RESUMEN
The ability of bacteria to colonize diverse environmental niches is often linked to their competence in biofilm formation. It depends on the individual characteristics of a strain, the nature of the colonized surface (abiotic or biotic), or the availability of certain nutrients. Pseudomonas donghuensis P482 efficiently colonizes the rhizosphere of various plant hosts, but a connection between plant tissue colonization and the biofilm formation ability of this strain has not yet been established. We demonstrate here that the potential of P482 to form biofilms on abiotic surfaces and the structural characteristics of the biofilm are influenced by the carbon source available to the bacterium, with glycerol promoting the process. Also, the type of substratum, polystyrene or glass, impacts the ability of P482 to attach to the surface. Moreover, P482 mutants in genes associated with motility or chemotaxis, the synthesis of polysaccharides, and encoding proteases or regulatory factors, which affect biofilm formation on glass, were fully capable of colonizing the root tissue of both tomato and maize hosts. Investigating the role of cellular factors in biofilm formation using these plant-associated bacteria shows that the ability of bacteria to form biofilm on abiotic surfaces does not necessarily mirror its ability to colonize plant tissues. Our research provides a broader perspective on the adaptation of these bacteria to various environments.
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Biopelículas , Carbono , Pseudomonas , Biopelículas/crecimiento & desarrollo , Pseudomonas/fisiología , Pseudomonas/metabolismo , Pseudomonas/genética , Carbono/metabolismo , Raíces de Plantas/microbiología , Rizosfera , Solanum lycopersicum/microbiología , Zea mays/microbiología , Vidrio , Adhesión Bacteriana , Glicerol/metabolismo , PoliestirenosRESUMEN
We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binderâthe P3 peptideâwas used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 µg mL-1) with a detection limit (LOD = 3σ/S) of 0.34 µg mL-1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 µg mL-1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.
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Proteína C-Reactiva , Péptidos , Secuencia de Aminoácidos , Anticuerpos , Bacteriófago M13RESUMEN
BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. METHODS: The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. RESULTS: Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. CONCLUSIONS: UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5.
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BACKGROUND: Immunotherapy is emerging as a powerful treatment approach for several types of cancers. Modulating the immune system to specifically target cancer cells while sparing healthy cells, is a very promising approach for safer therapies and increased survival of cancer patients. Tumour-associated antigens are favorable targets for cancer immunotherapy, as they are exclusively expressed by the cancer cells, minimizing the risk of an autoimmune reaction. The ability to initiate the activation of the immune system can be achieved by virus-like particles (VLPs) which are safe and potent delivery tools. VLP-based vaccines have evolved dramatically over the last few decades and showed great potential in preventing infectious diseases. Immunogenic potency of engineered VLPs as a platform for the development of effective therapeutic cancer vaccines has been studied extensively. This study involves recombinant VLPs presenting multiple copies of tumour-specific mucin 1 (MUC1) epitope as a potentially powerful tool for future immunotherapy. RESULTS: In this report VLPs based on the structural protein of Norovirus (NoV VP1) were genetically modified to present multiple copies of tumour-specific MUC1 epitope on their surface. Chimeric MUC1 particles were produced in the eukaryotic Leishmania tarentolae expression system and used in combination with squalene oil-in-water emulsion MF59 adjuvant to immunize BALB/c mice. Sera from vaccinated mice demonstrated high titers of IgG and IgM antibodies which were specifically recognizing MUC1 antigen. CONCLUSIONS: The obtained results show that immunization with recombinant chimeric NoV VP1- MUC1 VLPs result in high titers of MUC1 specific IgG antibodies and show great therapeutic potential as a platform to present tumour-associated antigens.
