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  • ÍtemAcceso Abierto
    Efficient workflow for structural elucidation applied to solidagenone derivative: combining the speed of ML-J-DP4 screening with the precision of DP4+
    (Elsevier, 2025-03-06) Cortés, Iván; Tibaldi Bollati, María Luz; Nicotra, Viviana Estela; García, Manuela E.; Sarotti, Ariel Marcelo; https://orcid.org/0000-0002-8069-2793; https://orcid.org/0000-0002-7931-0639; https://orcid.org/0000-0002-5255-4721; https://orcid.org/0000-0002-6809-4723; https://orcid.org/0000-0002-8151-0306
    The derivatization of solidagenone through ring expansion, using the "Complexity to Diversity" synthetic strategy to obtain polyketones, yielded two products upon reaction of an intermediate with in situ-generated ruthenium tetroxide. As might happen, the spectroscopic and spectrometric data were inconclusive for the structural determination of one of these products. To address the issue, a ML-J-DP4//DP4+ workflow was studied, which allowed simultaneous determination of connectivity and relative configuration with high accuracy, while saving computational resources. Furthermore, during the calculation of NMR data at the DP4+ level, abnormally large errors were observed for the beta carbon of a polyhydroxylated conjugated enone. A literature analysis revealed that this behavior could be generalized, and after careful examination, it was determined to be the result of small geometric distortions caused by an overestimated network of intramolecular hydrogen bonds. To mitigate this common source of discrepancy, a heuristic solution was introduced as a proof of concept by in silico blocking of hydroxyl groups.
  • ÍtemAcceso Abierto
    Discovery of unusual dimeric piperazyl cyclopeptides encoded by a Lentzea flaviverrucosa DSM 44664 biosynthetic supercluster
    (National Academy of Sciences, 2022-04-19) Li, Chun-shun; Huc, Yifei; Wua, Xiaohua; Stumpf, Spencer D.; Qic, Yunci; D’Alessandro, John M.; Nepal, Keshav K.; Sarotti, Ariel Marcelo; Cao, Shugeng; Blodgett, Joshua A. V.; https://orcid.org/0000-0002-3134-8224; https://orcid.org/0000-0002-0150-1777; https://orcid.org/0000-0002-8991-716X; https://orcid.org/0000-0002-8151-0306; https://orcid.org/0000-0001-6684-8221; https://orcid.org/0000-0002-7080-5870
    Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B. Petrichorin B is a symmetric homodimer similar to the known compound chloptosin, but petrichorin A is unique among known piperazyl cyclopeptides because it is an asymmetric heterodimer. Due to the structural complexity of petrichorin A, solving its structure required a combination of several standard chemical methods plus in silico modeling, strain mutagenesis, and solving the structure of its biosynthetic intermediate petrichorin C for confident assignment. Furthermore, we found that the piperazyl cyclopeptides comprising each half of the petrichorin A heterodimer are made via two distinct nonribosomal peptide synthetase (NRPS) assembly lines, and the responsible NRPS enzymes are encoded within a contiguous biosynthetic supercluster on the L. flaviverrucosa chromosome. Requiring promiscuous cytochrome p450 crosslinking events for asymmetric and symmetric biaryl production, petrichorins A and B exhibited potent in vitro activity against A2780 human ovarian cancer, HT1080 fibrosarcoma, PC3 human prostate cancer, and Jurkat human T lymphocyte cell lines with IC50 values at low nM levels. Cyclic piperazyl peptides and their crosslinked derivatives are interesting drug leads, and our findings highlight the potential for heterodimeric bicyclic peptides such as petrichorin A for inclusion in future pharmaceutical design and discovery programs
  • ÍtemAcceso Abierto
    Synthesis and characterization of new bases derived from nitrophenylpyrazoles, coordination to palladium and antifungal activity and catalytic activity in Mizoroki–Heck reactions
    (MDPI, 2024-06-16) Londoño Salazar, Jennifer; Restrepo Acevedo, Andrés; Torres, John Eduard; Abonia, Rodrigo; Svetaz, Laura Andrea; Zacchino, Susana A.; Le Lagadec, Ronan; Cuenú Cabezas, Fernando; https://orcid.org/0000-0002-4974-3599; https://orcid.org/0000-0003-3256-0961; https://orcid.org/0000-0002-5679-2081; https://orcid.org/0000-0003-2997-6720
