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Ítem Acceso Abierto Neuroprotective effect of NSCs-derived extracellular vesicles in Parkinson's disease models(Springer Nature, 2025-02-19) Díaz Reyes, Mercyleidi; Gatti, Sabrina; Delgado Ocaña, Susana; Ortega, Hugo H.; Banchio, Claudia; Dr. Wolozin, Benjamin: provide the WT/A53T α-syn DNA constructParkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by both motor and non-motor symptoms, caused by the degeneration and loss of dopaminergic neurons in the substantia nigra. Current therapies are limited to symptom management, unable to prevent neuronal loss or halt the progression of the disease. A significant limitation to more effective treatments is the difficulty of crossing the blood-brain barrier (BBB). Extracellular vesicles (EVs) communication plays a crucial role in several physiological processes within the nervous system. Notably, EVs have the unique ability to cross the BBB, making them a highly promising vehicle for delivering therapeutic agents directly to the brain. Given the rising prevalence of PD, the need for therapies that prevent neuronal death and promote cell survival is urgent. This study explores the potential of neural stem cell-derived extracellular vesicles (NSC-EVs) using two in vitro models of PD. Our findings demonstrate that NSC-EVs significantly enhance the survival of dopaminergic neurons by reducing apoptosis and showing strong neuroprotective effects. Notably, the natural extracellular vesicles used in this study are enriched with Catalase, a potent scavenger protein with antioxidant properties. This natural enrichment further strengthens their neuroprotective capacity, enabling them to mitigate oxidative stress and protect vulnerable neurons. The use of such naturally enriched extracellular vesicles represents a promising approach for developing innovative therapies to effectively combat Parkinson’s disease.Ítem Acceso Abierto Expression of the gene encoding secretor type galactoside 2 α fucosyltransferase (FUT2) and ABH antigens in patients with oral lesions(Sociedad Española de Medicina Oral, 2012) Campi, Carlos; Escovich, Livia; Moreno, Alejandra Soledad; Racca, Liliana; Cotorruelo, Carlos; Biondi, Claudia SilviaObjective: The aim of this work was to evaluate the expression of FUT2 gene in saliva and histo ABH antigens of patients with oral lesions. Study Design: In total 178 subjects were examined, half of whom suffered from oral pre-cancerous and cancerous lesions, while the other half were the healthy control group We analyzed the FUT 2 polymorphism by ASO-PCR (allele specific oligonucleotid – polymerase chain reaction) with specific primers for G428 allele and the wild type allele of FUT2 gene. To reveal A, B and H antigens in tissue sections of the patients (n= 89) we used a modified specific red cell adherence technique. Results: We found a high intensity of oral disease in the non-secretor group (OR = 2.43). A total of 58% of the patients with oral pre-cancerous and cancerous lesions was non secretors (se_/_), in contrast with the healthy population (21.5%). A strongly positive reaction was defined as a sheet of indicator erythrocytes adhered to the epithelial cells. In 31 of the 54 samples analyzed the test showed slightly positive results on atypical areas, and there was a complete antigen deletion in areas affected by neoplasia. Nineteen samples showed a total absence of ABH antigens in both histologically normal and pathological areas. Blood group antigens were expressed at a high level in benign and highly differentiated malignant tumors. In poorly differentiated malignant tumors, they were mostly absent. Conclusion: Considering these results we suggest the use of this method to monitor probable preneoplastic lesions in risk population, especially in those with no secretor status (absence of FUT2 gene).