Microbial Small RNAs – The Missing Link in the Nitrogen Cycle?

Non-coding small RNAs (sRNAs) regulate a wide range of physiological processes in microorganisms that allow them to rapidly respond to changes in environmental conditions. sRNAs have predominantly been studied in a few model organisms, however it is becoming increasingly clear that sRNAs play a crucial role in environmentally relevant pathways. Several sRNAs have been shown to control important enzymatic processes within the nitrogen cycle and many more have been identified in model nitrogen cycling organisms that remain to be characterized. Alongside these studies meta-transcriptomic data indicates both known and putative sRNA are expressed in microbial communities and are potentially linked to changes in environmental processes in these habitats. This review describes the current picture of the function of regulatory sRNAs in the nitrogen cycle. Anthropogenic influences have led to a shift in the nitrogen cycle resulting in an increase in microbial emissions of the potent greenhouse gas nitrous oxide (N2O) into the atmosphere. As the genetic, physiological, and environmental factors regulating the microbial processes responsible for the production and consumption of N2O are not fully understood, this represents a critical knowledge gap in the development of future mitigation strategies.This work was funded by the Biotechnology and Biological Sciences Research Council (United Kingdom) (BB/L022796/1, BB/M00256X/1, BB/S008942/1) and a University of East Anglia studentship as well as a Generalitat Valenciana (Spain) studentship (grant ACIF/2018/200), “Programa Propio para el Formento de la I+D+I del Vicerrectorado de Investigación y Transferencia de Conociemiento (GRE20-02-C)” University of Alicante

Essentiality of the glnA gene in Haloferax mediterranei: gene conversion and transcriptional analysis

Glutamine synthetase is an essential enzyme in ammonium assimilation and glutamine biosynthesis. The Haloferax mediterranei genome has two other glnA-type genes (glnA2 and glnA3) in addition to the glutamine synthetase gene glnA. To determine whether the glnA2 and glnA3 genes can replace glnA in nitrogen metabolism, we generated deletion mutants of glnA. The glnA deletion mutants could not be generated in a medium without glutamine, and thus, glnA is an essential gene in H. mediterranei. The glnA deletion mutant was achieved by adding 40 mM glutamine to the selective medium. This conditional HM26-ΔglnA mutant was characterised with different approaches in the presence of distinct nitrogen sources and nitrogen starvation. Transcriptomic analysis was performed to compare the expression profiles of the strains HM26-ΔglnA and HM26 under different growth conditions. The glnA deletion did not affect the expression of glnA2, glnA3 and nitrogen assimilation genes under nitrogen starvation. Moreover, the results showed that glnA, glnA2 and glnA3 were not expressed under the same conditions. These results indicated that glnA is an essential gene for H. mediterranei and, therefore, glnA2 and glnA3 cannot replace glnA in the conditions analysed.This work was funded by MICINN Grant Number BIO2013-42921P (to MJB), Generalitat Valenciana Grant Number ACIF/2018/200 (to GP) and Universidad de Alicante (VIGROB-016)

Small RNAs of Haloferax mediterranei: Identification and Potential Involvement in Nitrogen Metabolism

Small RNAs have been studied in detail in domains Bacteria and Eukarya but, in the case of the domain Archaea, the knowledge is scarce and the physiological function of these small RNAs (sRNAs) is still uncertain. To extend the knowledge of sRNAs in the domain Archaea and their possible role in the regulation of the nitrogen assimilation metabolism in haloarchaea, Haloferax mediterranei has been used as a model microorganism. The bioinformatic approach has allowed for the prediction of 295 putative sRNAs genes in the genome of H. mediterranei, 88 of which have been verified by means of RNA-Sequencing (RNA-Seq). The secondary structure of these sRNAs and their possible targets have been identified. Curiously, some of them present as possible target genes relating to nitrogen assimilation, such as glutamate dehydrogenase and the nitrogen regulatory PII protein. Analysis of RNA-Seq data has also revealed differences in the expression pattern of 16 sRNAs according to the nitrogen source. Consequently, RNomic and bioinformatic approaches used in this work have allowed for the identification of new sRNAs in H. mediterranei, some of which show different expression patterns depending on the nitrogen source. This suggests that these sRNAs could be involved in the regulation of nitrogen assimilation and can constitute an important gene regulatory network.This work was supported by grant BIO2013-42921-P from the Spanish Ministry of Economy and Competitiveness. Funds from “Programa Propio para el fomento de la I+D+i del Vicerrectorado de Investigación y Transferencia de Conocimiento” de la Universidad de Alicante are also acknowledged

