Hydrogen peroxide modulates the expression of the target of rapamycin (TOR) and cell division in Arabidopsis thaliana.

Hydrogen peroxide (H2O2) is naturally produced by plant cells during normal development and serves as a messenger that regulates cell metabolism. Despite its importance, the relationship between hydrogen peroxide and the target of rapamycin (TOR) pathway, as well as its impact on cell division, has been poorly analyzed. In this study, we explore the interaction of H2O2 with TOR, a serine/threonine protein kinase that plays a central role in controlling cell growth, size, and metabolism in Arabidopsis thaliana. By applying two concentrations of H2O2 exogenously (0.5 and 1 mM), we could correlate developmental traits, such as primary root growth, lateral root formation, and fresh weight, with the expression of the cell cycle gene CYCB1;1, as well as TOR expression. When assessing the expression of the ribosome biogenesis-related gene RPS27B, an increase of 94.34% was noted following exposure to 1 mM H2O2 treatment. This increase was suppressed by the TOR inhibitor torin 2. The elimination of H2O2 accumulation with ascorbic acid (AA) resulted in decreased cell division as well as TOR expression. The potential molecular mechanisms associated with the effects of H2O2 on the cell cycle and TOR expression in roots are discussed in the context of the results.

Sucrose supplements modulate the Pseudomonas chlororaphis-Arabidopsis thaliana interaction via decreasing the production of phenazines and enhancing the root auxin response.

Management of the plant microbiome may help support food needs for the human population. Bacteria influence plants through enhancing nutrient uptake, metabolism, photosynthesis, biomass production and/or reinforcing immunity. However, information into how these microbes behave under different growth conditions is missing. In this work, we tested how carbon supplements modulate the interaction of Pseudomonas chlororaphis with Arabidopsis thaliana. P. chlororaphis streaks strongly repressed primary root growth, lateral root formation and ultimately, biomass production. Noteworthy, increasing sucrose availability into the media from 0 to 2.4% restored plant growth and promoted lateral root formation in bacterized seedlings. This effect could not be observed by supplementing sucrose to leaves only, indicating that the interaction was strongly modulated by bacterial access to sugar. Total phenazine content decreased in the bacteria grown in high (2.4%) sucrose medium, and conversely, the expression of phzH and pslA genes were diminished by sugar supply. Pyocyanin antagonized the promoting effects of sucrose in lateral root formation and biomass production in inoculated seedlings, indicating that this virulence factor accounts for growth repression during the plant-bacterial interaction. Defence reporter transgenes PR-1::GUS and LOX2::GUS were induced in leaves, while the expression of the auxin-inducible, synthetic reporter gene DR5::GUS was enhanced in the roots of bacterized seedlings at low and high sucrose treatments, which suggests that growth/defence trade-offs in plants are critically modulated by P. chlororaphis. Collectively, our data suggest that bacterial carbon nutrition controls the outcome of the relation with plants.

The HGF/Met Receptor Mediates Cytotoxic Effect of Bacterial Cyclodipeptides in Human Cervical Cancer Cells.

BACKGROUND: Human cervix adenocarcinoma (CC) caused by papillomavirus is the third most common cancer among female malignant tumors. Bioactive compounds such as cyclodipeptides (CDPs) possess cytotoxic effects in human cervical cancer HeLa cells mainly by blocking the PI3K/Akt/mTOR pathway and subsequently inducing gene expression by countless transcription regulators. However, the upstream elements of signaling pathways have not been well studied. METHODS: To elucidate the cytotoxic and antiproliferative responses of the HeLa cell line to CDPs by a transcriptomic analysis previously carried out, we identified by immunochemical analyses, differential expression of genes related to the hepatocyte growth factor/mesenchymal-epithelial transition factor (HGF/MET) receptors. Furthermore, molecular docking was carried out to evaluate the interactions of CDPs with the EGF and MET substrate binding sites. RESULTS: Immunochemical and molecular docking analyses suggest that the HGF/MET receptor participation in CDPs cytotoxic effect was independent of the protein expression levels. However, protein modulation of downstream Met-targets occurred due to the inhibition of phosphorylation of the HGF/MET receptor. Results suggest that the antiproliferative and cytotoxicity of CDPs in HeLa cells involve the HGF/MET receptor upstream of PI3K/Akt/mTOR pathway; assays with the human breast cancer MCF-7 and MDA-MB-231cell lines supported the finding. CONCLUSION: Data provide new insights into the molecular mechanisms involved in CDPs cytotoxicity and antiproliferative effects, suggesting that the signal transduction mechanism may be related to the inhibition of the phosphorylation of the EGF/MET receptor at the level of substrate binding site by an inhibition mechanism similar to that of Gefitinib and foretinib anti-neoplastic drugs.

