RNA Preparation and RNA-Seq Bioinformatics for Comparative Transcriptomics.

The principal transcriptome analysis is the determination of differentially expressed genes across experimental conditions. For this, the next-generation sequencing of RNA (RNA-seq) has several advantages over other techniques, such as the capability of detecting all the transcripts in one assay over RT-qPCR, such as its higher accuracy and broader dynamic range over microarrays or the ability to detect novel transcripts, including non-coding RNA molecules, at nucleotide-level resolution over both techniques. Despite these advantages, many microbiology laboratories have not yet applied RNA-seq analyses to their investigations. The high cost of the equipment for next-generation sequencing is no longer an issue since this intermediate part of the analysis can be provided by commercial or central services. Here, we detail a protocol for the first part of the analysis, the RNA extraction and an introductory protocol to the bioinformatics analysis of the sequencing data to generate the differential expression results.

Retrospective and Prospective Analysis on "Sexting": Indicators of Productivity, Dispersion, and Content (2009-2019).

In the last decade, research on "sexting" has undergone an exponential increase, giving rise to the publication of numerous studies clarifying its meaning, offering information of educational value, and favoring a good use of technology to prevent problems caused by this exchange of sexual information. The aim of this article was to analyze the production, performance, impact, and content of scientific articles evaluating the "sexting" thesaurus (title, abstract, and/or keywords) between 2009 and September 2019. Articles were sourced from two internationally recognized databases: Scopus and Web of Science. A scientometric study was then carried out on a sample of 641 articles that met the established inclusion criteria. The main findings indicate that "sexting" is a very recent research focus, but one in full growth phase, with scientific production related to the topic likely to double over the next few years. Although "sexting" has been researched worldwide, the scientific production of the US and American authors is the most notable. There were also some differences between the Scopus and Web of Science databases, mainly in the volume of production and the trend. However, the studies do show a common research line, "cyberbullying," and a common target population: adolescents. Therefore, the content analysis reveals that research on "sexting" is mostly carried out with adolescents and takes into consideration other themes such as cyberbullying, dating violence, and sexuality.

Modulation of Multiple Gene Clusters' Expression by the PAS-LuxR Transcriptional Regulator PteF.

PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from Streptomyces avermitilis. Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in S. avermitilis DpteF and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces.

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus: Pushing the Limits of Nanopore Sequencing.

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.

Multi-omics Study of Planobispora rosea, Producer of the Thiopeptide Antibiotic GE2270A.

Planobispora rosea is the natural producer of the potent thiopeptide antibiotic GE2270A. Here, we present the results of a metabolomics and transcriptomics analysis of P. rosea during production of GE2270A. The data generated provides useful insights into the biology of this genetically intractable bacterium. We characterize the details of the shutdown of protein biosynthesis and the respiratory chain associated with the end of the exponential growth phase. We also provide the first description of the phosphate regulon in P. rosea. Based on the transcriptomics data, we show that both phosphate and iron are limiting P. rosea growth in our experimental conditions. Additionally, we identified and validated a new biosynthetic gene cluster associated with the production of the siderophores benarthin and dibenarthin in P. rosea. Together, the metabolomics and transcriptomics data are used to inform and refine the very first genome-scale metabolic model for P. rosea, which will be a valuable framework for the interpretation of future studies of the biology of this interesting but poorly characterized species. IMPORTANCE Planobispora rosea is a genetically intractable bacterium used for the production of GE2270A on an industrial scale. GE2270A is a potent thiopeptide antibiotic currently used as a precursor for the synthesis of two compounds under clinical studies for the treatment of Clostridium difficile infection and acne. Here, we present the very first systematic multi-omics investigation of this important bacterium, which provides a much-needed detailed picture of the dynamics of metabolism of P. rosea while producing GE2270A.

Coupled Transcriptomics for Differential Expression Analysis and Determination of Transcription Start Sites: Design and Bioinformatics.

The term coupled transcriptomics is coined to describe a design of an RNA-seq experiment intended for both differential expression analysis and genome-wide determination of the transcription start sites (TSS). The minimal requirements for the first analysis are two experimental conditions with at least two biological replicates enabling statistical tests. The second analysis involves the bioinformatics comparison of the data generated from a control RNA-seq library with another library enriched in primary transcripts using Terminator™ 5'-phosphate-dependent exonuclease, in an experiment denominated differential RNA-seq (dRNA-seq). Usually, dRNA-seq is carried out with specific protocols for library construction, different of those used for common differential expression analysis. Our experimental design allows to use the same data for both analyses, reducing the number of libraries to be generated and sequenced. This is a guide for designing a coupled transcriptomics experiment and for the subsequent bioinformatics procedures. The proposed methods can be applied to the detection and study of small RNA genes.