    In this study, we report the synthesis of eight Schiff bases (3–10) type N-heterocycle (N-het) using conventional refluxing conditions as well as different eco-friendly techniques such as grinding, thermal fusion, microwave irradiation (MWI) and ultrasound, all of them in the presence of a catalytic amount of acetic acid. These procedures had the additional advantage of being environmentally friendly and high-yield, making these protocols an alternative for Schiff-base syntheses. The obtained Schiff bases were coordinated to palladium, generating new complexes of type [Pd2Cl4(N-het)2]. Complexes [Pd2Cl4(5)2] and [Pd2Cl4(9)2] showed high activity and selectivity for a model Mizoroki–Heck C-C coupling reaction of styrene with iodobenzene and bromobenzaldehydes. All compounds and complexes were evaluated for antifungal activity against clinically important fungi such as Candida albicans and Cryptococcus neoformans. Although the Schiff bases (3–10) showed low antifungal activity against both fungi, some of their palladium complexes such as [Pd2Cl4(3)2], [Pd2Cl4(5)2], [Pd2Cl4(8)2] and [Pd2Cl4(10)2] showed comparatively higher antifungal effects mainly against C. neoformans. The product of the Mizoroki–Heck-type C-C coupling reactions, 4-styrylbenzaldehyde, was isolated and purified to be later used in the synthesis of four new nitrophenylpyrazole derivatives of styrylimine, which also displayed antifungal activity, especially against C. neoformans.
  • ÍtemAcceso Abierto
    Disentangling the regulatory response of Agrobacterium tumefaciens CHLDO to glyphosate for engineering whole-cell phosphonate biosensors
    (American Chemical Society, 2024-09-30) Masotti, Fiorella; Krink, Nicolas; Lencina, Nicolas; Gottig, Natalia; Ottado, Jorgelina; Nikel, Pablo I.; https://orcid.org/0000-0002-9313-7481
    Phosphonates (PHTs), organic compounds with a stable C−P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, onsite methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as a host and the source of genetic parts for constructing PHT biosensors. In this bacterial species, the phn gene cluster, encoding the C−P lyase pathway, is regulated by the PhnF transcriptional repressor. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise genetic optimization of the transcriptional cascade, we created a whole-cell biosensor capable of detecting GP in the 0.25−50 μM range in various samples, including soil and water.
  • ÍtemAcceso Abierto
    1,3,4-oxadiazoles as inhibitors of the atypical member of the BET family bromodomain factor 3 from Trypanosoma cruzi (TcBDF3)
    (Frontiers, 2024-10-01) Alonso, Victoria Lucía; Escalante, Andrea Marta; Rodríguez Araya, Elvio; Frattini, Gianfranco; Tavernelli, Luis Emilio; Moreno, Diego M.; Furlán, Ricardo Luis Eugenio; Serra, Esteban Carlos
    Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions globally, with increasing urban cases outside of Latin America. Treatment is based on two compounds, namely, benznidazole (BZ) and nifurtimox, but chronic cases pose several challenges. Targeting lysine acetylation, particularly bromodomain-containing proteins, shows promise as a novel antiparasitic target. Our research focuses on TcBDF3, a cytoplasmic protein, which is crucial for parasite differentiation that recognizes acetylated alpha-tubulin. In our previous study, A1B4 was identified as a high-affinity binder of TcBDF3, showing significant trypanocidal activity with low host toxicity in vitro. In this report, the binding of TcBDF3 to A1B4 was validated using differential scanning fluorescence, fluorescence polarization, and molecular modeling, confirming its specific interaction. Additionally, two new 1,3,4-oxadiazoles derived from A1B4 were identified, which exhibited improved trypanocide activity and cytotoxicity profiles. Furthermore, TcBDF3 was classified for the first time as an atypical divergent member of the bromodomain extraterminal family found in protists and plants. These results make TcBDF3 a unique target due to its localization and known functions not shared with higher eukaryotes, which holds promise for Chagas disease treatment.