Ítem Acceso Abierto Efecto de la respuesta inmune celular y humoral en parejas infértiles y su relación con infecciones genitales(INIESTARES S. A., 2017-09-28) Brufman, Adriana Silvia; Colombo, Laura G.R.; Streiger, Esteban; Pusillico, RocíoOBJETIVO: El propósito de este estudio fue determinar la prevalencia de los diferentes microorganismos aislados del tracto urogenital de hombres infértiles y evaluar si existen diferencias en los parámetros seminales según la presencia o ausencia de infecciones genitales. MÉTODOS: Se analizaron en forma retrospectiva los parámetros del semen en 280 muestras de hombres infértiles de acuerdo a los criterios de la Organización Mundial de la Salud (OMS 2010). El análisis microbiológico se realizó según la modificación del método de Stamey-Meares propuesta por Santoianni et al. RESULTADOS: Los estudios microbiológicos mostraron ausencia de microorganismos o presencia de colonizantes habituales no jerarquizables en el 67,86% (GRUPO 1) de las muestras, y presencia de al menos un patógeno o patógeno potencial en 32,14% del total (GRUPO 2). No se observaron diferencias significativas en volumen de eyaculado (p=0,353), valor de pH (p=0,801), movilidad (p>0,30), concentración de ácido cítrico (p=0,383) y viscosidad (p=0,948) entre los dos grupos. El recuento relativo de espermatozoides en los pacientes infectados fue significativamente menor que en aquellos que no presentaban patógenos (p=0,05). Se evaluó además el índice de teratozoospermia (IT). Las muestras de pacientes con infección presentaron valores de IT mayores (p<0,0001). CONCLUSIONES: Un tercio de la población estudiada presentó infecciones genitales. En base a nuestros resultados, consideramos fundamental la realización de un espermocultivo en las primeras etapas del estudio del paciente infértil para contribuir a un adecuado tratamiento de la pareja con fallas reproductivas.Ítem Acceso Abierto Carbapenem‑resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU)(Nature Research, 2024-08-19) Escalante, Jenny; Hamza, Mase; Nishimura, Brent; Melecio, Meghan; Davies Sala, Carol; Tuttobene, Marisel Romina; Subils, Tomás; Traglia, German M.; Pham, Chloe; Sieira, Rodrigo; Actis, Luis A.; Bonomo, Robert A.; Tolmasky, Marcelo E.; Ramírez, María SoledadCarbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in diferent body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host’s urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the diferential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and bioflm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These changes presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB’s metabolic adaptations to human fuids, as well as expand knowledge on bacterial responses to distinct human fuids containing diferent concentrations of human serum albumin (HSA).Ítem Acceso Abierto Genetic and phenotypic changes related to the development of mec-independent oxacillin non-susceptibility in ST8 Staphylococcus aureus recovered after antibiotic therapy in a patient with bacteremia(MDPI, 2024-06-13) Di Gregorio, Sabrina; Weltman, Gabriela; Fabbri, Carolina; Fernández, Silvina; Zárate, Soledad; Smayevsky, Jorgelina; Power, Pablo; Campos, Josefina; Llarrull, Leticia Irene; Mollerach, Marta; https://orcid.org/0000-0003-3787-3318; https://orcid.org/0000-0002-7051-9954; https://orcid.org/0000-0002-5679-4343; https://orcid.org/0000-0002-5735-8471The mec-independent oxacillin non-susceptible S. aureus (MIONSA) strains represent a great clinical challenge, as they are not easily detected and can lead to treatment failure. However, the responsible molecular mechanisms are still very little understood. Here, we studied four clinical ST8-MSSA-t024 isolates recovered during the course of antibiotic treatment from a patient suffering successive episodes of bacteremia. The first isolates (SAMS1, SAMS2, and SAMS3) were susceptible to cefoxitin and oxacillin. The last one (SA2) was susceptible to cefoxitin, resistant to oxacillin, lacked mec genes, and had reduced susceptibility to teicoplanin. SA2 showed higher β-lactamase activity than SAMS1. However, β-lactamase hyperproduction could not be linked to oxacillin resistance as it was not inhibited by clavulanic acid, and no genetic changes that could account for its hyperproduction were found. Importantly, we hereby report the in vivo acquisition and coexistence of different adaptive mutations in genes associated with peptidoglycan synthesis (pbp2, rodA, stp1, yjbH, and yvqF/vraT), which is possibly related with the development of oxacillin resistance and reduced susceptibility to teicoplanin in SA2. Using three-dimensional models and PBP binding assays, we demonstrated the high contribution of the SA2 PBP2 Ala450Asp mutation to the observed oxacillin resistance phenotype. Our results should be considered as a warning for physicians and microbiologists in the region, as MIONSA detection and treatment represent an important clinical challenge.