Nomophobia and the influence of time to REST among nursing students. A descriptive, correlational and predictive research.

The inclusion of the smartphone in society has brought many advantages, but also disadvantages, such as nomophobia, considered as a digital disease generated by the excessive use of the smartphone. The general objective of the research is to know and analyse the prevalence of nomophobia among nursing students and knowing the influence of time to rest. The research design is descriptive, correlational, transversal and predictive with a quantitative research methodology. The sample consist of nursing students from the University of Granada (N = 880) in Spain. A descriptive analysis has been carried out, as well as a bivariate correlation of Pearson, the student T test and a multiple linear regression. The results show an average level of nomophobia among nursing students. Although the students can recognize that the use of the smartphone can reduce their rest period, mainly focused on the hours they spend sleeping, there is no significant relationship between the two facts, confirming also that the levels of nomophobia are significantly more related in those who claim not to have problems in their rest due to the use of the mobile phone. A large percentage of them claim to rest less time due to excessive use of their smartphone.

Do Age and Educational Stage Influence No-Mobile-Phone Phobia?

Technological progress not only brings with it resources that improve and facilitate the day-to-day life of the people who make up society but also entails health risks, with the emergence of terms, such as nomophobia, which is considered an anxiety disorder produced by the fear that not having a mobile phone generates in a person. This research aims to identify the relationship and influence between levels of nomophobia and the age or educational stage of students. The research method is based on a correlational and predictive design of quantitative methodology. The instrument used is the NMP-Q questionnaire. The study population is students from different educational stages (obligatory secondary education, baccalaureate, vocational training and university). The results show that students over 12 years old present an average level of "nomophobia" (no-mobile-phone phobia), namely, not being able to communicate with the family where the highest levels are presented. We conclude that students over 12 years of age and of any educational stage present an average level of nomophobia, and it cannot be determined that either the educational stage or the age are determining factors in the presentation of this problem. This can occur at any age and at any level of the different educational stages, although there are risk indicators that we should bear in mind to avoid the appearance of nomophobia.

Eficacia contrastada de la Realidad Aumentada en el aprendizaje de la reanimación cardiopulmonar / Compared effectiveness of Augmented Reality in learning cardiopulmonary resuscitation

Introducción: La incidencia de la tecnología en la educación ha propiciado el desarrollo de nuevos recursos innovadores para afrontar los procesos de enseñanza y aprendizaje, por lo que la Realidad Aumentada se muestra como una de las tecnologías con mayor proyección, tanto en el sector educativo, en general, como en la educación médica, en particular. Objetivo: Determinar el nivel de eficacia de una metodología sustentada en Realidad Aumentada frente a una tradicional en el aprendizaje de la reanimación cardiopulmonar, en dos grupos de estudiantes universitarios. Métodos: El estudio comenzó en el curso 2017-2018. Se estableció un diseño experimental con grupo control, de tipo descriptivo y correlacional, en una muestra de 58 discentes de un centro universitario de España. Se desplegó un programa formativo en dos grupos con metodologías diferentes -Realidad Aumentada frente a tradicional-. Se empleó un cuestionario -derivado de la Escala de Autoeficacia general en reanimación cardiopulmonar- validado por el método Delphi y de pertinente fiabilidad (α = 0,86) para recoger los datos. Resultados: La metodología innovadora con Realidad Aumentada aplicada en el grupo experimental obtuvo mejores resultados en la motivación, la participación, la interacción con los contenidos, el rol activo de los participantes y mayores resultados de aprendizaje sobre el grupo que siguió una metodología tradicional sin empleo de la tecnología educativa. Conclusiones: Los hallazgos demostraron la eficacia de la Realidad Aumentada en el aprendizaje de las pautas de reanimación cardiopulmonar en discentes universitarios. Por tanto, se recomienda el uso de esta tecnología educativa para alcanzar la efectividad en el aprendizaje de este tipo de contenidos relacionados con la educación sanitaria(AU)

Introduction: The impact of technology on education has led to the development of new innovative resources to cope with the teaching and learning processes, a reason why augmented reality is shown as one of the technologies with the greatest projection, both in the educational sector, in general, as well as in medical education, in particular. Objective: To identify the level of effectiveness of a methodology based on augmented reality compared to a traditional one in learning cardiopulmonary resuscitation, in two groups of university students. Methods: The study began in the academic year 2017-2018. An experimental design of descriptive and correlational type and with a control group was established in a sample of 58 students from a university center in Spain. A training program was implemented in two groups with different methodologies: augmented reality versus a traditional one. A questionnaire, derived from the Generalized Self-Efficacy Scale in cardiopulmonary resuscitation, validated using the Delphi method with relevant reliability α=0.86, was used to collect the data. Results: The innovative methodology with augmented reality applied in the experimental group showed better results regarding motivation, participation, interaction with the contents, and the active role of the participants; as well as higher learning results over the group that followed a traditional methodology without using any educational technologies. Conclusions: The findings showed the effectiveness of augmented reality in learning cardiopulmonary resuscitation guidelines among university students. Therefore, the use of this educational technology is recommended to achieve effectiveness in learning this type of content related to health education(AU)

The EYCN and the Power of Networking.