Prevalence of Lynch Syndrome among Patients with Newly Diagnosed Endometrial Cancers

Background: Lynch syndrome (LS) is a hereditary condition that increases the risk for endometrial and other cancers. The identification of endometrial cancer (EC) patients with LS has the potential to influence life-saving interventions. We aimed to study the prevalence of LS among EC patients in our population. Methods: Universal screening for LS was applied for a consecutive series EC. Tumor testing using microsatellite instability (MSI), immunohistochemistry (IHC) for mismatch-repair (MMR) protein expression and MLH1-methylation analysis, when required, was used to select LS-suspicious cases. Sequencing of corresponding MMR genes was performed. Results: One hundred and seventy-three EC (average age, 63 years) were screened. Sixty-one patients (35%) had abnormal IHC or MSI results. After MLH1 methylation analysis, 27 cases were considered suspicious of LS. From these, 22 were contacted and referred for genetic counseling. Nineteen pursued genetic testing and eight were diagnosed of LS. Mutations were more frequent in younger patients (<50 yrs). Three cases had either intact IHC or MSS and reinforce the need of implement the EC screening with both techniques. Conclusion: The prevalence of LS among EC patients was 4.6% (8/173); with a predictive frequency of 6.6% in the Spanish population. Universal screening of EC for LS is recommended.This study was supported by Conselleria Sanidad Comunidad Valenciana, Spain (AP/177/10) (http://www.san.gva.es/); Biomedical Research Foundations of the Alicante University Hospital (PI14/2006 and NI02/2011) (http://www.dep19.san.gva.es/); and the Elche University Hospital, Spain (FIBElx-CO11/03) (http://www.dep20.san.gva.es/)

TECNOGAP. Herramientas tecnológicas corporativas en el Grado en Gestión y Administración Pública

El Grado en Gestión y Administración Pública de la Universidad de Alicante posee un alto componente multidisciplinar que mezcla materias vinculadas a ciencias jurídicas con otras provenientes de áreas como la economía, la sociología, la estadística o los lenguajes y sistemas informáticos. Esto hace que las tareas de coordinación de trabajos y metodologías docentes incorporen nuevos retos y dificultades. A pesar de las contantes mejoras del Campus Virtual (ahora UA Cloud) como herramienta esencial de gestión administrativa y académica, los recursos que proporciona como complemento a la docencia (materiales, pruebas, controles, sesiones,…) son a veces insuficientes o inadecuados para cubrir necesidades concretas. Por ello, la Universidad también ha adoptado y adaptado nuevas herramientas corporativas (Moodle, RUA, OCW) con el fin de complementar esas carencias. El objetivo de esta red es fomentar el uso de esas herramientas para facilitar las tareas académicas y mejorar los procesos de adquisición de conocimiento, buscando, además un encuentro entre docentes de diferentes áreas de conocimiento para posibilitar sinergias e intercambio de experiencias

Functional analysis of Lsm protein under multiple stress conditions in the extreme haloarchaeon Haloferax mediterranei

