. aegypti fed exclusively on blood (BF) or sugar (SF) were trypsin hydrolyzed (off-gel) and analyzed by the reverse-phase nano-liquid chromatography coupled with hybrid mass spectrometry. A total of 1139 proteins were identified in female heads, representing 7.4% of the predicted proteins in Ae. aegypti genome (total = 15 419 active genes). Gene ontology annotation and categories showed that, in this insect, the head was rich in proteins involved in the metabolic process, proton transport, organelle, macromolecular complex, structural molecule activity, antioxidant activity, and catalytic activity. Our report is the first indicating that many of the annotated genes are translated into functional proteins in heads of adult female Ae. aegypti. Interestingly, we identified 8.7 times more exclusively expressed proteins involved in signal transduction, replication-transcription-translation (5.5 x), and transport (2.9 x) activity in BF than in SF groups. This paper discusses the protein profile of Ae. aegypti female heads and its implications for blood ingestion and carbohydrate intake.

Unravelling potential virulence factor candidates in Xanthomonas citri. subsp. citri by secretome analysis.

Citrus canker is a major disease affecting citrus production in Brazil. It's mainly caused by Xanthomonas citri subsp. citri strain 306 pathotype A (Xac). We analysed the differential expression of proteins secreted by wild type Xac and an asymptomatic mutant for hrpB4 (ΔhrpB4) grown in Nutrient Broth (NB) and a medium mimicking growth conditions in the plant (XAM1). This allowed the identification of 55 secreted proteins, of which 37 were secreted by both strains when cultured in XAM1. In this secreted protein repertoire, the following stand out: Virk, Polyphosphate-selective porin, Cellulase, Endoglucanase, Histone-like protein, Ribosomal proteins, five hypothetical proteins expressed only in the wild type strain, Lytic murein transglycosylase, Lipoprotein, Leucyl-tRNA synthetase, Co-chaperonin, Toluene tolerance, C-type cytochrome biogenesis membrane protein, Aminopeptidase and two hypothetical proteins expressed only in the ΔhrpB4 mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isomerase and seven hypothetical proteins were detected in both strains, suggesting that there was no relationship with the secretion mediated by the type III secretory system, which is not functional in the mutant strain. Also worth mentioning is the Elongation factor Tu (EF-Tu), expressed only the wild type strain, and Type IV pilus assembly protein, Flagellin (FliC) and Flagellar hook-associated protein, identified in the wild-type strain secretome when grown only in NB. Noteworthy, that FliC, EF-Tu are classically characterized as PAMPs (Pathogen-associated molecular patterns), responsible for a PAMP-triggered immunity response. Therefore, our results highlight proteins potentially involved with the virulence. Overall, we conclude that the use of secretome data is a valuable approach that may bring more knowledge of the biology of this important plant pathogen, which ultimately can lead to the establishment of new strategies to combat citrus canker.

Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity.

BACKGROUND: The arthropod-borne Mayaro virus (MAYV) causes 'Mayaro fever', a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. METHODS: Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. RESULTS: The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 µg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. CONCLUSIONS: B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus.

Comparative proteomic analysis reveals that T3SS, Tfp, and xanthan gum are key factors in initial stages of Citrus sinensis infection by Xanthomonas citri subsp. citri.

The bacteria Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. The disease symptoms are characterized by localized host cell hyperplasia followed by tissue necrosis at the infected area. An arsenal of bacterial pathogenicity- and virulence-related proteins is expressed to ensure a successful infection process. At the post-genomic stage of Xac, we used a proteomic approach to analyze the proteins that are displayed differentially over time when the pathogen attacks the host plant. Protein extracts were prepared from infectious Xac grown in inducing medium (XAM1) for 24 h or from host citrus plants for 3 or 5 days after infection, detached times to evaluate the adaptation and virulence of the pathogen. The protein extracts were proteolyzed, and the peptides derived from tryptic digestion were investigated using liquid chromatography and tandem mass spectrometry. Changes in the protein expression profile were compared with the Xac genome and the proteome recently described under non-infectious conditions. An analysis of the proteome of Xac under infectious conditions revealed proteins directly involved in virulence such as the type III secretion system (T3SS) and effector proteins (T3SS-e), the type IV pilus (Tfp), and xanthan gum biosynthesis. Moreover, four new mutants related to proteins detected in the proteome and with different functions exhibited reduced virulence relative to the wild-type proteins. The results of the proteome analysis of infectious Xac define the processes of adaptation to the host and demonstrate the induction of the virulence factors of Xac involved in plant-pathogen interactions.

Four conventional soybean [Glycine max (L.) Merrill] seeds exhibit different protein profiles as revealed by proteomic analysis.

Soybeans have several functional properties due to their composition and may exert beneficial health effects that are attributed to proteins and their derivative peptides. The present study aimed to analyze the protein profiles of four new conventional soybean seeds (BRS 257, BRS 258, BRS 267, and Embrapa 48) with the use of proteomic tools. Two-dimensional (2D) and one-dimensional (1D) gel electrophoreses were performed, followed by MALDI-TOF/TOF and ESI-Q-TOF mass spectrometry analyses, respectively. These two different experimental approaches allowed the identification of 117 proteins from 1D gels and 46 differentially expressed protein spots in 2D gels. BRS 267 showed the greatest diversity of identified spots in the 2D gel analyses. In the 1D gels, the major groups were storage (25-40%) and lipid metabolism (11-25%) proteins. The differences in protein composition between cultivars could indicate functional and nutritional differences and could direct the development of new cultivars.

Proteome of the phytopathogen Xanthomonas citri subsp. citri: a global expression profile.

