Riboflavin kinase and pyridoxine 5'-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery

Enzymes catalysing sequential reactions have developed different mechanisms to control the transport and flux of reactants and intermediates along metabolic pathways, which usually involve direct transfer of metabolites from an enzyme to the next one in a cascade reaction. Despite the fact that metabolite or substrate channelling has been widely studied for reactant molecules, such information is seldom available for cofactors in general, and for flavins in particular. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) act as cofactors in flavoproteins and flavoenzymes involved in a wide range of physiologically relevant processes in all type of organisms. Homo sapiens riboflavin kinase (RFK) catalyses the biosynthesis of the flavin mononucleotide cofactor, and might directly interplay with its flavin client apo-proteins prior to the cofactor transfer. Non-etheless, none of such complexes has been characterized at molecular or atomic level so far. Here, we particularly evaluate the interaction of riboflavin kinase with one of its potential FMN clients, pyridoxine-5′-phosphate oxidase (PNPOx). The interaction capacity of both proteins is assessed by using isothermal titration calorimetry, a methodology that allows to determine dissociation constants for interaction in the micromolar range (in agreement with the expected transient nature of the interaction). Moreover, we show that; i) both proteins become thermally stabilized upon mutual interaction, ii) the tightly bound FMN product can be transferred from RFK to the apo-form of PNPOx producing an efficient enzyme, and iii) the presence of the apo-form of PNPOx slightly enhances RFK catalytic efficiency. Finally, we also show a computational study to predict likely RFK-PNPOx binding modes that can envisage coupling between the FMN binding cavities of both proteins for the potential transfer of FMN

Aberrant DNA methylation in breast cancer cells

The epigenome is regulated by a large number of macromolecular machines that are dynamically involved in various processes, including DNA methylation, histone modification and non-coding RNA signals, all of them working together to regulate the proper expression of the genome. Thus, in contrast with the genome, whose sequence is carefully conserved during cell life, the epigenome is highly dynamic. The epigenomic modifications are acquired during normal cell differentiation, replicated during mitosis and passed to daughter cells. A fundamental epigenetic attribute is that this plasticity occurs in response to environmental signals. It is therefore now accepted that the environment influences modifications in the cellular transcriptome through the epigenome. In developmental and evolutionary terms, the regulation of gene expression through epigenomic modifications is an advantageous shortcut and a highly conserved mechanism. However, it implies an increased risk for misregulation, as, for example, aberrant epigenomic modifications associate with the development of different human diseases, i.e. lupus, asthma, neurological diseases and cancer. Although epigenetic alterations in breast cancer have been deeply studied and discussed in the last decades, apparently contradictory results are yet often observed. Consequently, in this review, we will briefly discuss the latest findings of aberrant DNA methylation in breast tumorigenesis. Emphasis will be given to the discussion of the idea that different environments could explain paradoxical biological and pathobiological behaviors in individual patients and thus should be taken into consideration for the design and implementation of diagnosis, prognosis and predictive biomarkers.Fil: Campoy, Emanuel Martin. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; ArgentinaFil: Laurito, Sergio Roberto. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; ArgentinaFil: Urrutia, Guillermo. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; ArgentinaFil: Branham, Maria Teresita. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; ArgentinaFil: Roque Moreno, Maria. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; Argentin

Oral lacosamide for the treatment of refractory trigeminal neuralgia: A retrospective analysis of 86 cases

Background and Objectives First-line treatment for trigeminal neuralgia (TN) is limited to carbamazepine and oxcarbazepine, and in refractory cases, alternatives are scarce. Lacosamide has been suggested as a valid option. In this study, we describe a series of patients who received oral lacosamide as treatment for TN after first-line drug failure.Methods In this retrospective descriptive cohort study, we included patients with refractory TN who attended a tertiary center between 2015 and 2021 and were prescribed oral lacosamide after first-line treatment failure. The primary endpoints were pain relief and adverse effects. We secondarily analyzed clinical outcomes and compared responders versus nonresponders in the search for potential predictors of response.Results Eighty-six patients were included (mean age: 62 [SD 15.6] years; 54/86 [63%] female). The TN etiology was secondary in 16/86 (19%) patients. Concomitant continuous pain was present in 29/86 (34%) patients. The mean number of previous treatments was 2.7 [SD 1.5]. Pain relief was achieved in 57/86 (66%) cases, with 28/86 (33%) patients presenting adverse effects, all of which were mild. No statistically significant differences were observed between responders and nonresponders, but subtle clinical differences suggested potential predictors of response.Conclusion Lacosamide may be an effective and relatively safe treatment for refractory pain in TN patients after first-line treatment failure

PAX6 promoter methylation correlates with MDA-MB-231 cell migration, and expression of MMP2 and MMP9

