The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p

The pol3-t Hyperrecombination Phenotype and DNA Damage-Induced Recombination in Saccharomyces cerevisiae Is RAD50 Dependent

The DNA polymerase δ (POL3/CDC2) allele pol3-t of Saccharomyces cerevisiae has previously been shown to be sensitive to methylmethanesulfonate (MMS) and has been proposed to be involved in base excision repair. Our results, however, show that the pol3-t mutation is synergistic for MMS sensitivity with MAG1, a known base excision repair gene, but it is epistatic with rad50Δ, suggesting that POL3 may be involved not only in base excision repair but also in a RAD50 dependent function. We further studied the interaction of pol3-t with rad50Δ by examining their effect on spontaneous, MMS-, UV-, and ionizing radiation-induced intrachromosomal recombination. We found that rad50Δ completely abolishes the elevated spontaneous frequency of intrachromosomal recombination in the pol3-t mutant and significantly decreases UV- and MMS-induced recombination in both POL3 and pol3-t strains. Interestingly, rad50Δ had no effect on γ-ray-induced recombination in both backgrounds between 0 and 50 Gy. Finally, the deletion of RAD50 had no effect on the elevated frequency of homologous integration conferred by the pol3-t mutation. RAD50 is possibly involved in resolution of replication forks that are stalled by mutagen-induced external DNA damage, or internal DNA damage produced by growing the pol3-t mutant at the restrictive temperature

A recombination-based method to characterize human BRCA1 missense variants

Purpose. Many missense variants in BRCA1 are of unclear clinical significance. Functional and genetic approaches have been proposed for elucidating the clinical significance of such variants. The purpose of the present study was to evaluate BRCA1 missense variants for their effect on both Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). Methods. HR frequency evaluation: HeLaG1 cells, containing a stably integrated plasmid that allows to measure HR events by gene conversion events were transfected with the pcDNA3β expression vector containing the BRCA1-wild type (BRCA1-WT) or the BRCA1-Unclassified Variants (BRCA1-UCVs). The NHEJ was measured by a random plasmid integration assay. Results. This assays suggested a BRCA1 involvement mainly in the NHEJ. As a matter of fact, the Y179C and the A1789T variant altered significantly the NHEJ activity as compared to the wild type, suggesting that they may be related to BRCA1 associated pathogenicity by affecting this function. The variants N550H and I1766S, and the mutation M1775R did not alter the NHEJ frequency. Conclusions. These data, beside proposing a method for the study of BRCA1 variants effect on HR and NHEJ, highlighted the need for a range of functional assays to be performed in order to identify variants with altered function

Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress

Characterisation of gene expression profiles of yeast cells expressing BRCA1 missense variants

Germline mutations in breast cancer susceptibility gene 1 (BRCA1) confer high risk of developing breast and ovarian cancers. Even though most BRCA1 cancer-predisposing mutations produce a non-functional truncated protein, 5-10% of them cause single amino acid substitutions. This second type of mutations represents a useful tool for examining BRCA1 molecular functions. Human BRCA1 inhibits cell proliferation in transformed Saccharomyces cerevisiae cells and this effect is abolished by disease-associated mutations in the BRCT domain. Moreover, BRCA1 mutations located both inside and outside the BRCT domain may induce an increase in the homologous recombination frequency in yeast cells. Here we present a microarray analysis of gene expression induced in yeast cells transformed with five BRCA1 missense variants, in comparison with gene expression induced by wildtype BRCA1. Data analysis was performed by grouping the BRCA1 variants into three sets: Recombination (R)-set (Y179C and S1164I), Recombination and Proliferation (RP)-set(I1766S and M1775R) and Proliferation (P)-set (A1789T), according to their effects on yeast cell phenotype. We found 470, 740 and 1136 differentially expressed genes in R-, P- and RP-set, respectively. Our results point to some molecular mechanisms critical for the control of cell proliferation and of genome integrity providing support to a possible pathogenic role of the analysed mutations. They also confirm that yeast, despite the absence of a BRCA1 homologue, represents a valid model system to examine BRCA1 molecular functions, as the molecular pathways activated by BRCA1 variants are conserved in humans

MULTILEVEL SPINAL EPIDURAL EMPYEMA: SERIES OF CLINICAL CASES AND BIBLIOGRAPHIC

ABSTRACT Multilevel spinal epidural empyema (SEE) is a rare and serious infection of the spine with a high rate of morbidity and mortality. Although abscesses or empyema of the spine sector are well studied, this pathology is surprising due to its rarity and diagnostic and therapeutic challenge. It stands out for being more common in adulthood and in males and is associated with predisposing pathologies. The bacteriological agent responsible in most cases is Staphylococcus aureus. Early treatment is essential and is based on two pillars: antibiotic therapy and decompressive surgery. We present two clinical cases with multilevel involvement that evolved favorably both infectiously and neurologically without causing spine instability and we carried out a bibliographic review of the subject. Level of Evidence IV; Case Report

