(FBIOyF) Departamento de Química Orgánica - Artículos
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Ítem Acceso 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 CesarBackground: 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.Ítem Acceso 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, JorgelinaLike 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.Ítem Acceso 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/0000-0001-9282-1575NMR 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.Ítem Acceso 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 G.; https://orcid.org/0000-0002-0162-2771; https://orcid.org/0000-0001-8148-936XPersicaria 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.Ítem Acceso Abierto Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor(BMC, 2013-01-29) Comba, Santiago; Menendez Bravo, Simón M.; Arabolaza, Ana Lorena; Gramajo, Hugo CesarBackground: Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. Results: We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. Conclusions: The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these results provide new elements and tools for future cell engineering for next-generation biofuels production.Ítem Acceso Abierto Efficient Buchwald–Hartwig and nitrene-mediated five-membered ring closure approaches to the total synthesis of quindoline. Unexpected direct conversion of a nitro group into a phosphazene(Royal Society of Chemistry, 2023-05-05) Thobokholt, Elida N.; Simonetti, Sebastián Osvaldo; Kaufman, Teodoro Saúl; Larghi, Enrique Leandro; Bracca, Andrea Beatriz Juana; https://orcid.org/0000-0001-5780-6433; https://orcid.org/0000-0003-4236-7380; https://orcid.org/0000-0003-3173-2178; https://orcid.org/0000-0002-9791-4831; https://orcid.org/0000-0003-2647-475XTwo total syntheses of quindoline, which take place through the intermediacy of 3-nitroquinoline derivatives, are reported. The general synthetic sequence involves construction of the latter by mechanochemical condensation of benzaldehydes with 2-amino-nitrostyrene, followed either by reduction of the nitro group of the heterocycle and Buchwald–Hartwig cyclization or by a nitrenemediated cyclization under solventless conditions. Use of PPh3 to generate the nitrene resulted in the unprecedented formation of a phosphazene in place of quindoline. This unexpected transformation was explained by means of DFT computations.Ítem Acceso Abierto A coiled coil switch mediates cold sensing by the thermosensory protein DesK(Wiley, 2015-10-08) Saita, Emilio Adolfo; Abriata, Luciano Andrés; Tsai, Yi-Ting; Trajtenberg, Felipe; Lemmin, Thomas; Buschiazzo, Alejandro; Dal Peraro, Matteo; De Mendoza, Diego; Albanesi, DanielaThe thermosensor histidine kinase DesK from Bacillus subtilis senses changes in membrane fluidity initiating an adaptive response. Structural changes in DesK have been implicated in transmembrane signaling, but direct evidence is still lacking. On the basis of structure-guided mutagenesis, we now propose a mechanism of DesK-mediated signal sensing and transduction. The data indicate that stabilization/destabilization of a 2-helix coiled coil, which connects the transmembrane sensory domain of DesK to its cytosolic catalytic region, is crucial to control its signaling state. Computational modeling and simulations reveal couplings between protein, water and membrane mechanics. We propose that membrane thickening is the main driving force for signal sensing and that it acts by inducing helix stretching and rotation prompting an asymmetric kinase-competent state. Overall, the known structural changes of the sensor kinase, as well as further dynamic rearrangements that we now predict, consistently link structure determinants to activity modulation.Ítem Acceso Abierto The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci(Wiley, 2016-11-25) Hernández, Martín Alejandro; Lara, María Julia; Gago, Gabriela; Gramajo, Hugo Cesar; Álvarez, Héctor ManuelThe regulatory mechanisms involved in lipogenesis and triacylglycerol (TAG) accumulation are largely unknown in oleaginous rhodococci. In this study a regulatory protein (here called NlpR: Nitrogen lipid Regulator), which contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci was identified. Under nitrogen deprivation conditions, in which TAG accumulation is stimulated, the nlpR gene was significantly upregulated, whereas a significant decrease of its expression and TAG accumulation occurred when cerulenin was added. The nlpR disruption negatively affected the nitrate/nitrite reduction as well as lipid biosynthesis under nitrogen-limiting conditions. In contrast, its overexpression increased TAG production during cultivation of cells in nitrogen-rich media. A putative ‘NlpR-binding motif’ upstream of several genes related to nitrogen and lipid metabolisms was found. The nlpR disruption in RHA1 strain led to a reduced transcription of genes involved in nitrate/nitrite assimilation, as well as in fatty acid and TAG biosynthesis. Purified NlpR was able to bind to narK, nirD, fasI, plsC and atf3 promoter regions. It was suggested that NlpR acts as a pleiotropic transcriptional regulator by activating of nitrate/nitrite assimilation genes and others genes involved in fatty acid and TAG biosynthesis, in response to nitrogen deprivation.