Restriction-Modification and CRISPR-Cas Systems: Cooperation Between Innate and Adaptive Immunity in Prokaryotes

Bacteria have evolved numerous mechanisms to resist the constant assault of viruses (called bacteriophages, or simply phages) that can infect and kill them. Restriction-modification (RM) systems represent one such strategy. Generally, these systems provide defense by coordinating the activities of two distinct enzymes: a restriction endonuclease and a methyltransferase. Both enzymes recognize the same short DNA sequences. The methyltransferase modifies these target sites in the host chromosome, which prevents the restriction endonuclease from cleaving the host’s own DNA. In contrast, foreign phage DNA is usually not methylated at these sequences. Consequently, upon injection into the host, the viral DNA is recognized and cleaved by the restriction endonuclease, preventing the progression of the phage’s life cycle. Therefore, RM systems are considered a part of the innate immune response because they can provide defense against any phage, including ones that have never been encountered previously, as long as they harbor RM target sites. Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated genes (cas) form another defense system that destroys foreign DNA. The CRISPR array consists of a series of repetitive DNA sequences separated by unique DNA sequences known as spacers. During phage infection, short DNA fragments are taken from the viral DNA and integrated into the CRISPR locus to form new spacers. These sequences are then transcribed into CRISPR RNAs (crRNAs). In type II-A CRISPRCas systems, the crRNAs guide the Cas9 nuclease to a matching viral DNA target for cleavage. As such, unlike RM systems, CRISPR-Cas systems represent an adaptive immune response because they require an initial exposure to a virus in order to become successfully immunized through the acquisition of new spacer sequences. CRISPR-Cas and RM are two of the most prevalent types of defense systems found in bacteria and often co-exist together in a single host. Yet, how they may interact with each other in the context of immunity during bacteriophage infection is poorly understood. Here, in my thesis work, I investigate the interplay between RM and type II-A CRISPR-Cas systems. First, I demonstrate that RM systems provide a weak and temporary protection that stimulates CRISPR spacer acquisition, enabling the cells to survive the viral infection. Then, I go on to show that the restriction activity of the RM system is critical for this process and that the rate of spacer acquisition is correlated to the number of RM target sites in the phage genome. To further uncover the mechanistic link between restriction and the acquisition of new spacers, I implement next-generation sequencing to demonstrate that spacers are preferentially extracted at the dsDNA breaks (DSBs) generated by the restriction endonuclease. Additionally, I show that the host DNA repair complex, AddAB, can process these breaks, which further enhances spacer acquisition. Finally, I follow the dynamics between RM and CRISPR-Cas during the chain of events that occur upon viral infection. I demonstrate that although the RM system provides an immediate line of defense due to its ability to recognize a broad range of foreign invaders, it is ultimately overcome by the rapid emergence of methylated phages, resulting in the death of much of the bacterial population. However, the early RM immune response creates substrates for spacer acquisition by the CRISPR-Cas system in a subset of cells. By using these newly acquired spacers which specify the viral sequences for lethal cleavage by Cas9, these cells can now extinguish the methylated phages, resulting in the survival and regrowth of the population. Collectively, my thesis reveals the molecular mechanisms connecting RM and CRISPR-Cas systems in providing a synergistic anti-phage defense. Reminiscent of eukaryotic immunity, I demonstrate that RM systems provide an initial, short-lived innate immune response, which stimulates a secondary, more robust adaptive immune response by CRISPR-Cas. This work highlights an example of cooperation between RM and CRISPR-Cas, which are two of the most common bacterial defense systems. However, prokaryotes have been shown to harbor a multitude of other putative antiphage defense systems, which can often exist together in a single host. I predict that future studies will likely uncover many more fascinating instances of immune interaction among other sets of defense systems

Strategies for the page and strategies for the stage. The interplay of image and language

RESUMEN: Este estudio se centra en ciertos rasgos comunes de las estrategias utilizadas en los libros de emblemas y en los dramas concebidos para su representación en los tablados del Renacimiento inglés. Estos rasgos incluyen tanto la pantomima por señas expresiva como la enigmática, las intervenciones del coro, retablos alegóricos, y los usos de sentencias en el discurso dramático. PALABRAS CLAVES: Shakespeare, pantomima por señas, intervenciones del coro, retablos alegóricos, sentencias. ABSTRACT: The present study dwells on certain common features of the strategies followed by emblem books and by drama composed for the stages of the English Renaissance. These features include the expressive as well as the enigmatic dumb show, choric interventions, allegorical tableaux, and the uses of sententiæ in dramatic discourse. KEYWORDS: Shakespeare; Dumb Show; Choric Interventions; Allegorical Tableaux; Sentential.

LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit

DNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70, RPA32 and RPA14. We have previously shown that the LIM protein Ajuba associates with RPA, and that depletion of Ajuba leads to potent activation of ATR. In this study, we provide evidence that the Ajuba-RPA interaction occurs through direct protein contact with RPA70, and that their association is cell cycle-regulated and is reduced upon DNA replication stress. We propose a model in which Ajuba negatively regulates the ATR pathway by directly interacting with RPA70, thereby preventing inappropriate ATR activation. Our results provide a framework to further our understanding of the mechanism of ATR regulation in human cells in the context of cellular transformation

Disfunción familiar como factor asociado a abuso de alcohol en mayores de 15 años

Objetivo: Identificar si la disfunción familiar es factor de riesgo para abuso de alcohol. Material y Método: Se llevó a cabo un estudio de tipo, analítico, observacional de casos y controles retrospectivo, el cual tuvo una muestra de estudio constituida por 210 personas mayores de 15 años (105 casos y 105 controles) atendidas por Consultorios Externos de psiquiatría del Hospital Regional Docente de Trujillo en el periodo comprendido entre los años 2012 - 2018 , para establecer la relación entre variables se empleó el test Chi cuadrado, las asociaciones fueron significativas cuando p < 0.05 y se calculó el odds ratio (OR) del factor predictor en estudio. Resultados Se encontró que en los pacientes mayores de 15 años con abuso de alcohol el 90% presentaron disfunción familiar y en los que no abusan del consumo de alcohol el 42 % presento disfunción familiar OR = 11,847, IC 95% [5,680 -24.709], p = 0.001, la edad promedio de los casos fue 34,31 ± 11,54 y de los controles 37,30 ± 17,12, (p = 0,139), los que presentaron abuso de alcohol el 90% fueron de sexo masculino frente a los que no abusan del alcohol el 58% fueron de sexo masculino (p = 0,001), 68% proceden de área Urbana y tienen Diagnostico de abuso de alcohol, mientras que 67% proceden de área Urbana y no tienen abuso de alcohol (p = 0.001). Conclusiones: La disfunción familiar y el sexo masculino son factores de riesgo para abuso de alcohol en mayores de 15 años. por tal motivo debe realizarse investigaciones a nivel nacional para poder ampliar el panorama estadístico y poder intervenir adecuadamente a nivel familiar, además es necesario ampliar la búsqueda de más factores que podrían estar relacionados al abuso de alcohol.Objective: Identify if family dysfunction is a risk factor for alcohol abuse. Material and Method: A retrospective, analytical, observational and case-type study was carried out, which included a study sample consisting of 210 people over 15 years of age (105 cases and 105 controls) attended by outpatient psychiatric offices of the Regional Teaching Hospital. of Trujillo in the period between the years 2012 - 2018, to establish the relationship between variables the Chi square test was used, the associations were significant when p <0.05 and the odds ratio (OR) of the predictor factor in the study was calculated. Results: It was found that in patients older than 15 years with alcohol abuse 90% had family dysfunction and in those who do not abuse alcohol consumption 42% presented family dysfunction OR = 11,847, 95% CI [5,680 -24,709], p = 0.001, the average age of the cases was 34.31 ± 11.54 and of the controls 37.30 ± 17.12, (p = 0.139), those who presented abuse of alcohol 90% were male versus male. those who do not abuse alcohol 58% were male (p = 0.001), 68% come from Urban area and have Diagnosis of alcohol abuse, while 67% come from Urban area and do not have alcohol abuse (p = 0.001). Conclusions: Family dysfunction and male sex are risk factors for alcohol abuse in people over 15 years of age. For this reason, research must be carried out at the national level in order to expand the statistical panorama and be able to intervene appropriately at the family level, and it is necessary to broaden the search for more factors that could be related to alcohol abuse.Tesi

PinR mediates the generation of reversible population diversity in Streptococcus zooepidemicus

Opportunistic pathogens must adapt to and survive in a wide range of complex ecosystems. Streptococcus zooepidemicus is an opportunistic pathogen of horses and many other animals, including humans. The assembly of different surface architecture phenotypes from one genotype is likely to be crucial to the successful exploitation of such an opportunistic lifestyle. Construction of a series of mutants revealed that a serine recombinase, PinR, inverts 114 bp of the promoter of SZO_08560, which is bordered by GTAGACTTTA and TAAAGTCTAC inverted repeats. Inversion acts as a switch, controlling the transcription of this sortase-processed protein, which may enhance the attachment of S. zooepidemicus to equine trachea. The genome of a recently sequenced strain of S. zooepidemicus, 2329 (Sz2329), was found to contain a disruptive internal inversion of 7 kb of the FimIV pilus locus, which is bordered by TAGAAA and TTTCTA inverted repeats. This strain lacks pinR and this inversion may have become irreversible following the loss of this recombinase. Active inversion of FimIV was detected in three strains of S. zooepidemicus, 1770 (Sz1770), B260863 (SzB260863) and H050840501 (SzH050840501), all of which encoded pinR. A deletion mutant of Sz1770 that lacked pinR was no longer capable of inverting its internal region of FimIV. The data highlight redundancy in the PinR sequence recognition motif around a short TAGA consensus and suggest that PinR can reversibly influence the wider surface architecture of S. zooepidemicus, providing this organism with a bet-hedging solution to survival in fluctuating environments

AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system

We have implemented a genome annotation system for prokaryotes called AGMIAL. Our approach embodies a number of key principles. First, expert manual annotators are seen as a critical component of the overall system; user interfaces were cyclically refined to satisfy their needs. Second, the overall process should be orchestrated in terms of a global annotation strategy; this facilitates coordination between a team of annotators and automatic data analysis. Third, the annotation strategy should allow progressive and incremental annotation from a time when only a few draft contigs are available, to when a final finished assembly is produced. The overall architecture employed is modular and extensible, being based on the W3 standard Web services framework. Specialized modules interact with two independent core modules that are used to annotate, respectively, genomic and protein sequences. AGMIAL is currently being used by several INRA laboratories to analyze genomes of bacteria relevant to the food-processing industry, and is distributed under an open source license

Work engagement y compromiso laboral de los colaboradores del servicio Front 123 de Konecta BTO S.L., distrito del Callao, 2019

La presenta investigación, la cual lleva como título Work engagement y compromiso laboral de los colaboradores del servicio Front 123 de Konecta BTO S.L., distrito del Callao, 2019, tuvo como objetivo general determinar la relación que existe entre el work engagement y el compromiso laboral de los colaboradores del servicio Front 123 de Konecta BTO S.L., por lo cual el desarrollo del estudio se enfocó en alcanzar el objetivo mencionado, se planteó como hipótesis que, existe relación directa entre work engagement y el compromiso labora. Las teorías bases con las que se desarrolló la investigación fue la Teoría de las necesidades desarrollada por McClelland la cual se consideró para la variable work engagemente y la teoría Tridimensional del compromiso organizacional la cual fue desarrollada por Meyer y Allen. La investigación fue de tipo aplicada bajo un diseño no experimental y transversal ya que el objetivo fue entender y correlacionar las variables de estudios para al término se presenten recomendaciones que aporten soluciones al problema identificado. La muestra estuvo conformada por 320 colaboradores la cual es toda la población, se utilizó la población completa ya que tuvo la facilidad de aplicar los instrumentos a todos los colaboradores. La técnica utilizada fue la encuesta y los instrumentos fueron dos cuestionarios estandarizados los cuales se adaptaron al contexto de la empresa, se realizó la validación con expertos y para la confiabilidad se utilizó el Alfa de Cronbach que tuvo como resultado un nivel de confiabilidad muy alta. El procesamiento de datos se realizó haciendo uso del programa SPSS versión 25, los resultados obtenidos confirmaron la hipótesis planteada, por lo que se concluyó que, entre las variables, así como entre las dimensiones de work engagement con la variable Compromiso laboral, existe una relación alta, lo cual será útil para la toma de decisiones que buscan mejorar el compromiso laboral

Dampak Potensial Perubahan Iklim Terhadap Dinamika Penularan Penyakit DBD Di Kota Mataram

Mataram City is an endemic area of DHF because cases are always found in 3 consecutive years with the number of cases that fluctuated and tended to increase. DHF is related to climate factors because of vector life, pathogen, behavior and the physiology of the human body is influenced by climate. The impact of climate change on the dynamics of dengue transmission in the city of Mataram is very important to study because it can be used as a basis for knowing the pattern of the occurrence of dengue cases related to temperature, humidity, rainfall and wind speed. This study used a retrospective cohort design from BMKG secondary climate data and dengue cases at the Mataram City Health Office in the last 5 years (2013-2017). Data were analyzed based on monthly and annual patterns assuming normal data distribution to be carried out correlation and regression tests with α = 0.05. The results showed that climatic elements such as: humidity, rainfall, and temperature had a strong enough influence on the incidence of DHF, but the wind speed was not related to the incidence of DHF in the city of Mataram. The linear regression equation model found was DBD Case = -439.403 + 5.809 * humidity (R2 = 18.9%) assuming all linear regression was fulfilled.This model can be used to assist in efforts to mitigate dengue transmission through the determination of the timing of implementation of prevention and the provision of infrastructure facilities for the DHF treatment