Ítem Acceso Abierto Hetero-antagonism of avibactam and sulbactam with cefiderocol in carbapenem-resistant Acinetobacter spp(American Society for Microbiology, 2024-08-20) Wong, Olivia; Mezcord, Vyanka; Lopez, Christina; Traglia, German M.; Pasteran, Fernando; Tuttobene, Marisel Romina; Corso, Alejandra; Tolmasky, Marcelo E.; Bonomo, Robert A.; Ramírez, María Soledad; https://orcid.org/0000-0002-4780-5311; https://orcid.org/0000-0001-5840-5869; https://orcid.org/0000-0003-0234-4643; https://orcid.org/0000-0002-6298-7811; https://orcid.org/0000-0002-3299-894X; https://orcid.org/0000-0002-9904-7890Cefiderocol, a siderophore-cephalosporine conjugate antibiotic, shows promise as a therapeutic option for carbapenem-resistant (CR) Acinetobacter infections. While resistance has already been reported in A. baumannii, combination therapies with avibactam or sulbactam reduce MICs of cefiderocol, extending its efficacy. However, careful consideration is necessary when using these combinations. In our experiments, exposure of A. baumannii and A. lwoffii to cefiderocol and sulbactam or avibactam led to the selection of cefiderocol-resistant strains. Three of those were subjected to whole genome sequencing and transcriptomic analysis. The strains all possessed synonymous and non-synonymous substitutions and short deletions. The most significant mutations affected efflux pumps, transcriptional regulators, and iron homeostasis genes. Transcriptomics showed significant alterations in expression levels of outer membrane proteins, iron homeostasis, and β-lactamases, suggesting adaptive responses to selective pressure. This study underscores the importance of carefully assessing drug synergies, as they may inadvertently foster the selection of resistant variants and complicate the management of CR Acinetobacter infections.Ítem Acceso Abierto Cerebrospinal fuid (CSF) augments metabolism and virulence expression factors in Acinetobacter baumannii(Nature Research, 2021-02-26) Martínez, Jasmine; Razo Gutiérrez, Chelsea; Razo Gutiérrez, Chelsea; Courville, Robert; Pimentel, Camila; Liu, Christine; Fung, Sammie E.; Tuttobene, Marisel Romina; Phan, Kimberly; Vila, Alejandro J.; Shahrestani, Parvin; Jimenez, Veronica; Tolmasky, Marcelo E.; Becka, Scott A.; Papp Wallace, Krisztina M.; Bonomo, Robert A.; Soler Bistue, Alfonso; Sieira, Rodrigo; Ramírez, María SoledadIn a recent report by the Centers for Disease Control and Prevention (CDC), multidrug resistant (MDR) Acinetobacter baumannii is a pathogen described as an “urgent threat.” Infection with this bacterium manifests as diferent diseases such as community and nosocomial pneumonia, bloodstream infections, endocarditis, infections of the urinary tract, wound infections, burn infections, skin and soft tissue infections, and meningitis. In particular, nosocomial meningitis, an unwelcome complication of neurosurgery caused by extensivelydrug resistant (XDR) A. baumannii, is extremely challenging to manage. Therefore, understanding how A. baumannii adapts to diferent host environments, such as cerebrospinal fuid (CSF) that may trigger changes in expression of virulence factors that are associated with the successful establishment and progress of this infection is necessary. The present invitro work describes, the genetic changes that occur during A. baumannii infltration into CSF and displays A. baumannii’s expansive versatility to persist in a nutrient limited environment while enhancing several virulence factors to survive and persist. While a hypervirulent A. baumannii strain did not show changes in its transcriptome when incubated in the presence of CSF, a lowvirulence isolate showed signifcant diferences in gene expression and phenotypic traits. Exposure to 4% CSF caused increased expression of virulence factors such as fmbriae, pilins, and iron chelators, and other virulence determinants that was confrmed in various model systems. Furthermore, although CSF’s presence did not enhance bacterial growth, an increase of expression of genes encoding transcription, translation, and the ATP synthesis machinery was observed. This work also explores A. baumannii’s response to an essential component, human serum albumin (HSA), within CSF to trigger the diferential expression of genes associated with its pathoadaptibility in this environment.