  • ÍtemAcceso Abierto
    Larrea nitida extract-loaded nanodispersions as a novel bio-stimulant for tomato plants
    (Elsevier, 2024-12-05) Rocha, Felipe; Svetaz, Laura Andrea; Sortino, Maximiliano Andrés; Campos Bermúdez, Valeria Alina; Rius, Sebastián Pablo
    Larrea nitida Cav. (Zygophyllaceae) hydrophobic extract (LE) is rich in antimicrobial and antioxidant compounds. In our previous study, water-soluble nanodispersions without (PZ) and with LE (PZLE) were produced to facilitate their application in agriculture. The foliar treatment was carried out with water (control), PZ and PZLE thrice a week at concentrations of 33 and 100 mg.L− 1 on 1-week-old tomato seedlings (Solanum lycopersicum L. variety perita platensis). PZLE at a concentration of 100 mg.L− 1 significantly induced a growth promotion effect and decreased the activity of the antioxidant enzymes guaiacol peroxidase (GPx) and catalase (CAT), probably due to the presence of antioxidant compounds in PZLE. However, 5 days after infection with Pseudomona syringae pv. tomato, the activity of CAT increased by 73 % compared to the control, as a response to the stress caused by the infection. Additionally, plants treated with PZLE at 33 and 100 mg.L− 1 exhibited reductions in infection symptoms of 44 % and 76 %, respectively. As for both concentrations of PZ, no significant differences were found when compared to the control, suggesting a protective effect of PZLE. The photosynthetic parameters of the plants, such as Phi2 (φPSII), non-photochemical quenching (NPQ) and the relative chlorophyll in the leaves of these plants remained unaffected, suggesting no detrimental effects of PZLE on photosynthetic efficiency. PZLE demonstrates potential as a bio-stimulant for tomato plants, offering a complementary approach to traditional agricultural inputs.
  • ÍtemAcceso Abierto
    Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro
    (Frontiers, 2023-06-22) Ruatta, Santiago M.; Prada Gori, Denis N.; Fló Díaz, Martín; Carlucci, Renzo; Medrán, Noelia Soledad; Labadie, Guillermo Roberto; Martínez Amezaga, Maitena; Delpiccolo, Carina M. L.; Mata, Ernesto Gabino; Talevi, Alan; Comini, Marcelo A.; Dr. Hilgenfeld, R. provide the MPro expression vector
    Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence. Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy –performed in a large and diverse chemolibrary– complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening. Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 μM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12–20 μM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7–45 μM). Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known “garbage in, garbage out” machine learning principle.
  • ÍtemAcceso Abierto
    Antitumor activity of new chemicalcompounds in triple negative mammaryadenocarcinoma models
    (Future Science, 2020-02-23) Giolito, María V.; Camacho, Cristián Matías; Martínez Amezaga, Maitena; Traficante, Carla Inés; Giordano, Rocío A.; Cornier, Patricia Griselda; Mata, Ernesto Gabino; Delpiccolo, Carina M. L.; Boggián, Dora Bernarda; Del Giúdice, Antonela; Mainetti, Leandro; Scharovsky, Olga G.; Rozados, Viviana; Rico, María José
    Aim: According to the need for the development of new anticancer agents, we have synthetized novel bioactive compounds and aimed to determine their antitumor action. Materials & methods: We describe in vitro studies evaluating the effect of 35 novel chemical compounds on two triple negative murine mammary adenocarcinoma tumors. Results & conclusion: Three compounds were selected because of their high antitumor activity and their low toxicity to normal cells. Their effect on tumor cells apoptosis, clonogenicity and migratory capacity, were determined. We found that the selected compounds showed inhibition of viability and clonogenic capacity, and promotion of apoptosis. They also decreased the migratory capacity of tumor cells. The results obtained suggest the likely hood of their future use as antitumor and/or antimetastatic agents. Lay abstract: In spite of the important progress achieved in cancer therapeutics, the percentage of people dying because of cancer is still high. Hence, we need to develop new, effective and nontoxic anticancer agents. We synthetized novel compounds and tested their antitumor effect and toxicity, in order to choose those that are effective and do not affect normal cells and therefore, are suitable for human cancer therapies. We selected three out of 35 compounds that show high antitumor action and low toxicity. Also, we studied the mechanisms by which that effect was achieved. Our next goal is to develop experiments with animals in order to have preclinical data that, hopefully, will lead to the clinical use of one or more of the selected compounds.