Characterization of consumers of aromatic and medicinal plants in Argentina

Las plantas aromáticas y medicinales (PAM) son utilizadas desde la antigüedad para tratar la salud de las personas; ya sea por considerarse remedios naturales, por su bajo costo económico o por costumbre popular. Numerosos trabajos muestran las relaciones entre los humanos y las plantas de su entorno, los patrones de consumo, el uso en las comunidades y los sitios de expendio en distintas regiones de nuestro país. Sin embargo, no existe un relevamiento nacional actualizado que describa el consumo de PAM en la población argentina. Durante el aislamiento por la pandemia de Covid-19 el INTA desde el equipo del proyecto de mejoramiento genético de plantas ornamentales, aromáticas y medicinales definió actualizar la información sobre el mercado nacional de estas plantas. Se realizó un estudio de tipo exploratorio descriptivo de alcance nacional, mediante un muestreo no probabilístico, a través de una encuesta online, con el objetivo de caracterizar el perfil de consumidores de plantas aromáticas y medicinales e indagar sobre sus preferencias, formas de consumo y de abastecimiento de las principales PAM utilizadas. Los resultados indicaron que la población encuestada tiene un elevado consumo de PAM y utiliza una gran diversidad de plantas con distintos niveles de industrialización y origen. Se destacó la aceptación y el interés del consumidor por parte de estos productos naturales. Este trabajo definirá las necesidades actuales del mercado y las nuevas líneas de investigación y desarrollo que contribuyan a la sustentabilidad del sector.Aromatic and medicinal plants (AMP) have been used since ancient times to treat people’s health, either because they are considered natural remedies for their low cost or by popular knowledge. Numerous studies show the relationships between humans and the plants in their environment, the consumption patterns, the use in the communities and the places where they are sold in different regions of our country. However, there are no up-to-date national surveys describing the consumption of AMP in the Argentine population. During the isolation due to the Covid-19 pandemic, INTA's project team for the genetic improvement of ornamental, aromatic and medicinal plants decided to update the information on the national market of these plants. An exploratory descriptive study was carried out through an online nationwide survey, by non-probabilistic sampling, with the objective of characterizing the profile of consumers of aromatic and medicinal plants and to inquire about their preferences, forms of consumption and supply of the main AMP used. The results indicated that the surveyed population has a high consumption of AMP and uses a great diversity of plants with different levels of industrialization and origin. The consumer acceptance and interest in natural products is remarkable. This work will define the current market needs and new lines of research and development that will contribute to the sustainability of the sector.Fil: Fuentes Baluzzi, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Direccion Nacional Inta. Estacion Experimental Agropecuaria Area Metropolitana de Buenos Aires.; ArgentinaFil: Balsamo, Maricel. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Misiones. Estacion Experimental Agropecuaria Cerro Azul.; ArgentinaFil: Galli, María Carolina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional La Pampa-San Luis; ArgentinaFil: Guariniello, Julián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Jaldo Alvaro, Mariana. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Risso, Oscar Ariel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional La Pampa-San Luis; ArgentinaFil: Nagahama, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Sur. Estación Experimental Agropecuaria Esquel; ArgentinaFil: Mazzoni, Ariel Omar. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; Argentin

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders

Functional Interaction Between BRCA1 and DNA Repair in Yeast May Uncover a Role of RAD50, RAD51, MRE11A, and MSH6 Somatic Variants in Cancer Development

In this study, we determined if BRCA1 partners involved in DNA double-strand break (DSB) and mismatch repair (MMR) may contribute to breast and ovarian cancer development. Taking advantage the functional conservation of DNA repair pathways between yeast and human, we expressed several BRCA1 missense variants in DNA repair yeast mutants to identify functional interaction between BRCA1 and DNA repair in BRCA1-induced genome instability. The pathogenic p.C61G, pA1708E, p.M775R, and p.I1766S, and the neutral pS1512I BRCA1 variants increased intra-chromosomal recombination in the DNA-repair proficient strain RSY6. In the mre11, rad50, rad51, and msh6 deletion strains, the BRCA1 variants p.C61G, pA1708E, p.M775R, p.I1766S, and pS1215I did not increase intra-chromosomal recombination suggesting that a functional DNA repair pathway is necessary for BRCA1 variants to determine genome instability. The pathogenic p.C61G and p.I1766S and the neutral p.N132K, p.Y179C, and p.N550H variants induced a significant increase of reversion in the msh2Δ strain; the neutral p.Y179C and the pathogenic p.I1766S variant induced gene reversion also, in the msh6Δ strain. These results imply a functional interaction between MMR and BRCA1 in modulating genome instability. We also performed a somatic mutational screening of MSH6, RAD50, MRE11A, and RAD51 genes in tumor samples from 34 patients and identified eight pathogenic or predicted pathogenic rare missense variants: four in MSH6, one in RAD50, one in MRE11A, and two in RAD51. Although we found no correlation between BRCA1 status and these somatic DNA repair variants, this study suggests that somatic missense variants in DNA repair genes may contribute to breast and ovarian tumor development