Ítem Acceso Abierto Effects of lactoferrin, a protein present in the female reproductive tract, on parameters of human sperm capacitation and gamete interaction(Wiley, 2015-10-07) Zumoffen, Carlos María; Massa, Estefanía; Caille, Adriana María; Munuce, María José; Ghersevich, Sergio AlbinoIn a recent study, lactoferrin (LF) was detected in human oviductal secretion. The protein was able to bind to oocytes and sperm, and modulated gamete interaction. The aim of the present study was to investigate the effect of LF on parameters related to human sperm capacitation and sperm–zona pellucida interaction. Semen samples were obtained from healthy normozoospermic donors (n = 7). Human follicular fluids and oocytes were collected from patients undergoing in vitro fertilization. Motile sperm obtained by swim-up were incubated for 6 or 22 h under capacitating conditions with LF (0–100 μg/mL). After incubations, viability, motility, presence of α-d-mannose receptors (using a fluorescent probe on mannose coupled to bovine serum albumin), spontaneous and induced acrosome reaction (assessed with Pisum sativum agglutinin conjugated to fluorescein isothiocyanate), and tyrosine phosphorylation of sperm proteins were evaluated. Sperm–zona pellucida interaction in the presence of LF was investigated using the hemizone assay. The presence of LF did not affect sperm viability or motility, but caused a dose-dependent significant decrease in sperm α-d-mannose-binding sites, and the effect was already significant with the lowest concentration of the protein used after 22 h incubation. Dose-dependent significant increases in both induced acrosome reaction and tyrosine phosphorylation of sperm proteins were observed in the presence of LF. The present data indicate that LF modulates parameters of sperm function. The inhibition of gamete interaction by LF could be partially explained by the decrease in sperm d-mannose-binding sites. The presence of the LF promoted sperm capacitation in vitro.Ítem Acceso Abierto On the offensive: the role of outer membrane vesicles in the successful dissemination of New Delhi Metallo-β-lactamase (NDM-1)(American Society for Microbiology, 2021-09-28) Martínez, Melina María Belén; Bonomo, Robert A.; Vila, Alejandro J.; Maffía, Paulo César; González, Lisandro Javier; https://orcid.org/0000-0002-4007-8721; https://orcid.org/0000-0002-3299-894X; https://orcid.org/0000-0002-7978-3233; https://orcid.org/0000-0001-7423-2646; https://orcid.org/0000-0002-0575-1810The emergence and worldwide dissemination of carbapenemase-producing Gram-negative bacteria are a major public health threat. Metallo-β-lactamases (MBLs) represent the largest family of carbapenemases. Regrettably, these resistance determinants are spreading worldwide. Among them, the New Delhi metallo-β-lactamase (NDM-1) is experiencing the fastest and largest geographical spread. NDM-1 β-lactamase is anchored to the bacterial outer membrane, while most MBLs are soluble, periplasmic enzymes. This unique cellular localization favors the selective secretion of active NDM-1 into outer membrane vesicles (OMVs). Here, we advance the idea that NDM-containing vesicles serve as vehicles for the local dissemination of NDM-1. We show that OMVs with NDM-1 can protect a carbapenem-susceptible strain of Escherichia coli upon treatment with meropenem in a Galleria mellonella infection model. Survival curves of G. mellonella revealed that vesicle encapsulation enhances the action of NDM-1, prolonging and favoring bacterial protection against meropenem inside the larva hemolymph. We also demonstrate that E. coli cells expressing NDM-1 protect a susceptible Pseudomonas aeruginosa strain within the larvae in