Blood Serum Stimulates the Virulence Potential of Mucorales through Enhancement in Mitochondrial Oxidative Metabolism and Rhizoferrin Production.

This study analyzed the role of blood serum in enhancing the mitochondrial metabolism and virulence of Mucorales through rhizoferrin secretion. We observed that the spores of clinically relevant Mucorales produced in the presence of serum exhibited higher virulence in a heterologous infection model of Galleria mellonella. Cell-free supernatants of the culture broth obtained from spores produced in serum showed increased toxicity against Caenorhabditis elegans, which was linked with the enhanced secretion of rhizoferrin. Spores from Mucoralean species produced or germinated in serum showed increased respiration rates and reactive oxygen species levels. The addition of non-lethal concentrations of potassium cyanide and N-acetylcysteine during the aerobic or anaerobic growth of Mucorales decreased the toxicity of the cell-free supernatants of the culture broth, suggesting that mitochondrial metabolism is important for serum-induced virulence. In support of this hypothesis, a mutant strain of Mucor lusitanicus that lacks fermentation and solely relies on oxidative metabolism exhibited virulence levels comparable to those of the wild-type strain under serum-induced conditions. Contrary to the lower virulence observed, even in the serum, the ADP-ribosylation factor-like 2 deletion strain exhibited decreased mitochondrial activity. Moreover, spores produced in the serum of M. lusitanicus and Rhizopus arrhizus that grew in the presence of a mitophagy inducer showed low virulence. These results suggest that serum-induced mitochondrial activity increases rhizoferrin levels, making Mucorales more virulent.

Study of peripheral domains in structure-function of isocitrate lyase (ICL) from Pseudomonas aeruginosa.

The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.

Effect of Multi-walled Carbon Nanotubes Functionalized with Indol-3-butyric Acid on the Development of Avena sativa.

In pioneering research, it has been documented that the CNT influences the development of plants through the balance of phytoregulators. Therefore, in this work the objective is to evaluate the effects of CNT functionalized by non-covalent method with indole-3-butyric acid that they have on Avena sativa. The CNT was characterized by FTIR and Raman to confirm functionalization. It was observed that in the germination stage the seeds treated with IBA inhibited germination, however, when functionalizing the CNT with IBA it was observed that the CNT is contributing to counteract this inhibition.

Comparison of the Rhizobacteria Serratia sp. H6 and Enterobacter sp. L7 on Arabidopsis thaliana Growth Promotion.

The genera Serratia and Enterobacter belong to the Enterobacteriaceae family and several members have been described as plant growth-promoting rhizobacteria (PGPR). However, how these bacteria influence growth and development is unclear. We performed in vitro interaction assays between either Serratia sp. H6 or Enterobacter sp. L7 with Arabidopsis thaliana seedlings to analyze their effects on plant growth. In experiments of co-cultivation distant from the root tip, Enterobacter sp. decreased root length, markedly increased lateral root number, and slightly increased plant biomass by 33%, 230%, and 69%, respectively, and relative to the control. The volatile organic compounds (VOCs) emitted from Serratia sp. H6 but not those from Enterobacter sp. L7 promoted Arabidopsis growth. A blend of volatile compounds from the two bacteria had effects on plant growth that were similar to those observed for volatile compounds from H6 only. At several densities, the direct contact of roots with Serratia sp. H6 had phytostimulant properties but Enterobacter sp. L7 had clear deleterious effects. Together, these results suggest that direct contact and VOCs of Serratia sp. H6 were the main mechanisms to promote plant growth of A. thaliana, while diffusible compounds of Enterobacter sp. L7 were predominant in their PGPR activity.