Objective: Breast cancer is a heterogeneous disease characterized by an accumulation of genetic and epigenetic alterations that lead tumor cells to acquire characteristics like the capacity for invasion and metastasis. Metastasis remains a major challenge in cancer management and understanding of its molecular basis should result in improved prevention, diagnosis, and treatment of breast cancer patients. The aim of this study was to investigate how promoter DNA methylation regulates PAX6 gene expression and influences breast carcinoma cell migration. Methods: PAX6 promoter methylation was detected by Methyl Specific-Multiplex Ligation Probe Amplification (MS-MLPA). Gene expression was evaluated using qRT-PCR, while the effect of PAX6 on migration was ssessed by wound healing assay. In addition, MMP2 and MMP9 genes were studied using different bioinformatic tools. Results: The PAX6 promoter is methylated in breast cancer cell lines and methylation in this region impacts on its expression. Migration assays revealed that PAX6 overexpression promotes cell migration, while PAX6 inhibition decreases it. More importantly, we found that migration is affected by PAX6 methylation status. Employing bioinformatic analysis, binding sites for PAX6 on the regulatory regions of the MMP2 and MMP9 genes were established, PAX6 overexpression increasing MMP2 and MMP9 expression at the mRNA level. Conclusion: Our study provides novel insights into epigenetic events that regulate PAX6 expression and molecular mechanisms by which PAX6 modifies the migration capacity of breast cancer cells.Fil: Urrutia, Guillermo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Laurito, Sergio Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Campoy, Emanuel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Nasif, Daniela Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Branham, Maria Teresita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Roque Moreno, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Timing verification of automotive communication architectures using quantile estimation

Early stage timing verification on CAN traditionally relies on simulation and schedulability analysis, also known as worst-case response time (WCRT) analysis. Despite recent progresses, the latter technique remains pessimistic espe cially in complex networking architectures with gateways and heterogeneous communication stacks. Indeed, there are practical cases where no exact WCRT analysis is available, and merely upper bounds on the response times can be derived, on the basis of which unnecessary conservative design choices may be made. Simulation, on the other hand, does not provide any guarantees per se and, in the context of critical networks, should only be used along with an adequate methodology. In this paper, we argue for the use of quantiles of the response time distribution as performance metrics providing an adjustable trade-off between safety and resource usage optimization. We discuss how the exact value of the quantile to consider should be chosen with regard to the criticality of the frames, and illustrate the approach on two typical automotive use -cases

Epigenetic regulation of ID4 in breast cancer: Tumor suppressor or oncogene?

Background: Inhibitor of differentiation protein 4 (ID4) is a dominant negative regulator of the basic helix-loop-helix (bHLH) family of transcription factors. During tumorigenesis, ID4 may act as a tumor suppressor or as an oncogene in different tumor types. However, the role of ID4 in breast cancer is not clear where both an oncogenic and a tumor suppressor function have been attributed. Here, we hypothesize that ID4 behaves as both, but its role in breast differs according to the estrogen receptor (ER) status of the tumor. Methods: ID4 expression was retrieved from TCGA database using UCSC Xena. Association between overall survival (OS) and ID4 was assessed using Kaplan-Meier plotter. Correlation between methylation and expression was analyzed using the MEXPRESS tool. In vitro experiments involved ectopic expression of ID4 in MCF-7, T47D, and MDA-MB231 breast cancer cell lines. Migration and colony formation capacity were assessed after transfection treatments. Gene expression was analyzed by ddPCR and methylation by MSP, MS-MLPA, or ddMSP. Results: Data mining analysis revealed that ID4 expression is significantly lower in ER+ tumors with respect to ER- tumors or normal tissue. We also demonstrate that ID4 is significantly methylated in ER+ tumors. Kaplan-Meier analysis indicated that low ID4 expression levels were associated with poor overall survival in patients with ER+ tumors. In silico expression analysis indicated that ID4 was associated with the expression of key genes of the ER pathway only in ER+ tumors. In vitro experiments revealed that ID4 overexpression in ER+ cell lines resulted in decreased migration capacity and reduced number of colonies. ID4 overexpression induced a reduction in ER levels in ER+ cell lines, while estrogen deprivation with fulvestrant did not induce changes neither in ID4 methylation nor in ID4 expression. Conclusions: We propose that ID4 is frequently silenced by promoter methylation in ER+ breast cancers and functions as a tumor suppressor gene in these tumors, probably due to its interaction with key genes of the ER pathway. Our present study contributes to the knowledge of the role of ID4 in breast cancer.Fil: Nasif, Daniela Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo; ArgentinaFil: Campoy, Emanuel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo; ArgentinaFil: Laurito, Sergio Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo; ArgentinaFil: Branham, Richard Lacy. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Urrutia, Guillermo Alejandro. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo; ArgentinaFil: Roque Moreno, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo; ArgentinaFil: Branham, Maria Teresita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo; Argentin

Division of labour brings greater benefits to clones of Carpobrotus edulis in the non-native range: evidence for rapid adaptive evolution