the presence of meropenem. By using E. coli variants engineered to secrete variable amounts of NDM-1, we demonstrate that the protective effect correlates with the amount of NDM-1 secreted into vesicles. We conclude that secretion of NDM-1 into OMVs contributes to the survival of otherwise susceptible nearby bacteria at infection sites. These results disclose that OMVs play a role in the establishment of bacterial communities, in addition to traditional horizontal gene transfer mechanisms. IMPORTANCE: Resistance to carbapenems, last-resort antibiotics, is spreading worldwide, raising great concern. NDM-1 is one of the most potent and widely disseminated carbapenem-hydrolyzing enzymes spread among many bacteria and is secreted to the extracellular medium within outer membrane vesicles. We show that vesicles carrying NDM-1 can protect carbapenem-susceptible strains of E. coli and P. aeruginosa upon treatment with meropenem in a live infection model. These vesicles act as nanoparticles that encapsulate and transport NDM-1, prolonging and favoring its action against meropenem inside a living organism. Secretion of NDM-1 into vesicles contributes to the survival of otherwise susceptible nearby bacteria at infection sites. We propose that vesicles play a role in the establishment of bacterial communities and the dissemination of antibiotic resistance, in addition to traditional horizontal gene transfer mechanisms.Ítem Acceso Abierto Slow protein dynamics elicits new enzymatic functions by means of epistatic interactions(Oxford University Press, 2022-09-22) Rossi, María Agustina; Palzkill, Timothy; Almeida, Fabio C. L.; Vila, Alejandro J.; https://orcid.org/0000-0002-7978-3233Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M β-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried β-sheet. The two mutations located in this β-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M β-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.Ítem Acceso Abierto Fibroblast growth factor receptors (FGFRs) in human sperm: expression, functionality and involvement in motility regulation(Public Library of Science (PLOS), 2015-05-13) Saucedo, Lucía; Buffa, Gabriela Natalia; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrián; Munuce, María José; Vázquez Levin, Mónica Hebe; Marín Briggiler, Clara; Dr. Baldi, Alberto provide the FGF2Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.Ítem Acceso Abierto Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase(eLife Sciences Publications, 2023-02-24) Zhao, Zhuoran; Shen, Xiayu; Chen, Shuang; Gu, Jing; Wang, Haun; Mojica, María F.; Samanta, Moumita; Bhowmik, Debsindhu; Vila, Alejandro J.; Bonomo, Robert A.; Haider, Shozeb; http://orcid.org/0000-0002-1380-9824; http://orcid.org/0000-0001-7770-9091; http://orcid.org/0000-0002-7978-3233; http://orcid.org/0000-0003-2650-2925β-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of β-lactamases among pathogenic bacteria limits the efficacy of β-lactam antibiotics. This has created a major public health crisis. The use of β-lactamase inhibitors has proven useful in restoring the activity of β-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-β-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the β-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-β7 and β12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cellbased experiments. The results demonstrate that there are extremely significant gating interactions between α3-β7 and β12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.Ítem Acceso Abierto Arabidopsis thaliana Hcc1 is a Sco-like metallochaperonefor Cu A assembly in Cytochrome c Oxidase(Wiley, 2020-12) Llases, María Eugenia; Lisa, María Natalia; Morgada, Marcos Nicolás; Giannini, Estefanía; Alzari, Pedro M.; Vila, Alejandro J.The assembly of the CuA site in Cytochrome c Oxidase (COX) is a criticalstep for aerobic respiration in COX-dependent organisms. Several geneproducts have been associated with the assembly of this copper site, themost conserved of them belonging to the Sco family of proteins, whichhave been shown to perform different roles in different organisms. Plantsexpress two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known