Networking is a symbiosis-it is about establishing, building and cultivating relationships that you will maintain over a long time and which may lead to mutually beneficial exchanges in your future. Your professional contacts might also become your close friends, since you frequently share the same experiences or merely have similar scientific and general interests or nerdy humour. Chemical societies are a fantastic way to expand your network and engaging or following the activities of the European Young Chemists Network (EYCN) might be the perfect starting point for you.

Nomophobia: impact of cell phone use and time to rest among teacher students.

Nomophobia is one of the modern pathologies that has been born as a consequence of the impact that portable technologies have had on society and the dependence generated among citizens, especially towards smartphones. This phobia manifests itself and is intensified by the loss of immediate access to information, to the network of contacts, as well as by the impossibility of contacting or being contacted by other people. All this ends up interfering with the development of the person's daily life (physical, physiological, psychological, social problems, among others). Although the research is in an incipient phase, the problem has not been studied with the teaching population, so we present a pioneering study with this group, the main objective being to analyze the prevalence of nomophobia in future teachers of Early Childhood and Primary Education, as well as to check the incidence of rest time in the levels of nomophobia. The study follows a descriptive, correlational, transversal and predictive design and a quantitative methodology. The standardized nomophobia questionnaire NMP-Q was used on a sample of n = 849 future teachers. The results show average levels of nomophobia in most of the variables. However, the higher levels of nervousness, fear or anxiety stand out due to the inability to communicate instantaneously. Also, a higher prevalence of the problem is observed in the sector of the sample that claims to sacrifice rest time due to the use of their mobile phone. Although these numbers are not alarming, we must take into account that in some variables the prevalence is slightly higher, making it necessary to make educational interventions in this regard and to promote education for the responsible and critical use of media and technologies.

Do Age, Gender and Poor Diet Influence the Higher Prevalence of Nomophobia among Young People?

The use of Information and Communication Technologies (ICT) is generating the emergence of new pathologies such as nomophobia. The aim of this research was to analyze the prevalence of nomophobia among young people, as well as to check whether the level of nomophobia is higher in males or females and in those students who claim to have less healthy nutrition due to the use of their mobile phones. The research method was based on a correlational and predictive design with a quantitative methodology. The measurement tool used is the Nomophobia Questionnaire (NMP-Q). The participating sample was 1743 students between 12 and 20 years old from different educational stages of the Autonomous City of Ceuta (Spain). The results show that highest rates of nomophobia were found in relation to the inability to communicate and contact others immediately. About gender, women have higher rates of nomophobia than men. In relation to age, no significant differences were found; thus, the problem may affect all ages equally. Finally, students who think that their smartphone use is detrimental to their good nutrition show higher levels on the scale provided.

Nomophobia: An Individual's Growing Fear of Being without a Smartphone-A Systematic Literature Review.

This review examines the current literature focused on nomophobia (objectives, methodological design, main variables, sample details, and measurement methods) in the Scopus and Web of Science databases. To this end, we conducted a systematic literature review in accordance with the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines. The initial sample consisted of 142 articles, of which 42 met the inclusion criteria and were analyzed in detail. The findings show that the current research is in an exploratory phase, with a greater predominance of descriptive, nonexperimental, and cross-sectional studies that explore the prevalence of nomophobia mainly in adolescents and university students. The most widely used measurement instrument is the Nomophobia Questionnaire (NMP-Q) proposed by Yildrim and Correia. In addition, the research suggests that nomophobia negatively affects personality, self-esteem, anxiety, stress, academic performance, and other physical and mental health problems. We are therefore faced with a health problem, which negatively affects a person, causing psychological problems and physical and behavioral changes.

Activation of Secondary Metabolite Gene Clusters in Streptomyces clavuligerus by the PimM Regulator of Streptomyces natalensis.

Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex.

Genome-wide transcriptome response of Streptomyces tsukubaensis to N-acetylglucosamine: effect on tacrolimus biosynthesis.

Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic-producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces tsukubaensis NRRL 18488. Using microarrays technology, we found that GlcNAc represses the transcription of fkbN, encoding the main transcriptional activator of the tacrolimus biosynthetic cluster, and of ppt1, encoding a phosphopantheteinyltransferase involved in tacrolimus biosynthesis. On the contrary, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems, in the physiology of S. tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.