  • ÍtemAcceso Abierto
    Bacterial and plant natriuretic peptides improve plant defence responses against pathogens
    (BSPP and Wiley, 2018-03-14) Ficarra, Florencia Andrea; Grandellis, Carolina; Garavaglia, Betiana Soledad; Gottig, Natalia; Ottado, Jorgelina
    Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP-like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP-A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over-expressing XacPNP, lines over-expressing AtPNP-A and AtPNP-A-deficient plants were generated. Plants over-expressing XacPNP or AtPNP-A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP-deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP-A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over-expressing XacPNP or AtPNP-A were more resistant to Pst infection than control plants, whereas PNP-deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non-host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.
  • ÍtemAcceso Abierto
    Water extract from inflorescences of industrial hemp Futura 75 variety as a source of anti-inflammatory, anti-proliferative and antimycotic agents: results from in silico, in vitro and ex vivo studies
    (MDPI, 2020-05-17) Orlando, Giustino; Recinella, Lucia; Chiavaroli, Annalisa; Brunetti, Luigi; Leone, Sheila; Carradori, Simone; Di Simone, Simonetta; Ciferri, Maria Chiara; Zengin, Gokhan; Ak, Gunes; Abdullah, Hassan H.; Cordisco, Estefanía; Sortino, Maximiliano Andrés; Svetaz, Laura Andrea; Politi, Matteo; Angelini, Paola; Covino, Stefano; Venanzoni, Roberto; Cesa, Stefania; Menghini, Luigi; Ferrante, Claudio
    Industrial hemp (Cannabis sativa) is traditionally cultivated as a valuable source of fibers and nutrients. Multiple studies also demonstrated antimicrobial, anti-proliferative, phytotoxic and insecticide effects of the essential oil from hemp female inflorescences. On the other side, only a few studies explored the potential pharmacological application of polar extracts from inflorescences. In the present study, we investigated the water extract from inflorescences of industrial hemp Futura 75 variety, from phytochemical and pharmacological point of view. The water extract was assayed for phenolic compound content, radical scavenger/reducing, chelating and anti-tyrosinase effects. Through an ex vivo model of toxicity induced by lipopolysaccharide (LPS) on isolated rat colon and liver, we explored the extract effects on serotonin, dopamine and kynurenine pathways and the production of prostaglandin (PG)E2. Anti-proliferative effects were also evaluated against human colon cancer HCT116 cell line. Additionally, antimycotic effects were investigated against Trichophyton rubrum, Trichophyton interdigitale, Microsporum gypseum. Finally, in silico studies, including bioinformatics, network pharmacology and docking approaches were conducted in order to predict the putative targets underlying the observed pharmacological and microbiological effects. Futura 75 water extract was able to blunt LPS-induced reduction of serotonin and increase of dopamine and kynurenine turnover, in rat colon. Additionally, the reduction of PGE2 levels was observed in both colon and liver specimens, as well. The extract inhibited the HCT116 cell viability, the growth of T. rubrum and T. interdigitale and the activity of tyrosinase, in vitro, whereas in silico studies highlighting the inhibitions of cyclooxygenase-1 (induced by carvacrol), carbonic anhydrase IX (induced by chlorogenic acid and gallic acid) and lanosterol 14-α-demethylase (induced by rutin) further support the observed pharmacological and antimycotic effects. The present findings suggest female inflorescences from industrial hemp as high quality by-products, thus representing promising sources of nutraceuticals and cosmeceuticals against inflammatory and infectious diseases.