Pathogenesis in Pseudomonas aeruginosa PAO1 Biofilm-Associated Is Dependent on the Pyoverdine and Pyocyanin Siderophores by Quorum Sensing Modulation.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals, and plants, through producing different molecular factors such as biofilm, siderophores, and other virulence factors which favor bacterial establishment and infection in the host. In P. aeruginosa PAO1, the production of these factors is regulated by the bacterial quorum sensing (QS) mechanisms. From them, siderophores are involved in iron acquisition, transport, and homeostasis. They are also considered some of the main virulence factors in P. aeruginosa; however, detailed mechanisms to induce bacterial pathogenesis are poorly understood. In this work, through reverse genetics, we evaluated the function of bacterial pathogenesis in the pvd cluster genes, which are required for synthesizing the siderophore pyoverdine (PVD). Single pvdI, pvdJ, pvdL, and double mutant strains were analyzed, and contrary to expected, the pvdL and pvdI mutations increased the concentration of PVD and other phenazines, such as pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA) and also an increased biofilm production and morphology depending on the autoinducer 2-alkyl-4-quinolone (PQS) and the QS molecules acyl-homoserine lactones. Consequently, in the in vivo pathogenicity model of Caenorhabditis elegans, the mutations in pvdI, pvdJ, and pvdL increased the survival of the worms exposed to supernatants or biofilms of the bacterial cultures. However, the double mutant pvdI/pvdJ increased its toxicity in agreeing with the biofilm production, PVD, PYO, and PCA. The findings indicate that the mutations in pvd genes encode non-ribosomal peptide synthetases impacted the biofilm's structure, but suppressively also of the phenazines, confirming that the siderophores contribute to the bacterial establishment and pathogenicity of P. aeruginosa PAO1.

Linolenic Acid Plus Ethanol Exacerbates Cell Death in Saccharomyces cerevisiae by Promoting Lipid Peroxidation, Cardiolipin Loss, and Necrosis.

Polyunsaturated fatty acids (PUFA) hypersensitize yeast to oxidative stress. Ethanol accumulation during fermentation is another factor that induces oxidative stress via mitochondrial dysfunction and ROS overproduction. Since this microorganism has raised growing interest as a PUFA factory, we have studied if the combination of PUFA plus ethanol enhances yeast death. Respiration, ROS generation, lipid peroxidation, mitochondrial cardiolipin content, and cell death were assessed in yeast grown in the presence of 10% ethanol (ETOH) or linolenic acid (C18:3), or ethanol plus C18:3 (ETOH+C18:3). Lipid peroxidation and cardiolipin loss were several-fold higher in cells with ETOH+C18:3 than with C18:3. On the contrary, ETOH tended to increase cardiolipin content without inducing changes in lipid peroxidation. This was consistent with a remarkable diminution of cell growth and an exacerbated propidium iodide staining in cells with only ETOH+C18:3. The respiration rate decreased with all the treatments to a similar degree, and this was paralleled with similar increments in ROS between all the treatments. These results indicate that PUFA plus ethanol hypersensitize yeast to necrotic cell death by exacerbating membrane damage and mitochondrial cardiolipin loss, independent of mitochondrial dysfunction and ROS generation. The implications of these observations for some biotechnological applications in yeast and its physiology are discussed.

Bacterial cyclodipeptides elicit Arabidopsis thaliana immune responses reducing the pathogenic effects of Pseudomonas aeruginosa PAO1 strains on plant development.