Why some species become invasive while others do not is a central research request in biological invasions. Clonality has been suggested as an attribute that could contribute to plant invasiveness. Division of labor is an important advantage of clonal growth, and it seems reasonable to anticipate that clonal plants may intensify this clonal attribute in an invaded range because of positive selection on beneficial traits. To test this hypothesis, we collected clones of Carpobrotus edulis from native and invasive populations, grew pairs of connected and severed ramets in a common garden and under negative spatial covariance of nutrients and light to induce division of labor, and measured biomass allocation ratios, final biomass, and photochemical efficiency. Our results showed that both clones from the native and invaded range develop a division of labor at morphological and physiological level. However, the benefit from the division of labor was significantly higher in apical ramets from the invaded range than in ramets from the native area. This is a novel and outstanding result because it provides the first evidence that the benefit of a key clonal trait such as division of labor may have been subjected to evolutionary adaptation in the invaded range. The division of labor can therefore be considered an important trait in the invasiveness of C. edulis. An appropriate assessment of the influence of clonal traits in plant invasions seems key for understanding the underlying mechanisms behind biological invasions of new environments

Beyond a platform protein for the degradosome assembly: the apoptosis-inducing factor as an efficient nuclease involved in chromatinolysis

The Apoptosis-Inducing Factor (AIF) is a moonlighting flavoenzyme involved in the assembly of mitochondrial respiratory complexes in healthy cells, but also able to trigger DNA cleavage and parthanatos. Upon apoptotic-stimuli, AIF redistributes from the mitochondria to the nucleus, where upon association with other proteins such as endonuclease CypA and histone H2AX, it is proposed to organize a DNA–degradosome complex. In this work, we provide evidence for the molecular assembly of this complex as well as for the cooperative effects among its protein components to degrade genomic DNA into large fragments. We have also uncovered that AIF has nuclease activity that is stimulated in the presence of either Mg2+ or Ca2+. Such activity allows AIF by itself and in cooperation with CypA to efficiently degrade genomic DNA. Finally, we have identified TopIB and DEK motifs in AIF as responsible for its nuclease activity. These new findings point, for the first time, to AIF as a nuclease able to digest nuclear dsDNA in dying cells, improving our understanding of its role in promoting apoptosis and opening paths for the development of new therapeutic strategies

Aplicación de biología molecular en veterinaria: PCR como método diagnóstico de brucelosis canina. Implicancia en salud pública y en reproducción con fines comerciales

La brucelosis canina, producida por Brucella canis, es una enfermedad ampliamente distribuida a nivel mundial. Esta enfermedad se reconoce como una entidad que produce importantes pérdidas económicas en los criaderos, como consecuencia de las alteraciones reproductivas. Tiene carácter zoonótico. Se han descrito alteraciones genitales asociadas a la infección por B. canis, destacando la infertilidad en los machos, como una entidad progresiva e irreversible. En las hembras, el signo más común es el aborto súbito al día 45-55 de gestación que ocurre en un elevado porcentaje (sobre 75%). Esta bacteria se transmite entre los caninos por contacto de las mucosas con material infectado (flujo vaginal, semen, otros fluidos y tejidos asociados con abortos), que contienen gran concentración de bacterias. Otros materiales que contienen estos microorganismos son orina, sangre, leche, saliva y heces. Además, B. canis se puede propagar por fómites. Brucella canis se considera una causa poco frecuente de la brucelosis humana; hay que considerar que la importancia clínica de esta infección puede haber sido subestimada. En el diagnóstico de rutina se usa la prueba serológica de aglutinación rápida en placa 2ß-mercaptoetanol, que detecta anticuerpos específicos, con el problema de que hay una proporción significativa de resultados falsos negativos. El objetivo es utilizar reacción en cadena de la polimerasa PCR como método de diagnóstico de brucelosis canina a partir de distintas muestras como sangre, orina y saliva en perros que asisten a la UPV; incorporar normas de Bioseguridad a protocolos de trabajo en la Unidad de Prácticas Veterinarias (UPV) de la Universidad Juan Agustín Maza para reducir el riesgo biológico al que están expuestos alumnos y docentes. Realizar análisis de riesgo frente a casos positivos a brucelosis en la Unidad de Práctica Veterinaria. Desarrollar recursos humanos para mejorar habilidades técnicas e interpersonales para el pensamiento creativo y el liderazgo, estimulando el desarrollo del pensamiento científico, trabajo en grupo, responsabilidad y capacitación en los becarios y el resto del equipo. La metodología es extracción de ADN de las muestras de sangre, leche, orina o saliva y detección de brucelosis canina por la técnica de PCR. En la segunda etapa se realizará la toma de muestras y detección de brucelosis canina por PCR

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteinsparticipateinawidevarietyofphysiologicallyrelevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FADmoleculespermonomerinredoxcommunicationwithanactive disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein “DDOR” (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-basedtransferofreducingequivalentsinbacterialmembranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.Spanish Ministerio de Economía, Industria y Competitividad BFU2016-80343-P, BIO2016-75634-