tobe essential for plant development and for COX maturation, but its precisefunction has not been addressed until now. Here, we report the biochemi-cal, structural and functional characterization of Arabidopsis thaliana Hcc1protein (here renamed Sco1). We solved the crystal structure of the Cu+1-bound soluble domain of this protein, revealing a tri coordinated environ-ment involving a CxxxCxnH motif. We show that AtSco1 is able to workas a copper metallochaperone, inserting two Cu+1 ions into the CuA site ina model of CoxII. We also show that AtSco1 does not act as a thiol-disul-fide oxido-reductase. Overall, this information sheds new light on the bio-chemistry of Sco proteins, highlighting the diversity of functions amongthem despite their high structural similarities.Ítem Acceso Abierto Assessing endocrine and immune parameters in human immunodeficiency virus-infected patients before and after the immune reconstitution inflammatory syndrome(Brazilian Society of Endocrinology and Metabolism, 2018-02) Rateni, Liliana Beatriz; Lupo, Sergio; Racca, Liliana; Palazzi, Jorge; Ghersevich, Sergio AlbinoObjective: The present study compares immune and endocrine parameters between HIV-infected patients who underwent the Immune Reconstitution Inflammatory Syndrome (IRIS-P) during antiretroviral therapy (ART) and HIV-patients who did not undergo the syndrome (non-IRIS-P). Materials and methods: Blood samples were obtained from 31 HIV-infected patients (15 IRIS-P and 16 non-IRIS-P) before ART (BT) and 48 ± 2 weeks after treatment initiation (AT). Plasma Interleukin-6 (IL-6) and Interleukin-18 (IL-18) were determined by ELISA. Cortisol, dehydroepiandrosterone sulfate (DHEA-S) and thyroxin concentrations were measured using chemiluminescence immune methods. Results: Concentrations of IL-6 (7.9 ± 1.9 pg/mL) and IL-18 (951.5 ± 233.0 pg/mL) were significantly higher (p < 0.05) in IRIS-P than in non-IRIS-P (3.9 ± 1.0 pg/mL and 461.0 ± 84.4 pg/mL, respectively) BT. Mean T4 plasma level significantly decreased in both groups of patients after treatment (p < 0.05). In both groups cortisol levels were similar before and after ART (p > 0.05). Levels of DHEA-S in IRIS-P decreased AT (1080.5 ± 124.2 vs. 782.5 ± 123.8 ng/mL, p < 0.05) and they were significantly lower than in non-IRIS-P (782.5 ± 123.8 vs. 1203.7 ± 144.0 ng/mL, p < 0.05). IRIS-P showed higher values of IL-6 and IL-18 BT and lower levels of DHEA-S AT than in non-IRIS-P. Conclusion: These parameters could contribute to differentiate IRIS-P from non-IRIS-P. The significant decrease in DHEA-S levels in IRIS-P after ART might suggest a different adrenal responseÍtem Acceso Abierto Identification of key sequence features required for microRNA biogenesis in plants(Nature Research, 2020-10-21) Rojas, Arantxa María Larisa; Drusin, Salvador Iván; Chorostecki, Uciel Pablo; Mateos, Julieta L.; Moro, Belén; Bologna, Nicolás G.; Bresso, Edgardo Gabriel; Schapire, Arnaldo L.; Rasia, Rodolfo M.; Moreno, Diego M.; Palatnik, Javier F.; http://orcid.org/0000-0003-4316-4518; http://orcid.org/0000-0001-5350-514X; http://orcid.org/0000-0003-2229-6853; http://orcid.org/0000-0002-1156-6662; http://orcid.org/0000-0002-2810-3533; http://orcid.org/0000-0002-2161-7910; http://orcid.org/0000-0002-9798-459X; http://orcid.org/0000-0003-3940-067X; http://orcid.org/0000-0001-5493-8537; http://orcid.org/0000-0001-7996-5224MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.Ítem Acceso Abierto Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: crystallography supports stereoselective binding of cephem/carbapenem products(Elsevier, 2023-03-15) Hinchliffe, Philip; Calvopiña, Karina; Rabe, Patrick; Mojica, María F.; Schofield, Christopher J.; Dmitrienko, Gary I.; Bonomo, Robert A.; Vila, Alejandro J.; Spencer, James; https://orcid.org/0000-0002-1380-9824; https://orcid.org/0000-0002-7978-3233L1 is a dizinc subclass B3 metallo-β-lactamase (MBL) that hydrolyzes most β-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed β-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the β-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1β-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1β-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1β-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3′ leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (β) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and β-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.