Plants being sessile organisms are exposed to various biotic and abiotic factors, thus causing stress. The Pseudomonas aeruginosa bacterium is an opportunistic pathogen for animals, insects, and plants. Direct exposure of Arabidopsis thaliana to the P. aeruginosa PAO1 strain induces plant death by producing a wide variety of virulence factors, which are regulated mainly by quorum sensing systems. Besides virulence factors, P. aeruginosa PAO1 also produces cyclodipeptides (CDPs), which possess auxin-like activity and promote plant growth through activation of the target of the rapamycin (AtTOR) pathway. On the other hand, plant defense mechanisms are regulated through the production of phytohormones, such as salicylic acid (SA) and jasmonic acid (JA), which are induced in response to pathogen-associated molecular patterns (PAMPs), activating defense genes associated with SA and JA such as PATHOGENESIS-RELATED-1 (PR-1) and LIPOXYGENASE2 (LOX2), respectively. PR proteins are suggested to play critical roles in coordinating the Systemic Acquired Resistance (SAR). In contrast, LOX proteins (LOX2, LOX3, and LOX4) have been associated with the production of JA by producing its precursors, oxylipins. The activation of defense mechanisms involves signaling cascades such as Mitogen-Activated Protein Kinases (MAPKs) or the TOR pathway as a switch for re-directing energy towards defense or growth. In this work, we challenged A. thaliana (wild type, mpk6 or mpk3 mutants, and overexpressing TOR) seedlings with P. aeruginosa PAO1 strains to identify the role of bacterial CDPs in the plant immune response. Results showed that the pre-exposure of these Arabidopsis seedlings to CDPs significantly reduced plant infection of the pathogenic P. aeruginosa PAO1 strains, indicating that plants that over-express AtTOR or lack MPK3/MPK6 protein-kinases are more susceptible to the pathogenic effects. In addition, CDPs induced the GUS activity only in the LOX2::GUS plants, indicative of JA-signaling activation. Our findings indicate that the CDPs are molecules that trigger SA-independent and JA-dependent defense responses in A. thaliana; hence, bacterial CDPs may be considered elicitors of the Arabidopsis immune response to pathogens.

Transcriptomics Reveals the Mevalonate and Cholesterol Pathways Blocking as Part of the Bacterial Cyclodipeptides Cytotoxic Effects in HeLa Cells of Human Cervix Adenocarcinoma.

The incidence of human cervix adenocarcinoma (CC) caused by papillomavirus genome integration into the host chromosome is the third most common cancer among women. Bacterial cyclodipeptides (CDPs) exert cytotoxic effects in human cervical cancer HeLa cells, primarily by blocking the PI3K/Akt/mTOR pathway, but downstream responses comprising gene expression remain unstudied. Seeking to understand the cytotoxic and anti-proliferative effects of CDPs in HeLa cells, a global RNA-Seq analysis was performed. This strategy permitted the identification of 151 differentially expressed genes (DEGs), which were either up- or down-regulated in response to CDPs exposure. Database analysis, including Gene Ontology (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed differential gene expression on cancer transduction signals, and metabolic pathways, for which, expression profiles were modified by the CDPs exposure. Bioinformatics confirmed the impact of CDPs in the differential expression of genes from signal transduction pathways such as PI3K-Akt, mTOR, FoxO, Wnt, MAPK, P53, TGF-ß, Notch, apoptosis, EMT, and CSC. Additionally, the CDPs exposure modified the expression of cancer-related transcription factors involved in the regulation of processes such as epigenetics, DNA splicing, and damage response. Interestingly, transcriptomic analysis revealed the participation of genes of the mevalonate and cholesterol biosynthesis pathways; in agreement with this observation, total cholesterol diminished, confirming the blockage of the cholesterol synthesis by the exposure of HeLa cells to CDPs. Interestingly, the expression of some genes of the mevalonate and cholesterol synthesis such as HMGS1, HMGCR, IDI1, SQLE, MSMO1, SREBF1, and SOAT1 was up-regulated by CDPs exposure. Accordingly, metabolites of the mevalonate pathway were accumulated in cultures treated with CDPs. This finding further suggests that the metabolism of cholesterol is crucial for the occurrence of CC, and the blockade of the sterol synthesis as an anti-proliferative mechanism of the bacterial CDPs, represents a reasonable chemotherapeutic drug target to explore. Our transcriptomic study supports the anti-neoplastic effects of bacterial CDPs in HeLa cells shown previously, providing new insights into the transduction signals, transcription factors and metabolic pathways, such as mevalonate and cholesterol that are impacted by the CDPs and highlights its potential as anti-neoplastic drugs.