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Neoplasias , Escualeno , Animales , Epítopos , Humanos , Inmunización , Inmunoglobulina G , Ratones , Mucina-1 , Neoplasias/terapia , AguaRESUMEN
Dendrobium is one of the most species-rich genera of the Paleotropical orchids. It embraces more than 1000 species, most of which are epiphytes. The strong variation in floral characters causes many identification difficulties within this genus. One of the key structures, often sufficient in identification on a species level, is the labellum, which in many species of Dendrobium possesses a thickened callus and various types of trichomes and papillae. The aim of this study is to identify and describe the structures present on the labellum surface of the analyzed species, determine their distribution and density, as well as to check whether the obtained data have taxonomic value. In this paper, we present the results of a micromorphological study on the labellum of 21 species of Dendrobium, representing 13 sections, using scanning electron microscopy (SEM). Our studies revealed the presence of both uni- and multicellular structures on the surface of the labellum. We observed three types of trichomes (conical, cylindrical, ellipsoidal) and three types of papillae (conical, cylindrical, semicircular). Neither trichomes nor papillae were recorded for five species. In addition, we made diagrams showing the distribution and density of structures on the labellum. Based on the micromorphological results combined with the phylogenetic tree performed, we suggest that the presence/absence of labellum structures does not necessarily reflect the phylogenetic relationship and might be misleading, as in some cases, they arise due to convergence.
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Dendrobium , Orchidaceae , Flores/anatomía & histología , Microscopía Electrónica de Rastreo , Orchidaceae/anatomía & histología , Filogenia , TricomasRESUMEN
BACKGROUND: Noroviruses are a major cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. Unfortunately, the development of an effective norovirus vaccine has proven difficult and no prophylactic vaccine is currently available. Further research on norovirus vaccine development should be considered an absolute priority and novel vaccine candidates are needed. One of the recent approaches in safe vaccine development is the use of virus-like particles (VLPs). VLP-based vaccines show great immunogenic potential as they mimic the morphology and structure of viral particles without the presence of the virus genome. RESULTS: This study is the first report showing successful production of norovirus VLPs in the protozoan Leishmania tarentolae (L. tarentolae) expression system. Protozoan derived vaccine candidate is highly immunogenic and able to not only induce a strong immune response (antibody titer reached 104) but also stimulate the production of neutralizing antibodies confirmed by receptor blocking assay. Antibody titers able to reduce VLP binding to the receptor by > 50% (BT50) were observed for 1:5-1:320 serum dilutions. CONCLUSIONS: Norovirus VLPs produced in L. tarentolae could be relevant for the development of the norovirus vaccine.
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Anticuerpos Neutralizantes/sangre , Leishmania/genética , Leishmania/virología , Norovirus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/inmunología , Animales , Inmunización , Inmunoglobulina G/sangre , Leishmania/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Norovirus/genética , Desarrollo de Vacunas , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Temperature is one of the critical factors affecting gene expression in bacteria. Despite the general interest in the link between bacterial phenotypes and environmental temperature, little is known about temperature-dependent gene expression in plant pathogenic Pectobacterium atrosepticum, a causative agent of potato blackleg and tuber soft rot worldwide. In this study, twenty-nine P. atrosepticum SCRI1043 thermoregulated genes were identified using Tn5-based transposon mutagenesis coupled with an inducible promotorless gusA gene as a reporter. From the pool of 29 genes, 14 were up-regulated at 18 °C, whereas 15 other genes were up-regulated at 28 °C. Among the thermoregulated loci, genes involved in primary bacterial metabolism, membrane-related proteins, fitness-corresponding factors, and several hypothetical proteins were found. The Tn5 mutants were tested for their pathogenicity in planta and for features that are likely to remain important for the pathogen to succeed in the (plant) environment. Five Tn5 mutants expressed visible phenotypes differentiating these mutants from the phenotype of the SCRI1043 wild-type strain. The gene disruptions in the Tn5 transposon mutants caused alterations in bacterial generation time, ability to form a biofilm, production of lipopolysaccharides, and virulence on potato tuber slices. The consequences of environmental temperature on the ability of P. atrosepticum to cause disease symptoms in potato are discussed.