  • ÍtemAcceso Abierto
    Alkylhalovinylboranes: a new class of Diels-Alder dienophiles
    (Royal Society of Chemistry, 2018-10-02) Pisano, Pablo Luis; Pellegrinet, Silvina Carla; https://orcid.org/0000-0002-6368-9234; https://orcid.org/0000-0001-6854-7377
    The Diels–Alder reactions of alkylhalovinylboranes have been investigated theoretically and experimentally. Alkylhalovinylboranes presented higher reactivity than the corresponding dialkylvinylboranes. Although endo/exo selectivities were high for the reactions with cyclopentadiene, facial selectivities for the chiral analogues were low. Our results demonstrate that the replacement of an alkyl group on the boron atom by a halogen increases the dienophilicity considerably.
  • ÍtemAcceso Abierto
    Pleiotropic effect of AccD5 and AccE5 depletion in acyl-coenzyme a carboxylase activity and in lipid biosynthesis in mycobacteria
    (Public Library of Science, 2014-06-20) Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Cabruja, Matías Ezequiel; Bardou, Fabienne; Quémard, Annaïk; Gago, Gabriela; Gramajo, Hugo Cesar
    Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acylCoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.
  • ÍtemAcceso Abierto
    Metal-mediated synthesis of pyrrolines
    (Royal Society of Chemistry, 2019-02-27) Medrán, Noelia Soledad; La Venia, Agustina; Testero, Sebastián A.; https://orcid.org/0000-0002-0185-5751
    The five-membered, nitrogen-containing pyrroline ring is a privileged structure. This ring is present in many bioactive compounds from natural sources. Pyrrolines—the dihydro derivatives of pyrroles—have three structural isomer classes, depending on the location of the double bond: 1-pyrrolines (3,4-dihydro-2H-pyrroles), 2-pyrrolines (2,3-dihydro-1H-pyrroles) and 3-pyrrolines (2,5-dihydro-1H-pyrroles). This review aims to describe the latest advances for the synthesis of pyrrolines by transition metal-catalyzed cyclizations. Only reactions in which the pyrroline ring is formed by metal promotion are described. Transformations of the pyrroline ring in other heterocycles, and the structural manipulations of the pyrroline itself are not discussed. The review is organized into three parts, each covering the metal-mediated synthesis of the three pyrroline isomers. Each part is subdivided according to the metal involved, and concludes with a brief description of notable biological activities within the class.
  • ÍtemAcceso Abierto
    Structural requirements for the antifungal activities of natural drimane sesquiterpenes and analogues, supported by conformational and electronic studies
    (MDPI, 2013-02-05) Derita, Marcos Gabriel; Montenegro, Iván; Garibotto, Francisco; Enriz, Ricardo Daniel; Cuellar Fritis, Mauricio; Zacchino, Susana; https://orcid.org/0000-0002-7112-5500
    Seventeen drimanes including polygodial (1), isopolygodial (2), drimenol (3) and confertifolin (4) obtained from natural sources and the semi-synthetic derivatives 5–17 obtained from 1–3, were evaluated in vitro for antifungal properties against a unique panel of fungi with standardized procedures by using two end-points, MIC100 and MIC50. A SAR analysis of the whole series, supported by conformational and electronic studies, allowed us to show that the Δ7,8 -double bond would be one of the key structural features related to the antifungal activity. The MEPs obtained for active compounds exhibit a clear negative minimum value (deep red zone) in the vicinity of the Δ7,8 -double bond, which is not present in the inactive ones. Apart of this negative zone, a positive region (deep blue) appears in 1, which is not observed either in its epimer 2 nor in the rest of the active compounds. The LogP of active compounds varies between 2.33 and 3.84, but differences in MICs are not correlated with concomitant variations in LogP values.