Early sensing of phosphate deprivation triggers the formation of extra root cap cell layers via SOMBRERO through a process antagonized by auxin signaling.

KEY MESSAGE: The role of the root cap in the plant response to phosphate deprivation has been scarcely investigated. Here we describe early structural, physiological and molecular changes prior to the determinate growth program of the primary roots under low Pi and unveil a critical function of the transcription factor SOMBRERO in low Pi sensing. Mineral nutrient distribution in the soil is uneven and roots efficiently adapt to improve uptake and assimilation of sparingly available resources. Phosphate (Pi) accumulates in the upper layers and thus short and branched root systems proliferate to better exploit organic and inorganic Pi patches. Here we report an early adaptive response of the Arabidopsis primary root that precedes the entrance of the meristem into the determinate developmental program that is a hallmark of the low Pi sensing mechanism. In wild-type seedlings transferred to low Pi medium, the quiescent center domain in primary root tips increases as an early response, as revealed by WOX5:GFP expression and this correlates with a thicker root tip with extra root cap cell layers. The halted primary root growth in WT seedlings could be reversed upon transfer to medium supplemented with 250 µM Pi. Mutant and gene expression analysis indicates that auxin signaling negatively affects the cellular re-specification at the root tip and enabled identification of the transcription factor SOMBRERO as a critical element that orchestrates both the formation of extra root cap layers and primary root growth under Pi scarcity. Moreover, we provide evidence that low Pi-induced root thickening or the loss-of-function of SOMBRERO is associated with expression of phosphate transporters at the root tip. Our data uncover a developmental window where the root tip senses deprivation of a critical macronutrient to improve adaptation and surveillance.

Participation of Acyl-Coenzyme A Synthetase FadD4 of Pseudomonas aeruginosa PAO1 in Acyclic Terpene/Fatty Acid Assimilation and Virulence by Lipid A Modification.

The pathogenic bacterium Pseudomonas aeruginosa possesses high metabolic versatility, with its effectiveness to cause infections likely due to its well-regulated genetic content. P. aeruginosa PAO1 has at least six fadD paralogous genes, which have been implicated in fatty acid (FA) degradation and pathogenicity. In this study, we used mutagenesis and a functional approach in P. aeruginosa PAO1 to determine the roles of the fadD4 gene in acyclic terpene (AT) and FA assimilation and on pathogenicity. The results indicate that fadD4 encodes a terpenoyl-CoA synthetase utilized for AT and FA assimilation. Additionally, mutations in fadD paralogs led to the modification of the quorum-sensing las/rhl systems, as well as the content of virulence factors pyocyanin, biofilm, rhamnolipids, lipopolysaccharides (LPS), and polyhydroxyalkanoates. In a Caenorhabditis elegans in vivo pathogenicity model, culture supernatants from the 24-h-grown fadD4 single mutant increased lethality compared to the PAO1 wild-type (WT) strain; however, the double mutants fadD1/fadD2, fadD1/fadD4, and fadD2/fadD4 and single mutant fadD2 increased worm survival. A correlation analysis indicated an interaction between worm death by the PAO1 strain, the fadD4 mutation, and the virulence factor LPS. Fatty acid methyl ester (FAME) analysis of LPS revealed that a proportion of the LPS and FA on lipid A were modified by the fadD4 mutation, suggesting that FadD4 is also involved in the synthesis/degradation and modification of the lipid A component of LPS. LPS isolated from the fadD4 mutant and double mutants fadD1/fadD4 and fadD2/fadD4 showed a differential behavior to induce an increase in body temperature in rats injected with LPS compared to the WT strain or from the fadD1 and fadD2 mutants. In agreement, LPS isolated from the fadD4 mutant and double mutants fadD1/fadD2 and fadD2/fadD4 increased the induction of IL-8 in rat sera, but IL1-ß cytokine levels decreased in the double mutants fadD1/fadD2 and fadD1/fadD4. The results indicate that the fadD genes are implicated in the degree of pathogenicity of P. aeruginosa PAO1 induced by LPS-lipid A, suggesting that FadD4 contributes to the removal of acyl-linked FA from LPS, rendering modification in its immunogenic response associated to Toll-like receptor TLR4. The genetic redundancy of fadD is important for bacterial adaptability and pathogenicity over the host.