The Sm, like-Sm, and Hfq proteins belonging to the Sm superfamily of proteins are represented in all domains of life. These proteins are involved in several RNA metabolism pathways. The functions of bacterial Hfq and eukaryotic Sm proteins have been described, but knowledge about the in vivo functions of archaeal Sm proteins remains limited. This study aims to improve the understanding of Lsm proteins and their role using the haloarchaeon Haloferax mediterranei as a model microorganism. The Haloferax mediterranei genome contains one lsm gene that overlaps with the rpl37e gene. To determine the expression of lsm and rpl37e genes and the co-transcription of both, reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed under different standard and stress conditions. The results suggest that the expression of lsm and rpl37e is constitutive. Co-transcription occurs at sub-optimal salt concentrations and temperatures, depending on the growth phase. The halophilic Lsm protein contains two Sm motifs, Sm1 and Sm2, and the sequence encoding the Sm2 motif also constitutes the promoter of the rpl37e gene. To investigate their biological functions, the lsm deletion mutant and the Sm1 motif deletion mutant, where the Sm2 motif remained intact, were generated and characterised. Comparison of the lsm deletion mutant, Sm1 deletion mutant, and the parental strain HM26 under standard and stress growth conditions revealed growth differences. Finally, swarming assays in complex and defined media showed greater swarming capacity in the deletion mutants.This work was supported by the Spanish Ministry of Economy and Competitiveness [grant BIO2013-42921-P]; Programa Propio para el fomento de la I+D+I del Vicerrectorado de Investigación y Transferencia de Conocimiento” of the University of Alicante (VIGRO-016) and the Generalitat Valenciana (Spain) [ACIF/2018/200]

Comprehensive Bioinformatics Analysis of the Biodiversity of Lsm Proteins in the Archaea Domain

The Sm protein superfamily includes Sm, like-Sm (Lsm), and Hfq proteins. Sm and Lsm proteins are found in the Eukarya and Archaea domains, respectively, while Hfq proteins exist in the Bacteria domain. Even though Sm and Hfq proteins have been extensively studied, archaeal Lsm proteins still require further exploration. In this work, different bioinformatics tools are used to understand the diversity and distribution of 168 Lsm proteins in 109 archaeal species to increase the global understanding of these proteins. All 109 archaeal species analyzed encode one to three Lsm proteins in their genome. Lsm proteins can be classified into two groups based on molecular weight. Regarding the gene environment of lsm genes, many of these genes are located adjacent to transcriptional regulators of the Lrp/AsnC and MarR families, RNA-binding proteins, and ribosomal protein L37e. Notably, only proteins from species of the class Halobacteria conserved the internal and external residues of the RNA-binding site identified in Pyrococcus abyssi, despite belonging to different taxonomic orders. In most species, the Lsm genes show associations with 11 genes: rpl7ae, rpl37e, fusA, flpA, purF, rrp4, rrp41, hel308, rpoD, rpoH, and rpoN. We propose that most archaeal Lsm proteins are related to the RNA metabolism, and the larger Lsm proteins could perform different functions and/or act through other mechanisms of action.This research was funded by “Programa Propio para el Fomento de la I+D+I del Vicerrectorado de Investigación y Transferencia de Conocimiento” of the University of Alicante (VIGRO-016) and the Generalitat Valenciana (Spain) [ACIF/2018/200]

New proposal of nitrogen metabolism regulation by small RNAs in the extreme halophilic archaeon Haloferax mediterranei

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.This study was funded by the Spanish Ministry of Economy and Competitiveness and FEDER (grant BIO2013-42921-P); the Generalitat Valenciana (Spain) (grant ACIF/2018/200); and the “Programa Propio para el Fomento de la I + D + i del Vicerrectorado de Investigación y Transferencia de Conocimiento” of the University of Alicante (Spain)

Conjugation of a Novel Apaf-1 Inhibitor to Peptide-based Cell-Membrane Transporters. Effective Methods to Improve Inhibition of Mitochondria-Mediated Apoptosis

The definitive verison is available at http://www.sciencedirect.com/science//journal/01969781We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy.This work was supported by grants from Spanish Ministry of Science and Education (MEC) (BIO2004-998 and CTQ2005-00995), Fundación Centro de Investigación Príncipe Felipe and a Marie Curie Reintegration grant (MERG-2004-06307). Mar Orzáez thanks Bancaja for a postdoctoral fellowship. Laura Mondragón is supported by a FPI fellowship from MEC. The confocal microscope was supported by MEC through FEDER.Peer reviewe