  • ÍtemAcceso Abierto
    A penicillin derivative exerts an anti-metastatic activity in melanoma cells through the downregulation of integrin αvβ3 and Wnt/β-Catenin pathway
    (Frontiers Media, 2020-02-25) Barrionuevo, Elizabeth; Cayrol, María Florencia; Cremaschi, Graciela Alicia; Cornier, Patricia Griselda; Boggián, Dora Bernarda; Delpiccolo, Carina M. L.; Mata, Ernesto Gabino; Roguin, Leonor P.; Blank, Viviana C.
    The synthetic triazolylpeptidyl penicillin derivative, named TAP7f, has been previously characterized as an effective antitumor agent in vitro and in vivo against B16-F0 melanoma cells. In this study, we investigated the anti-metastatic potential of this compound on highly metastatic murine B16-F10 and human A375 melanoma cells. We found that TAP7f inhibited cell adhesion, migration and invasion in a dose-dependent manner. Additionally, we demonstrated that TAP7f downregulated integrin αvβ3 expression and Wnt/β-catenin pathway, a signaling cascade commonly related to tumor invasion and metastasis. Thus, TAP7f reduced both the enzymatic activity and the expression levels of matrix-metalloproteinases-2 and -9 in a time dependent manner. Moreover, TAP7f inhibited the expression of the transcription factor Snail and the mesenchymal markers vimentin, and N-cadherin, and up-regulated the expression of the epithelial marker E-cadherin, suggesting that the penicillin derivative affects epithelial–mesenchymal transition. Results obtained in vitro were supported by those obtained in a B16-F10-bearing mice metastatic model, that showed a significant TAP7f inhibition of lung metastasis. These findings suggest the potential of TAP7f as a chemotherapeutic agent for the treatment of metastatic melanoma.
  • ÍtemAcceso Abierto
    High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties
    (BMC (part of Springer Nature), 2016-10-14) Menendez Bravo, Simón M.; Roulet, Julia; Sabatini, Martín; Comba, Santiago; Dunn, Robert; Gramajo, Hugo Cesar; Arabolaza, Ana Lorena
    Background: microbial synthesis of oleochemicals derived from native fatty acid (FA) metabolism has presented significant advances in recent years. Even so, native FA biosynthetic pathways often provide a narrow variety of usually linear hydrocarbons, thus yielding end products with limited structural diversity. To overcome this limitation, we took advantage of a polyketide synthase-based system from Mycobacterium tuberculosis and developed an Escherichia coli platform with the capacity to synthesize multimethyl-branched long-chain esters (MBE) with novel chemical structures. Results: with the aim to initiate the characterization of these novel waxy compounds, here, we describe the chassis optimization of the MBE producer E. coli strain for an up-scaled oil production. By carrying out systematic metabolic engineering, we improved the final titer to 138.1 ± 5.3 mg MBE L−1 in batch cultures. Fed-batch microbial fermentation process was also optimized achieving a maximum yield of 790.2 ± 6.9 mg MBE L−1 with a volumetric productivity of 15.8 ± 1.1 mg MBE (L h)−1. Purified MBE oil was subjected to various physicochemical analyses, including differential scanning calorimetry (DSC) and pressurized-differential scanning calorimetry (P-DSC) studies. Conclusions: the analysis of the pour point, DSC, and P-DSC data obtained showed that bacterial MBE possess improved cold flow properties than several plant oils and some chemically modified derivatives, while exhibiting high oxidation stability at elevated temperatures. These encouraging data indicate that the presence of multiple methyl branches in these novel esters, indeed, conferred favorable properties which are superior to those of linear esters.
  • ÍtemAcceso Abierto
    Engineering a Streptomyces coelicolor biosynthesis pathway into Escherichia coli for high yield triglyceride production
    (BMC, 2014-12) Comba, Santiago; Sabatini, Martín; Menendez Bravo, Simón M.; Arabolaza, Ana Lorena; Gramajo, Hugo Cesar
    Background: Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations. Results: The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lppβ genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958 ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD600nm of 80 and a product titer of 722.1 mg TAG L-1 at the end of the process. Conclusions: This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.