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Elementos Transponibles de ADN/genética , Pectobacterium/genética , Enfermedades de las Plantas/genética , Solanum tuberosum/genética , Resistencia a la Enfermedad/genética , Regulación Bacteriana de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Pectinas/química , Pectinas/genética , Pectobacterium/patogenicidad , Enfermedades de las Plantas/microbiología , Solanum tuberosum/microbiología , Temperatura , Transposasas/genéticaRESUMEN
The high photodynamic effect of the Newman strain of the S. aureus and of clinical strains of S. aureus MRSA 12673 and E. coli 12519 are observed for new cationic light-activated phenosafranin polyhedral oligomeric silsesquioxane (POSS) conjugates in vitro. Killing of bacteria was achieved at low concentrations of silsesquioxanes (0.38 µM) after light irradiation (λem. max = 522 nm, 10.6 mW/cm2) for 5 min. Water-soluble POSS-photosensitizers are synthesized by chemically coupling a phenosafranin dye (PSF) (3,7-diamino-5-phenylphenazine chloride) to an inorganic silsesquioxane cage activated by attachment of succinic anhydride rings. The chemical structure of conjugates is confirmed by 1H, 13C NMR, HRMS, IR, fluorescence spectroscopy and UV-VIS analyzes. The APDI and daunorubicin (DAU) synergy is investigated for POSSPSFDAU conjugates. Confocal microscopy experiments indicate a site of intracellular accumulation of the POSSPSF, whereas iBuPOSSPSF and POSSPSFDAU accumulate in the cell wall or cell membrane. Results from the TEM study show ruptured S. aureus cells with leaking cytosolic mass and distorted cells of E. coli. Bacterial cells are eradicated by ROS produced upon irradiation of the covalent conjugates that can kill the bacteria by destruction of cellular membranes, intracellular proteins and DNA through the oxidative damage of bacteria.
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Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Fenazinas/farmacología , Antibacterianos/farmacología , Bacterias/metabolismo , Cationes/metabolismo , Membrana Celular/metabolismo , Escherichia coli/efectos de los fármacos , Fenazinas/metabolismo , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/farmacología , Porfirinas/química , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ÏA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ÏA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.
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Bacteriófagos , Regulación Bacteriana de la Expresión Génica , Mutación , Pectobacterium , Enfermedades de las Plantas/microbiología , Polisacáridos Bacterianos , Solanum tuberosum/microbiología , Factores de Virulencia/biosíntesis , Bacteriófagos/genética , Bacteriófagos/metabolismo , Pectobacterium/genética , Pectobacterium/metabolismo , Pectobacterium/patogenicidad , Pectobacterium/virología , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Factores de Virulencia/genéticaRESUMEN
Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL-1) and only weakly sensitive to their mixtures, i.e., extracts and extracts' fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.
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Planta Carnívora/química , Nanopartículas del Metal/química , Extractos Vegetales/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/efectos adversos , Antibacterianos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Naftoquinonas/efectos adversos , Naftoquinonas/química , Naftoquinonas/farmacología , Extractos Vegetales/química , Pseudomonas aeruginosa/patogenicidad , Metabolismo Secundario/efectos de los fármacos , Plata/química , Espectrofotometría UltravioletaRESUMEN
Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.