Ligninolytic activity of the Penicillium chrysogenum and Pleurotus ostreatus fungi involved in the biotransformation of synthetic multi-walled carbon nanotubes modify its toxicity.

Multi-walled carbon nanotubes (MWCNTs) are of multidisciplinary scientific interest due to their exceptional physicochemical properties and a broad range of applications. However, they are considered potentially toxic nanoparticles when they accumulate in the environment. Given their ability to oxidize resistant polymers, mycorremediation with lignocellulolytic fungi are suggested as biological alternatives to the mineralization of MWCNTs. Hence, this study involves the ability of two fungi specie to MWCNTs biotransformation by laccase and peroxidases induction and evaluation in vivo of its toxicity using Caenorhabditis elegans worms as a model. Results showed that the fungi Penicillium chrysogenum and Pleurotus ostreatus were capable to grow on media with MWCNTs supplemented with glucose or lignin. Activities of lignin-peroxidase, manganese-peroxidase, and laccase in cultures of both fungi were induced by MWCNTs. Raman, FTIR spectroscopy, HR-TEM, and TGA analyses of the residue from the cultures of both fungi revealed structural modifications on the surface of MWCNTs and its amount diminished, correlating the MWCNTs structural modifications with the laccase-peroxidase activities in the fungal cultures. Results indicate that the degree of toxicity of MWCNTs on the C. elegans model was enhanced by the structure modification associated with the fungal ligninolytic activity. The toxic effect of MWCNTs on the in vivo model of worms reveals the increment of reactive oxygen species as a mechanism of toxicity. Findings indicate that the MWCNTs can be subject in nature to biotransformation processes such as the fungal metabolism, which contribute to modify their toxicity properties on susceptible organisms and contributing to environmental elimination.

N-vanillyl-octanamide represses growth of fungal phytopathogens in vitro and confers postharvest protection in tomato and avocado fruits against fungal-induced decay.

Plant diseases caused by pathogenic fungi result in considerable losses in agriculture. The use of fungicides is an important alternative to combat these pathogens, but may affect both the environment and human health. Plants produce many bioactive compounds to defend themselves from biotic challenges and an increasing number of secondary metabolites have been identified, which may be used to control fungal infections. Here, the bioactivity of a synthetic capsaicinoid, N-vanillyl-octanamide, also termed ABX-I, in the growth of five phytopathogenic fungi was assessed in vitro. The compound inhibited growth of Colletotrichum gloeosporioides, Botrytis cinerea, Colletotrichum acutatum, Fusarium sp., and Rhizoctonia solani AG2, while the magnitude of this effect differed from capsaicin. To investigate if ABX-I could effectively protect crops against phytopathogens, fungal challenges were performed in tomato leaves and fruits, as well as avocado fruits co-infiltrated with Botrytis cinerea or Colletotrichum gloeosporioides, respectively. In both tomato leaves and fruits and avocado fruits, ABX-I decreased the fungal damage not only in vegetative but also in edible tissues, and diminished decay symptoms compared with untreated fruits, which were highly sensitive to the pathogens. Furthermore, ABX-I spray application to tomato or avocado plants did not compromise growth and development, whereas it repressed spore germination and growth of C. gloeosporioides, which suggests its potential as an affordable and promising resource to control fungal diseases in the agronomic sector.

Growth promotion in Arabidopsis thaliana by bacterial cyclodipeptides involves the TOR/S6K pathway activation.