  • ÍtemAcceso Abierto
    HrpE, the major component of the Xanthomonas type three protein secretion pilus, elicits plant immunity responses
    (Springer Nature, 2018-06-29) Gottig, Natalia; Vranych, Cecilia Verónica; Sgro, Germán Gustavo; Piazza, Ainelén; Ottado, Jorgelina
    Like several pathogenic bacteria, Xanthomonas infect host plants through the secretion of effector proteins by the Hrp pilus of the Type Three Protein Secretion System (T3SS). HrpE protein was identified as the major structural component of this pilus. Here, using the Xanthomonas citri subsp. citri (Xcc) HrpE as a model, a novel role for this protein as an elicitor of plant defense responses was found. HrpE triggers defense responses in host and non-host plants revealed by the development of plant lesions, callose deposition, hydrogen peroxide production and increase in the expression levels of genes related to plant defense responses. Moreover, pre-infiltration of citrus or tomato leaves with HrpE impairs later Xanthomonas infections. Particularly, HrpE C-terminal region, conserved among Xanthomonas species, was sufficient to elicit these responses. HrpE was able to interact with plant Glycine-Rich Proteins from citrus (CsGRP) and Arabidopsis (AtGRP-3). Moreover, an Arabidopsis atgrp-3 knockout mutant lost the capacity to respond to HrpE. This work demonstrate that plants can recognize the conserved C-terminal region of the T3SS pilus HrpE protein as a danger signal to defend themselves against Xanthomonas, triggering defense responses that may be mediated by GRPs.
  • ÍtemAcceso Abierto
    May the force (field) be with you: on the importance of conformational searches in the prediction of NMR chemical shifts
    (MDPI, 2022-11-08) Cuadrado, Cristina; Hernández Daranas, Antonio; Sarotti, Ariel Marcelo; https://orcid.org/0000-0001-9282-1575
    NMR data prediction is increasingly important in structure elucidation. The impact of force field selection was assessed, along with geometry and energy cutoffs. Based on the conclusions, we propose a new approach named mix-J-DP4, which provides a remarkable increase in the confidence level of complex stereochemical assignments—100% in our molecular test set—with a very modest increment in computational cost.
  • ÍtemAcceso Abierto
    Botanical control of citrus green mold and peach brown rot on fruits assays using a persicaria acuminata phytochemically characterized extract
    (MDPI, 2021-02-24) Di Liberto, Melina G.; Seimandi, Gisela; Fernández, Laura; Ruiz, Verónica; Svetaz, Laura Andrea; Derita, Marcos Gabriel; https://orcid.org/0000-0002-0162-2771; https://orcid.org/0000-0001-8148-936X
    Persicaria acuminata (Polygonaceae) is a perennial herb that grows in the central area of Argentina and it is commonly used by native populations to heal infected wounds and other conditions related to fungal infections. In this article, we explored the in vitro antifungal activity of its ethyl acetate extract against a panel of three fruit phytopathogenic fungi including: Penicillium digitatum, P. italicum, and Monilinia fructicola. The sesquiterpenes isolated from the extract were also evaluated against these strains, demonstrating that the dialdehyde polygodia was the responsible for this activity. In order to encourage the use of the extract rather than the pure compound, we displayed ex vivo assays using fresh oranges and peaches inoculated with P. digitatum and M. fructicola, respectively, and subsequently treated by immersion with an extract solution of 250 and 62.5 µg/mL, respectively. There were no statistically significant differences between the treatments with commercial fungicides and the extract over the control of both fruit rots. The concentration of the active compound present in the extract used on fruit experiments was determined by Gas Chromatography-Mass Spectroscopy. Finally, cytotoxicity evaluation against Huh7 cells showed that P. acuminata extract was less cytotoxic than the commercial fungicides at the assayed concentrations. After these findings we could conclude that a chemically characterized extract of P. acuminata should be further developed to treat fungal diseases in fruits from an agro-ecological model.