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Antibacterianos/farmacología , Cólera/dietoterapia , Isotiocianatos/farmacología , Mariposas Nocturnas/microbiología , Sulfóxidos/farmacología , Vibrio cholerae/efectos de los fármacos , Animales , Biopelículas/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , ADN/biosíntesis , Modelos Animales de Enfermedad , Guanosina Tetrafosfato/biosíntesis , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , ARN/biosíntesis , Células Vero , Vibrio cholerae/patogenicidad , Virulencia/efectos de los fármacos , Factores de Virulencia/biosíntesisRESUMEN
Ultrashort cationic lipopeptides (USCLs) are considered to be a promising class of antimicrobials with high activity against a broad-spectrum of microorganisms. However, the majority of these compounds are characterized by significant toxicity toward human cells, which hinders their potential application. To overcome those limitations, several approaches have been advanced. One of these is disulfide cyclization that has been shown to improve drug-like characteristics of peptides. In this article the effect of disulfide cyclization of the polar head of N-palmitoylated USCLs on in vitro biological activity has been studied. Lipopeptides used in this study consisted of three or four basic amino acids (lysine and arginine) and cystine in a cyclic peptide. In general, disulfide cyclization of the lipopeptides resulted in peptides with reduced cytotoxicity. Disulfide-cyclized USCLs exhibited improved selectivity between Candida sp., Gram-positive strains and normal cells in contrast to their linear counterparts. Interactions between selected USCLs and membranes were studied by molecular dynamics simulations using a coarse-grained force field. Moreover, membrane permeabilization properties and kinetics were examined. Fluorescence and transmission electron microscopy revealed damage to Candida cell membrane and organelles. Concluding, USCLs are strong membrane disruptors and disulfide cyclization of polar head can have a beneficial effect on its in vitro selectivity between Candida sp. and normal human cells.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proliferación Celular/efectos de los fármacos , Lipopéptidos/farmacología , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Candida/efectos de los fármacos , Ciclización , Disulfuros/química , Disulfuros/farmacología , Bacterias Grampositivas/efectos de los fármacos , Humanos , Lipopéptidos/química , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
Obstacles to effective therapies for mucopolysaccharidoses (MPSs) determine the need for continuous studies in order to enhance therapeutic strategies. Dimethyl sulfoxide (DMSO) is frequently utilised as a solvent in biological studies, and as a vehicle for drug therapy and the in vivo administration of water-insoluble substances. In the light of the uncertainty on the mechanisms of DMSO impact on metabolism of glycosaminoglycans (GAGs) pathologically accumulated in MPSs, in this work, we made an attempt to investigate and resolve the question of the nature of GAG level modulation by DMSO, the isoflavone genistein solvent employed previously by our group in MPS treatment. In this work, we first found the cytotoxic effect of DMSO on human fibroblasts at concentrations above 3%. Also, our results displayed the potential role of DMSO in the regulation of biological processes at the transcriptional level, then demonstrated a moderate impact of the solvent on GAG synthesis. Interestingly, alterations of lysosomal ultrastructure upon DMSO treatment were visible. As there is growing evidence in the literature that DMSO can affect cellular pathways leading to numerous changes, it is important to expand our knowledge concerning this issue.
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Dimetilsulfóxido/administración & dosificación , Genisteína/administración & dosificación , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Mucopolisacaridosis/tratamiento farmacológico , Línea Celular , Proliferación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/antagonistas & inhibidores , Humanos , Isoflavonas/metabolismo , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Mucopolisacaridosis/metabolismo , Mucopolisacaridosis/patologíaRESUMEN
Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes.
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Bacteriófago lambda/genética , Replicación del ADN , Regiones Promotoras Genéticas , Transcripción Genética , ADN Viral/biosíntesis , Mutación , Plásmidos/biosíntesis , Origen de Réplica , Proteínas Virales/metabolismoRESUMEN
The aquatic environment is constantly under threat due to the release of numerous pollutants. Among them, pharmaceuticals constitute a huge and diverse group. Non-steroidal anti-inflammatory drugs (NSAIDs) are increasingly found in water bodies, but knowledge about their potential toxicity is still low. In particular, there is a lack of information about their influences on aquatic plants and algae. We estimated the susceptibility of the microalgae Chlamydomonas reinhardtii to nabumetone (NBT) and flufenamic acid (FFA), focusing on photosynthesis. Due to the differences in the structures of these compounds, it was assumed that these drugs would have different toxicities to the tested green algae. The hypothesis was confirmed by determining the effective concentration values, the intensity of photosynthesis, the intensity of dark respiration, the contents of photosynthetic pigments, the fluorescence of chlorophyll a in vivo (OJIP test), and cell ultrastructure analysis. Assessment of the toxicity of the NSAIDs was extended by the calculation of an integrated biomarker response index (IBR), which is a valuable tool in ecotoxicological studies. The obtained results indicate an over six times higher toxicity of NBT compared to FFA. After analysis of the chlorophyll a fluorescence in vivo, it was found that NBT inhibited electron transport beyond the PS II. FFA, unlike NBT, lowered the intensity of photosynthesis, probably transforming some reaction centers into "silent centers", which dissipate energy as heat. The IBR estimated based on photosynthetic parameters suggests that the toxic effect of FFA results mainly from photosynthesis disruption, whereas NBT significantly affects other cellular processes. No significant alteration in the ultrastructure of treated cells could be seen, except for changes in starch grain number and autophagic vacuoles that appeared in FFA-treated cells. To the best of our knowledge, this is the first work reporting the toxic effects of NBT and FFA on unicellular green algae.