Cyclodipeptides (CDPs) are the smallest peptidic molecules that can be produced by diverse organisms such as bacteria, fungi, and animals. They have multiple biological effects. In this paper, we examined the CDPs produced by the bacteria Pseudomonas aeruginosa PAO1, which are known as opportunistic pathogens of humans and plants on TARGET OF RAPAMYCIN (TOR) signaling pathways, and regulation of root system architecture. This bacterium produces the bioactive CDPs: cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In a previous report, these molecules were found to modulate basic cellular programs not only via auxin mechanisms but also by promoting the phosphorylation of the S6 ribosomal protein kinase (S6K), a downstream substrate of the TOR kinase. In the present work, we found that the inoculation of Arabidopsis plants with P. aeruginosa PAO1, the non-pathogenic P. aeruginosa ΔlasI/Δrhll strain (JM2), or by direct exposure of plants to CDPs influenced growth and promoted root branching depending upon the treatment imposed, while genetic evidence using Arabidopsis lines with enhanced or decreased TOR levels indicated a critical role of this pathway in the bacterial phytostimulation.

Bacterial Cyclodipeptides Target Signal Pathways Involved in Malignant Melanoma.

Melanoma is an aggressive cancer that utilizes multiple signaling pathways, including those that involve oncogenes, proto-oncogenes, and tumor suppressors. It has been suggested that melanoma formation requires cross-talk of the PI3K/Akt/mTOR and Ras-ERK pathways. This pathway cross-talk has been associated with aggressiveness, drug resistance, and metastasis; thus, simultaneous targeting of components of the different pathways involved in melanoma may aid in therapy. We have previously reported that bacterial cyclodipeptides (CDPs) are cytotoxic to HeLa cells and inhibit Akt phosphorylation. Here, we show that CDPs decreased melanoma size and tumor formation in a subcutaneous xenografted mouse melanoma model. In fact, CDPs accelerated death of B16-F0 murine melanoma cells. In mice, antitumor effect was improved by treatment with CDPs using cyclodextrins as drug vehicle. In tumors, CDPs caused nuclear fragmentation and changed the expression of the Bcl-2 and Ki67 apoptotic markers and promoted restoration of hyperactivation of the PI3K/Akt/mTOR pathway. Additionally, elements of several signaling pathways such as the Ras-ERK, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, epithelial-mesenchymal transition, and cancer stem cell pathways were also modified by treatment of xenografted melanoma mice with CDPs. The findings indicate that the multiple signaling pathways implicated in aggressiveness of the murine B16-F0 melanoma line are targeted by the bacterial CDPs. Molecular modeling of CDPs with protein kinases involved in neoplastic processes suggested that these compounds could indeed interact with the active site of the enzymes. The results suggest that CDPs may be considered as potential antineoplastic drugs, interfering with multiple pathways involved in tumor formation and progression.

Antiproliferative effect of bacterial cyclodipeptides in the HeLa line of human cervical cancer reveals multiple protein kinase targeting, including mTORC1/C2 complex inhibition in a TSC1/2-dependent manner.

Cervix adenocarcinoma rendered by human papillomavirus (HPV) integration is an aggressive cancer that occurs by dysregulation of multiple pathways, including oncogenes, proto-oncogenes, and tumor suppressors. The PI3K/Akt/mTOR pathway, which cross-talks with the Ras-ERK pathway, has been associated with cervical cancers (CC), which includes signaling pathways related to carcinoma aggressiveness, metastasis, recurrence, and drug resistance. Since bacterial cyclodipeptides (CDPs) possess cytotoxic properties in HeLa cells with inhibiting Akt/S6k phosphorylation, the mechanism of CDPs cytotoxicity involved was deepened. Results showed that the antiproliferative effect of CDPs occurred by blocking the PI3K/Akt/mTOR pathway, inhibiting the mTORC1/mTORC2 complexes in a TSC1/TSC2-dependent manner. In addition, the CDPs blocked protein kinases from multiple signaling pathways involved in survival, proliferation, invasiveness, apoptosis, autophagy, and energy metabolism, such as PI3K/Akt/mTOR, Ras/Raf/MEK/ERK1/2, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, Wnt/ß-catenin, HSP27, EMT, CSCs, and receptors, such as EGF/ErbB2/HGF/Met. Thus, the antiproliferative effect of the CDPs made it possible to identify the crosstalk of the signaling pathways involved in HeLa cell malignancy and to suggest that bacterial CDPs may be considered as a potential anti-neoplastic drug in human cervical adenocarcinoma therapy.