Ítem Acceso Abierto Furo[3,2-c]coumarins carrying carbon substituents at C-2 and/or C-3. Isolation, biological activity, synthesis and reaction mechanisms(Royal Society of Chemistry, 2020) Cortés, Iván; Cala, Javier L.; Bracca, Andrea Beatriz Juana; Kaufman, Teodoro Saúl; http://orcid.org/0000-0002-8069-2793; http://orcid.org/0000-0003-2647-475X; http://orcid.org/0000-0003-3173-2178The isolation, biological activity and synthesis of natural furo[3,2-c]coumarins are presented, covering mainly the developments in the last 35 years. The most relevant approaches toward the synthesis of 2- substituted, 3-substituted and 2,3-disubstituted heterocycles are also discussed, with emphasis on the efficiency of the processes and their mechanisms.Ítem Acceso Abierto Crystal structure of cis-1-phenyl-8-(pyridin-2-yl-methyl)dibenzo[1,2-c:2,1-h]-2,14-dioxa-8-aza-1-borabicyclo[4.4.0]deca-3,8-diene(International Union of Crystallography, 2017-12-01) Ledesma, Gabriela N.; Signorella, Sandra R.; Back, Davi; Schulz Lang, ErnestoThe title compound, C26H23BN2O2, was obtained as by product during synthetic attempts of a complexation reaction between the tripodal ligand H2L [N,N-bis(2-hydroxybenzyl)(pyridin-2-yl)methylamine] and manganese(III) acetate in the presence of NaBPh4. The isolated B-phenyl dioxazaborocine contains an N→B dative bond with a cis conformation. In the crystal, C—H⋯O hydrogen bonds define chains parallel to the b-axis direction. A comparative analysis with other structurally related derivatives is also included, together with a rationalization of the unexpected production of this zwitterionic heterocycle.Ítem Acceso Abierto Antifungal activity and toxicity studies of flavanones isolated from Tessaria dodoneifolia aerial parts(Elsevier, 2020) Soberón, José R.; Sgariglia, Melina A.; Carabajal Torrez, José A.; Aguilar, Franco A.; Pero, Edgardo J.I.; Sampietro, Diego A.; Fernández de Luco, Julia; Labadie, Guillermo RobertoTessaria dodoneifolia [Asteraceae] is traditionally employed in Northwestern Argentina for fungal infections treatment. We report the antifungal activity guided isolation and identification of substances from aerial parts of this species, both individually and in combination with fluconazole (FLU), against Candida albicans strains. Two antifungal flavanones were identified as naringenin (NAR) and pinocembrin (PIN). These compounds could individually inhibit the growth of C. albicans strains. Combinations of NAR and PIN with FLU were synergistic against the FLU resistant and sensitive C. albicans strains. Genotoxic and cytotoxic evaluations were also performed. NAR, PIN and their combinations with FLU did not have a genotoxic effect on Bacillus subtilis rec strains. Finally, these compounds did not show cytotoxicity at concentrations below 80 μg/mL.Ítem Acceso Abierto Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine(Springer Nature, 2020-07-24) Lara, María Julia; Diacovich, Lautaro; Trajtenberg, Felipe; Larrieux, Nicole; Malchiodi, Emilio L.; Fernández, Marisa M.; Gago, Gabriela; Gramajo, Hugo Cesar; Buschiazzo, Alejandro; https://orcid.org/0000-0002-3339-0100; https://orcid.org/0000-0003-0427-5549; https://orcid.org/0000-0001-7501-3330; https://orcid.org/0000-0001-7668-0119; https://orcid.org/0000-0002-2509-6526Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.Ítem Acceso Abierto XacFhaB adhesin, an important Xanthomonas citri ssp. citri virulence factor, is recognized as a pathogen-associated molecular pattern(BSPP and Wiley, 2016-11-15) Garavaglia, Betiana Soledad; Zimaro, Tamara; Abriata, Luciano Andrés; Ottado, Jorgelina; Gottig, NataliaAdhesion to host tissue is one of the key steps of the bacterial pathogenic process. Xanthomonas citri ssp. citri possesses a non-fimbrial adhesin protein, XacFhaB, required for bacterial attachment, which we have previously demonstrated to be an important virulence factor for the development of citrus canker. XacFhaB is a 4753-residue-long protein with a predicted β-helical fold structure, involved in bacterial aggregation, biofilm formation and adhesion to the host. In this work, to further characterize this protein and considering its large size, XacFhaB was dissected into three regions based on bioinformatic and structural analyses for functional studies. First, the capacity of these protein regions to aggregate bacterial cells was analysed. Two of these regions were able to form bacterial