Ítem Acceso Abierto The H-NS regulator plays a role in the stress induced by carbapenemase expression in acinetobacter baumannii(American Society for Microbiology, 2020-08-26) Huang, Fanny; Fitchett, Noelle; Razo Gutiérrez, Chelsea; Le, Casin; Martínez, Jasmine; Ra, Grace; López, Carolina; González, Lisandro Javier; Sieira, Rodrigo; Vila, Alejandro J.; Bonomo, Robert A.; Ramírez, María Soledad; https://orcid.org/0000-0002-9904-7890Disruption of the histone-like nucleoid structuring protein (H-NS) was shown to affect the ability of Gram-negative bacteria to regulate genes associated with virulence, persistence, stress response, quorum sensing, biosynthesis pathways, and cell adhesion. Here, we used the expression of metallo--lactamases (MBLs), known to elicit envelope stress by the accumulation of toxic precursors in the periplasm, to interrogate the role of H-NS in Acinetobacter baumannii, together with other stressors. Using a multidrug-resistant A. baumannii strain, we observed that H-NS plays a role in alleviating the stress triggered by MBL toxic precursors and counteracts the effect of DNA-damaging agents, supporting its role in stress response.Ítem Acceso Abierto Structural determinants of Arabidopsis thaliana Hyponastic Leaves 1 function in vivo(Public Library of Science, 2014-11-19) Burdisso, Paula; Milia, Fernando; Schapire, Arnaldo L.; Bologna, Nicolás G.; Palatnik, Javier F.; Rasia, Rodolfo M.MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity.Ítem Acceso Abierto Acute diarrhoea in children: determination of duration using a combined bismuth hydroxide gel and oral rehydration solution therapy vs. oral rehydration solution(MDPI, 2016-12-21) Oviedo, Adriana; Díaz, Mirna; Valenzuela, María Laura; Vidal, Victoria; Racca, Liliana; Bottai, Hebe; Priore, Graciela; Peluffo, Graciela; Di Bartolomeo, Susana; Cabral, Graciela; Toca, María del CarmenOral rehydration salt (ORS) treatment in young children with acute diarrhoea (AD) has contributed to decrease mortality associated with dehydration although effective strategies to reduce morbidity associated with this disease are required. The aim of this study was to evaluate the diarrhoea duration when using combined colloidal bismuth hydroxide gel (CBHG) and oral rehydration salt treatment compared with ORS therapy in children with AD. We designed a double-blind, randomised prospective study with treatment and control groups. Patients aged one to 12 years, with no prior pathology and with AD of less than 48 h were included. The Chi-squared and Mann-Whitney tests were used, as well as the Cox proportional hazards model and the Kaplan-Meier estimator. Patients were randomised into an ORS and CBHG treatment group and a control group for ORS plus placebo. (Average age: 3.2 years). The result of the post-treatment evaluation with respect to the average duration of AD was 25.5 h for the treated group vs. 41.5 h for the control group (p = 0.015). The average number of stools was 4.8 in the treated group and 8.2 in the control group (p = 0.032). We conclude that the use of CBHG plus ORS significantly reduced the duration of AD, the number of stools and the percentage of children with persistent AD after 24 h of treatment compared to the control group. AD remitted almost twice as fast in patients treated with CBHG and ORS compared to those who received ORS plus placebo.Ítem Acceso Abierto An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance(Springer Nature, 2019-12-20) Belluzo, Bruno Salvador; Abriata, Luciano Andrés; Giannini, Estefanía; Mihovilcevic, Damila; Dal Peraro, Matteo; Llarrull, Leticia IreneThe treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to β-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to β-lactam antibiotics are activated only when the bacterium encounters a β-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the β-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how β-lactam binding culminates in the proteolytic degradation of MecI.