Multi-walled carbon nanotubes produced after forest fires improve germination and development of Eysenhardtia polystachya.

BACKGROUND: Multi-walled carbon nanotubes (MWCNTs) are nanoparticles with countless applications. MWCNTs are typically of synthetic origin. However, recently, the formation of MWCNTs in nature after forest fires has been documented. Previous reports have demonstrated the positive effects of synthetic MWCNTs on the germination and development of species of agronomic interest; nevertheless, there is practically no information on how synthetic or natural MWCNTs affect forest plant development. In this report, based on insights from dose-response assays, we elucidate the comparative effects of synthetic MWCNTs, amorphous carbon, and natural MWCNTs obtained after a forest fire on Eysenhardtia polystachya plant. METHODS: E. polystachya seeds were sown in peat moss-agrolite substrate and conserved in a shade house. Germination was recorded daily up to 17 days after sowing, and plant development (manifested in shoot and root length, stem diameter, foliar area, and root architecture parameters) was recorded 60 days after sowing. RESULTS: The treatments with natural MWCNTs accelerated the emergence and improved the germination of this plant, thus while untreated seeds achieve 100% of germination within 16th day, seeds supplemented with natural MWCNTs at doses of 20 µg/mL achieve the above percentage within the 4th day. Natural MWCNTs also promoted fresh and dry biomass in all applied treatments, specially at doses of 40 µg/mL where natural MWCNTs significantly promoted leaf number, root growth, and the dry and fresh weights of shoots and roots of seedlings. Seeds supplemented with doses between 20 and 40 µg/mL of amorphous carbon achieving 100% of germination within the 6th day; however, seeds supplemented either with doses of 60 µg/mL of the above carbon or with synthetic MWCNTs at all the tested concentrations could achieve at most 80 % and 70% of germination respectively within the 17 days. Finally, neither treatments added with amorphous carbon nor those added with synthetic MWCNTs, showed significant increases in the fresh and dry biomass of the tested plant. Likewise, the survival of seedlings was reduced between 10 and 20 % with 40 and 60 µg/mL of amorphous carbon, and with synthetic MWCNTs in all the doses applied was reduced at 30% of survival plants. CONCLUSIONS: These findings indicate that MWCNTs produced by wildfire act as plant growth promoters, contributing to the germination and development of adapted to fire-prone conditions species such as E. polystachya.

Omeprazole as a potent activator of human cytosolic aldehyde dehydrogenase ALDH1A1.

BACKGROUND: Accumulation of lipid aldehydes plays a key role in the etiology of human diseases where high levels of oxidative stress are generated. In this regard, activation of aldehyde dehydrogenases (ALDHs) prevents oxidative tissue damage during ischemia-reperfusion processes. Although omeprazole is used to reduce stomach gastric acid production, in the present work this drug is described as the most potent activator of human ALDH1A1 reported yet. METHODS: Docking analysis was performed to predict the interactions of omeprazole with the enzyme. Recombinant human ALDH1A1 was used to assess the effect of omeprazole on the kinetic properties. Temperature treatment and mass spectrometry were conducted to address the nature of binding of the activator to the enzyme. Finally, the effect of omeprazole was evaluated in an in vivo model of oxidative stress, using E. coli cells expressing the human ALDH1A1. RESULTS: Omeprazole interacted with the aldehyde binding site, increasing 4-6 fold the activity of human ALDH1A1, modified the kinetic properties, altering the order of binding of substrates and release of products, and protected the enzyme from inactivation by lipid aldehydes. Furthermore, omeprazole protected E. coli cells over-expressing ALDH1A1 from the effects of oxidative stress generated by H2O2 exposure, reducing the levels of lipid aldehydes and preserving ALDH activity. CONCLUSION: Omeprazole can be repositioned as a potent activator of human ALDH1A1 and may be proposed for its use in therapeutic strategies, to attenuate the damage generated during oxidative stress events occurring in different human pathologies.