aggregates, with the most amino-terminal region being dispensable for this activity. Moreover, XacFhaB shows features resembling pathogen-associated molecular patterns (PAMPs), which are recognized by plants. As PAMPs activate plant basal immune responses, the role of the three XacFhaB regions as elicitors of these responses was investigated. All adhesin regions were able to induce basal immune responses in host and non-host plants, with a stronger activation by the carboxyl-terminal region. Furthermore, pre-infiltration of citrus leaves with XacFhaB regions impaired X. citri ssp. citri growth, confirming the induction of defence responses and restraint of citrus canker. This work reveals that adhesins from plant pathogens trigger plant defence responses, opening up new pathways for the development of protective strategies for disease control.Ítem Acceso Abierto Langmuir-Blodgett monolayers holding a wound healing active compound and its effect in cell culture. A model for the study of surface mediated drug delivery systems(Elsevier, 2021-03-14) Fernández, Luciana Andrea; Reviglio, Ana Lucía; Heredia, Daniel Alejandro; Morales, Gustavo Marcelo; Santo, Marisa Rosana; Otero, Luis Alberto; Alustiza, Fabrisio Eduardo; Liaudat, Ana Cecilia; Bosch, Pablo; Larghi, Enrique Leandro; Bracca, Andrea Beatriz Juana; Kaufman, Teodoro SaúlLangmuir and Langmuir-Blodgett films holding a synthetic bioinspired wound healing active compound were used as drug-delivery platforms. Palmitic acid Langmuir monolayers were able to incorporate 2-methyltriclisine, a synthetic Triclisine derivative that showed wound healing activity. The layers proved to be stable and the nanocomposites were transferred to solid substrates. Normal human lung cells (Medical Research Council cell strain 5, MRC-5) were grown over the monomolecular Langmuir-Blodgett films that acted as a drug reservoir and delivery system. The proliferation and migration of the cells were clearly affected by the presence of 2 methyltriclisine in the amphiphilic layers. The methodology is proposed as a simple and reliable model for the study of the effects of bioactive compounds over cellular cultures.Ítem Acceso Abierto The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation(Springer Nature, 2014-04-18) Zimaro, Tamara; Sgro, Germán Gustavo; Garofalo, Cecilia Graciela; Ficarra, Florencia Andrea; Ottado, Jorgelina; Gottig, Natalia; Thomas, Ludivine; Marondedze, Claudius; Gehring, ChrisBackground: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. Results: The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. Conclusions: Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis.Ítem Acceso Abierto Synthesis of Novel Triazine-Based Chalcones and 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as Potential Leads in the Search of Anticancer, Antibacterial and Antifungal Agents(MDPI, 2024-03-23) Moreno, Leydi M.; Quiroga, Jairo; Abonia, Rodrigo; Crespo Ortíz, María del Pilar; Aranaga, Carlos Andrés; Martínez-Martínez, Luis; Sortino, Maximiliano Andrés; Barreto, Mauricio; Burbano, María E.; Insuasty, Braulio; https://orcid.org/0000-0002-9451-3063; https://orcid.org/0000-0003-3256-0961; https://orcid.org/0000-0003-2160-5167; https://orcid.org/0000-0003-1806-657X; https://orcid.org/0000-0002-6091-4045; https://orcid.org/0000-0002-4271-1690; https://orcid.org/0000-0001-5361-4901; https://orcid.org/0000-0001-8371-3310This study presents the synthesis of four series of novel hybrid chalcones (20,21)a–g and (23,24)a–g and six series of 1,3,5-triazine-based pyrimido[4,5-b][1,4]diazepines (28–33)a–g and the evaluation of their anticancer, antibacterial, antifungal, and cytotoxic properties. Chalcones 20b,d, 21a,b,d, 23a,d–g, 24a–g and the pyrimido[4,5-b][1,4]diazepines 29e,g, 30g, 31a,b,e–g, 33a,b,e–g exhibited outstanding anticancer activity against a panel of 60 cancer cell lines with GI50 values between 0.01 and 100 μM and LC50 values in the range of 4.09 μM to >100 μM, several of such derivatives showing higher activity than the standard drug 5-fluorouracil (5-FU). On the other hand, among the synthesized compounds, the best antibacterial properties against N. gonorrhoeae, S. aureus (ATCC 43300), and M. tuberculosis were exhibited by the pyrimido[4,5-b][1,4]diazepines (MICs: 0.25–62.5 µg/mL). The antifungal activity studies showed that triazinylamino-chalcone 29e and triazinyloxy-chalcone 31g were the most active compounds against T. rubrum and T. mentagrophytes and A. fumigatus, respectively (MICs = 62.5 μg/mL). Hemolytic activity studies and in silico toxicity analysis demonstrated that most of the compounds are safe.