ArgR of Streptomyces coelicolor Is a Pleiotropic Transcriptional Regulator: Effect on the Transcriptome, Antibiotic Production, and Differentiation in Liquid Cultures.

ArgR is a well-characterized transcriptional repressor controlling the expression of arginine and pyrimidine biosynthetic genes in bacteria. In this work, the biological role of Streptomyces coelicolor ArgR was analyzed by comparing the transcriptomes of S. coelicolor ΔargR and its parental strain, S. coelicolor M145, at five different times over a 66-h period. The effect of S. coelicolor ArgR was more widespread than that of the orthologous protein of Escherichia coli, affecting the expression of 1544 genes along the microarray time series. This S. coelicolor regulator repressed the expression of arginine and pyrimidine biosynthetic genes, but it also modulated the expression of genes not previously described to be regulated by ArgR: genes involved in nitrogen metabolism and nitrate utilization; the act, red, and cpk genes for antibiotic production; genes for the synthesis of the osmotic stress protector ectoine; genes related to hydrophobic cover formation and sporulation (chaplins, rodlins, ramR, and whi genes); all the cwg genes encoding proteins for glycan cell wall biosynthesis; and genes involved in gas vesicle formation. Many of these genes contain ARG boxes for ArgR binding. ArgR binding to seven new ARG boxes, located upstream or near the ectA-ectB, afsS, afsR, glnR, and redH genes, was tested by DNA band-shift assays. These data and those of previously assayed fragments permitted the construction of an improved model of the ArgR binding site. Interestingly, the overexpression of sporulation genes observed in the ΔargR mutant in our culture conditions correlated with a sporulation-like process, an uncommon phenotype.

Self-control of the PHO regulon: the PhoP-dependent protein PhoU controls negatively expression of genes of PHO regulon in Streptomyces coelicolor.

Phosphate control of the biosynthesis of secondary metabolites in Streptomyces is mediated by the two component system PhoR-PhoP. Linked to the phoR-phoP cluster, and expressed in the opposite orientation, is a phoU-like encoding gene with low identity to the phoU gene of Escherichia coli. Expression of this phoU-like gene is strictly dependent on PhoP activation. We have isolated a PhoU-null mutant and used transcriptomic and RNA-sequencing (RNA-seq) procedures to identify its transcription start site and regulation. RNA-seq studies identified two transcription start sites, one upstream of phoU and the second upstream of the mptA gene. Whereas transcription of PhoU is entirely dependent on PhoP, expression of the downstream mtpA gene is only partially dependent on PhoP activation. The phoU mutant grows more slowly than the parental strain, sporulates poorly and the spores lack pigmentation. Production of actinorhodin and undecylprodigiosin decreased in the phoU mutant, indicating that PhoU has a positive modulating effect on production of these antibiotics. Indeed, transcriptional studies of expression of the actII-ORF4 and redD genes indicated that the PhoU protein activates expression of these antibiotic regulators. Using the glpQ1 promoter as in vivo reporter of the activity of the PHO regulon genes, we observed that expression of glpQ1 is negatively modulated by PhoU. These results were confirmed by reverse transcription-PCR studies of three genes of the PHO regulon; that is, glpQ1, pstS and phoR. In conclusion, PhoU acts as a negative modulator of expression of the PHO regulon genes and as phoU expression is strictly dependent on PhoP activation, this mechanism appears to work as a feed-back control mechanism (self-regulation).The Journal of Antibiotics advance online publication, 1 November 2017; doi:10.1038/ja.2017.130.

Streptomyces tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources.

In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces tsukubaensis. A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. tsukubaensis and the model species Streptomyces coelicolor. A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus production.

Analysis of the Pho regulon in Streptomyces tsukubaensis.

Phosphate regulation of antibiotic biosynthesis in Streptomyces has been studied due to the importance of this genus as a source of secondary metabolites with biological activity. Streptomyces tsukubaensis is the main producer of tacrolimus (or FK506), an immunosuppressant macrolide that generates important benefits for the pharmaceutical market. However, the production of tacrolimus is under a negative control by phosphate and, therefore, is important to know the molecular mechanism of this regulation. Despite its important role, there are no reports about the Pho regulon in S. tsukubaensis. In this work we combined transcriptional studies on the response to phosphate starvation with the search for PHO boxes in the whole genome sequence of S. tsukubaensis. As a result, we identified a set of genes responding to phosphate starvation and containing PHO boxes that include common Pho regulon members but also new species-specific candidates. In addition, we demonstrate for the first time the functional activity of PhoP from S. tsukubaensis through complementation studies in a Streptomyces coelicolor ΔphoP strain. For this purpose, we developed an anhydrotetracycline inducible system that can be applied to the controlled expression of target genes.