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Chlamydomonas reinhardtii , Chlorophyta , Clorofila A , Clorofila , Nabumetona/farmacología , Ácido Flufenámico/toxicidad , Fotosíntesis , Antiinflamatorios no Esteroideos/farmacologíaRESUMEN
Herein, we report the synthesis of new compounds with demonstrated anticancer properties based on the 2,3,4,9-tetrahydro-1H-carbazole scaffold. The Fischer indolization method was used to close the heterocyclic motif. The synthesis method's scope and limitations were thoroughly assessed through a series of experiments. Biological assays revealed that two thioamide compounds exhibited significant anticancer activity against MCF-7, HTC116, and A596 cell lines. Comprehensive in vitro profiling included evaluation of cell cytotoxicity, morphological alterations, colony formation and cell adhesion in 3D cultures, cell cycle analysis, DNA damage induction, impact on mitochondria, and apoptosis. Ex ovo studies further demonstrated these compounds' potential to inhibit angiogenic processes. Our results indicate that the newly developed compounds activate processes leading to DNA damage and disruption of mitochondrial function.
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Background: Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Methods: Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L-1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy. Results: The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants. Conclusions: The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms.
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Antiinflamatorios no Esteroideos , Chlamydomonas reinhardtii , Diclofenaco , Potencial de la Membrana Mitocondrial , Mitocondrias , Consumo de Oxígeno , Especies Reactivas de Oxígeno , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Diclofenaco/toxicidad , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestructura , Antiinflamatorios no Esteroideos/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Cianuro de Potasio/toxicidad , Oxidorreductasas/metabolismo , Salicilamidas , Microscopía Electrónica de Transmisión , Proteínas de Plantas , Proteínas MitocondrialesRESUMEN
BACKGROUND: Compounds of natural origin are potent source of drugs with unique mechanisms of action. Among phytochemicals, trans-cinnamaldehyde (t-CA) exhibits a wide range of biological activity, thus has been used for centuries to fight bacterial and fungal infections. However, the molecular basis of these properties has not been fully covered. Considering that difficult-to-control infections are becoming a rising global problem, there is a need to elucidate the molecular potential of t-CA. PURPOSE: To evaluate the antibacterial activity of t-CA against Shiga-toxigenic E. coli strains and elucidate its mechanism of action based on the inhibition of the virulence factor expression. METHODS: The antimicrobial potential of t-CA was assessed with two-fold microdilution and time-kill assays. Further evaluation included bioluminescence suppression assays, quantification of reactive oxygen species (ROS) and assessment of NAD+/NADH ratios. Morphological changes post t-CA exposure were examined using transmission electron microscopy. RNA sequencing and radiolabeling of nucleotides elucidated the metabolic alterations induced by t-CA. Toxin expression level was monitored through the application of fusion proteins, monitoring of bacteriophage development, and fluorescence microscopy studies. Lastly, the therapeutic efficacy in vivo was assessed using Galleria mellonella infection model. RESULTS: A comprehensive study of t-CA's bioactivity showed unique properties affecting bacterial metabolism and morphology, resulting in significant bacterial cell deformation and effective virulence inhibition. Elucidation of the underlying mechanisms indicated that t-CA activates the global regulatory system, the stringent response, manifested by its alarmone, (p)ppGpp, overproduction mediated by the RelA enzyme, thereby inhibiting bacterial proliferation. Intriguingly, t-CA effectively downregulates Shiga toxin gene expression via alarmone molecules, indicating its potential for therapeutic effect. In vivo validation demonstrated a significant improvement in larval survival rates post- t-CA treatment with 50 mg/kg (p < 0.05), akin to the efficacy observed with azithromycin, thus indicating its effectiveness against EHEC infections (p < 0.05). CONCLUSIONS: Collectively, these results reveal the robust antibacterial capabilities of t-CA, warranting its further exploration as a viable anti-infective agent.