BACKGROUND: Citrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS). RESULTS: In order to gain insight into the metabolism of Xac, cells were grown on two different media (NB - Nutrient Broth and TSE - Tryptone Sucrose broth enriched with glutamic acid), and proteins were proteolyzed with trypsin and examined by 2D LC-MS/MS. Approximately 39% of all predicted proteins by annotation of Xac were identified with their component peptides unambiguously assigned to tandem mass spectra. The proteins, about 1,100, were distributed in all annotated functional categories. CONCLUSIONS: This is the first proteomic reference map for the most aggressive strain of Xanthomonas pathogen of all orange varieties. The compilation of metabolic pathways involved with bacterial growth showed that Xac expresses a complete central and intermediary metabolism, replication, transcription and translation machineries and regulation factors, distinct membrane transporters (ABC, MFS and pumps) and receptors (MCP, TonB dependent and metabolites acquisition), two-component systems (sensor and regulatory components) and response regulators. These data corroborate the growth curve in vitro and are the first reports indicating that many of these genome annotated genes are translated into operative in Xac. This proteomic analysis also provided information regarding the influence of culture medium on growth and protein expression of Xac.

Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data.

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.

Secretome of HepG2 cells infected with dengue virus: implications for pathogenesis.

Viral hemorrhagic fever is a clinical syndrome that poses serious global health threat. Among the causative agents, dengue virus (DV) has the highest incidence rate and its infection is the major cause of viral hemorrhagic fever in the world. Although the pathophysiological mechanisms of DV-induced diseases are not yet understood, it is well accepted that liver is a site of viral replication. In this study, we used proteomics to analyze infection of a hepatic cell lineage, HepG2, with DV, focusing on the secreted proteins. 1D-electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were used, allowing the identification of a total of 107 proteins, among which 35 were found only in control secretome and 24 only in infected cells secretome. To validate these data, we performed 2D-eletrophoresis followed by MALDI-TOF/TOF, resulting in the identification of 20 proteins, 8 of them confirming LC-MS/MS results. We discuss the results obtained taking into account the proteins previously described in the secretome of HepG2 cells, proteins present in human plasma and proteins of interest for dengue pathogenesis. Altogether the data presented here provide clues for the progress in the understanding of the role of liver secretion in the progression of the disease.

VP4 protein from human rhinovirus 14 is released by pressure and locked in the capsid by the antiviral compound WIN.

Rhinoviruses are the major causative agents of the common cold in humans. Here, we studied the stability of human rhinovirus type 14 (HRV14) under conditions of high hydrostatic pressure, low temperature, and urea in the absence and presence of an antiviral drug. Capsid dissociation and changes in the protein conformation were monitored by fluorescence spectroscopy, light scattering, circular dichroism, gel filtration chromatography, mass spectrometry and infectivity assays. The data show that high pressure induces the dissociation of HRV14 and that this process is inhibited by WIN 52084. MALDI-TOF mass spectrometry experiments demonstrate that VP4, the most internal viral protein, is released from the capsid by pressure treatment. This release of VP4 is concomitant with loss of infectivity. Our studies also show that at least one antiviral effect of the WIN drugs involves the locking of VP4 inside the capsid by blocking the dynamics associated with cell attachment.

Structure of a membrane-binding domain from a non-enveloped animal virus: insights into the mechanism of membrane permeability and cellular entry.

The gamma(1)-peptide is a 21-residue lipid-binding domain from the non-enveloped Flock House virus (FHV). Unlike enveloped viruses, the entry of non-enveloped viruses into cells is believed to occur without membrane fusion. In this study, we performed NMR experiments to establish the solution structure of a membrane-binding peptide from a small non-enveloped icosahedral virus. The three-dimensional structure of the FHV gamma(1)-domain was determined at pH 6.5 and 4.0 in a hydrophobic environment. The secondary and tertiary structures were evaluated in the context of the capacity of the peptide for permeabilizing membrane vesicles of different lipid composition, as measured by fluorescence assays. At both pH values, the peptide has a kinked structure, similar to the fusion domain from the enveloped viruses. The secondary structure was similar in three different hydrophobic environments as follows: water/trifluoroethanol, SDS, and membrane vesicles of different compositions. The ability of the peptide to induce vesicle leakage was highly dependent on the membrane composition. Although the gamma-peptide shares some structural properties to fusion domains of enveloped viruses, it did not induce membrane fusion. Our results suggest that small protein components such as the gamma-peptide in nodaviruses (such as FHV) and VP4 in picornaviruses have a crucial role in conducting nucleic acids through cellular membranes and that their structures resemble the fusion domains of membrane proteins from enveloped viruses.

Hydrolytic protein cleavage mediated by unusual mononuclear copper(II) complexes: X-ray structures and solution studies.

The crystal structures and redox and UV-vis/EPR spectroscopic properties of two new mononuclear copper(II) complexes, [Cu(HL1)Cl2] (1) and [Cu(L1)Cl] (2), prepared through the reaction between copper(II) chloride and the ligand 2-[(bis(pyridylmethyl)amino)methyl]-4-methyl-6-formylphenol (HL1) under distinct base conditions, are reported along with solution studies. Also, we demonstrate that these CuII complexes are able to cleave unactivated peptide bonds from bovine serum albumin (BSA) and the thermostable enzyme Taq DNA polymerase at micromolar concentration, under mild pH and temperature conditions. The cleavage activity seems to be specific with defined proteolytic fragments appearing after protein treatment. The location of the specific cleavage sites was tentatively assigned to solvent-accessible portions of the protein. These are two of the most active Cu(II) complexes described to date, since their cleavage activity is detected in minutes and evidence is here presented for a hydrolytic mechanism mediating protein cleavage by these complexes.