Ítem Acceso Abierto Endocannabinoid 2-arachidonoyl glycerol increases the transcription of daf-7 in ASI neurons(Caltech Library, 2018-10-19) Galles, Celina; Prez, Gastón M.; De Mendoza, DiegoThis article investigates the effect of the endocannabinoid 2-arachidonoyl glycerol (2-AG) on the expression of the daf-7 gene in ASI neurons of nematodes. It demonstrates that daf-7 expression levels increase notably when nematodes are exposed to 2-AG. This observation is supported by fluorescence microscopy images showing elevated GFP signal in ASI neurons treated with 2-AG compared to control groups. Statistical analysis confirms a significant difference in daf-7 expression between the 2-AG treatment and solvent control conditions. The study conducted three independent experiments, analyzing a total of 70 ASI neurons for each condition. Regarding the methods, worms were cultured on solid NGM plates supplemented with bacteria and specific supplements for each experimental condition. Daf-7 expression was monitored in synchronized L1 larvae fed with Escherichia coli supplemented with either 2-AG or carrier solvent. Fluorescence microscopy was utilized to visualize GFP intensity in ASI neurons, and image analysis software quantified the data. The study concludes a robust increase in daf-7 expression in response to 2-AG treatment.Ítem Acceso Abierto An introductory classroom exercise on protein molecular model visualization and detailed analysis of protein–ligand binding(Wiley, 2013-03-23) Poeylaut-Palena, Andrés A.; Laborde, María de los ÁngelesA learning module for molecular level analysis of protein structure and ligand/drug interaction through the visualization of X-ray diffraction is presented. Using DeepView as molecular model visualization software, students learn about the general concepts of protein structure. This Biochemistry classroom exercise is designed to be carried out by following the detailed instructions that make software handling straightforward. Students learn about protein structure and gain insight into the molecular level of the interaction of two active compounds with their receptor.Ítem Acceso Abierto Expression and purification of untagged GlnK proteins from actinobacteria(EXCLI, 2017-06-27) Gerhardt, Edileusa C. M.; Moure, Vivian R.; Souza, Andrey W.; Pedrosa, Fabio O.; Souza, Emanuel M.; Diacovich, Lautaro; Gramajo, Hugo Cesar; Huergo, Luciano F.The PII protein family constitutes one of the most conserved and well distributed family of signal transduction proteins in nature. These proteins play key roles in nitrogen and carbon metabolism. PII function has been well documented in Gram-negative bacteria. However, there are very few reports describing the in vitro properties and function of PII derived from Gram-positive bacteria. Here we present the heterologous expression and efficient purification protocols for untagged PII from three Actinobacteria of medical and biotechnological interest namely: Mycobacterium tuberculosis, Rhodococcus jostii and Streptomyces coelicolor. Circular dichroism and gel filtration analysis supported that the purified proteins are correctly folded. The purification protocol described here will facilitate biochemical studies and help to uncover the biochemical functions of PII proteins in Actinobacteria.Ítem Acceso Abierto First total synthesis of chromanone A, preparation of related compounds and evaluation of their antifungal activity against Candida albicans, a biofilm forming agent(Royal Society of Chemistry, 2021-06-01) Cortés, Iván; Cordisco, Estefanía; Kaufman, Teodoro Saúl ; Sortino, Maximiliano Andrés; Svetaz, Laura Andrea; Bracca, Andrea Beatriz Juana; https://orcid.org/0000-0002-8069-2793; https://orcid.org/0000-0002-6574-1702; https://orcid.org/0000-0003-3173-2178; https://orcid.org/0000-0002-4271-1690; https://orcid.org/0000-0002-1023-1641; https://orcid.org/0000-0003-2647-475XA straightforward and convenient approach for the first total syntheses of chromanone A and a related 7-OMe substituted natural product is reported. These unique C-3 substituted 2-hydroxymethyl chromones were recently isolated as fungal metabolites. Chromanone A was synthesized in 25.3% overall yield from the readily available pyrocatechol, whereas the second natural product was prepared in 39.7% global yield. A small library of chromones, including both natural products and some of their synthetic heterocyclic precursors, was evaluated against Candida albicans ATCC 10231, a biofilm forming agent. It was found that 8-methoxy-3-methyl-4-oxo-4H-chromene-2-carbaldehyde, a partially oxidized form of chromanone A, exhibited a minimum inhibitory concentration of 7.8 μg mL−1 and significantly inhibited the yeast's virulence factors, including the adherence to buccal epithelial cells and the secretion of phospholipases, as well as the formation of germ tubes and the generation of the hyphal pseudomycelium. In addition, despite the heterocycle exhibiting non-significant inhibition of the formation of the Candida biofilm, it completely inhibited the growth of C. albicans in preformed biofilms at 62.5 μg mL−1.