Asunto(s)
Acroleína , Antibacterianos , Escherichia coli Enterohemorrágica , Pruebas de Sensibilidad Microbiana , Acroleína/análogos & derivados , Acroleína/farmacología , Antibacterianos/farmacología , Escherichia coli Enterohemorrágica/efectos de los fármacos , Animales , Especies Reactivas de Oxígeno/metabolismo , Factores de VirulenciaRESUMEN
Podocytes are crucial for regulating glomerular permeability. They have foot processes that are integral to the renal filtration barrier. Understanding their energy metabolism could shed light on the pathogenesis of filtration barrier injury. Lactate has been increasingly recognized as more than a waste product and has emerged as a significant metabolic fuel and reserve. The recent identification of lactate transporters in podocytes, the expression of which is modulated by glucose levels and lactate, highlights lactate's relevance. The present study investigated the impact of lactate on podocyte respiratory efficiency and mitochondrial dynamics. We confirmed lactate oxidation in podocytes, suggesting its role in cellular energy production. Under conditions of glucose deprivation or lactate supplementation, a significant shift was seen toward oxidative phosphorylation, reflected by an increase in the oxygen consumption rate/extracellular acidification rate ratio. Notably, lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) isoforms, which are involved in lactate conversion to pyruvate, were detected in podocytes for the first time. The presence of lactate led to higher intracellular pyruvate levels, greater LDH activity, and higher LDHB expression. Furthermore, lactate exposure increased mitochondrial DNA-to-nuclear DNA ratios and resulted in upregulation of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor coactivator-1α and transcription factor A mitochondrial, regardless of glucose availability. Changes in mitochondrial size and shape were observed in lactate-exposed podocytes. These findings suggest that lactate is a pivotal energy source for podocytes, especially during energy fluctuations. Understanding lactate's role in podocyte metabolism could offer insights into renal function and pathologies that involve podocyte injury.
Asunto(s)
L-Lactato Deshidrogenasa , Ácido Láctico , Dinámicas Mitocondriales , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Podocitos , Podocitos/metabolismo , Podocitos/patología , Animales , Ratas , Ácido Láctico/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Glucosa/metabolismo , Metabolismo Energético , Lactato Deshidrogenasa 5/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , Consumo de Oxígeno , Células Cultivadas , Ácido Pirúvico/metabolismo , IsoenzimasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) presents a formidable challenge with high lethality and limited effective drug treatments. Its heightened metastatic potential further complicates the prognosis. Owing to the significant toxicity of current chemotherapeutics, compounds like [Met5]-enkephalin, known as opioid growth factor (OGF), have emerged in oncology clinical trials. OGF, an endogenous peptide interacting with the OGF receptor (OGFr), plays a crucial role in inhibiting cell proliferation across various cancer types. This in vitro study explores the potential anticancer efficacy of a newly synthesized OGF bioconjugate in synergy with the classic chemotherapeutic agent, gemcitabine (OGF-Gem). The study delves into assessing the impact of the OGF-Gem conjugate on cell proliferation inhibition, cell cycle regulation, the induction of cellular senescence, and apoptosis. Furthermore, the antimetastatic potential of the OGF-Gem conjugate was demonstrated through evaluations using blood platelets and AsPC-1 cells with a light aggregometer. In summary, this article demonstrates the cytotoxic impact of the innovative OGF-Gem conjugate on pancreatic cancer cells in both 2D and 3D models. We highlight the potential of both the OGF-Gem conjugate and OGF alone in effectively inhibiting the ex vivo pancreatic tumor cell-induced platelet aggregation (TCIPA) process, a phenomenon not observed with Gem alone. Furthermore, the confirmed hemocompatibility of OGF-Gem with platelets reinforces its promising potential. We anticipate that this conjugation strategy will open avenues for the development of potent anticancer agents.