Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli

Background: The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system. Results: To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a “landing pad” (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %. Conclusions: Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.Fil: Hernández Tamayo, Rogelio. Universidad Nacional Autónoma de México; MéxicoFil: Torres Tejerizo, Gonzalo Arturo. Universidad Nacional Autónoma de México; México. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Brom, Susana. Universidad Nacional Autónoma de México; MéxicoFil: Romero, David. Universidad Nacional Autónoma de México; Méxic

LA SUPERACIÓN DE POSTGRADO EN EL MARCO DE UN PROYECTO INSTITUCIONAL DE CIENCIAS EN BANES

Article describes the procedure for diagnosing the needs for improvement in different segments of the faculty of sciences and the strategy to solve a problem created in 2013: with the arrival of new endowments of sciences laboratory to schools of the territory, is evidence insufficient preparation of teachers to use these new resources. For this reason, was established an institutional science project with teachers in Physics, Biology, Chemistry and Natural Sciences, which helps meet the needs of educations related to the experiment. In coordinated work of the Municipal University Center and the Municipal Department of Education these demands are met and to date have developed nineteen including overcoming course, postgraduate course and training, with a positive impact on teacher performance and student learning.En el artículo se describen el procedimiento para diagnosticar las necesidades de superación en los diferentes segmentos del claustro de ciencias y la estrategia seguida para resolver un problema generado en 2013: con la llegada de nuevas dotaciones de laboratorio para ciencias a las escuelas del territorio, se evidencia la insuficiente preparación de los docentes para usar estos nuevos recursos. Entonces se constituye un proyecto institucional de ciencias con docentes de Física, Biología, Química y Ciencias Naturales, lo cual favorece enfrentar las necesidades de las educaciones relacionadas con el experimento. En trabajo coordinado del Centro Universitario Municipal y la Dirección Municipal de Educación se atienden estas demandas y hasta la fecha se han desarrollado diecinueve cursos; entre capacitaciones, postgrados y entrenamientos, con un impacto positivo en el desempeño de docentes y el aprendizaje de estudiantes

Regularities of the professional improvement process on Community Acquired Pneumonia for Internal Medicine teachers

Introducción: la superación de los profesionales de salud constituye una necesidad por el continuo desarrollo científico técnico.Objetivo: identificar las regularidades del proceso de superación profesional sobre Neumonía Adquirida en la Comunidad (NAC) para especialistas de Medicina Interna profesores en el Hospital Clínico Quirúrgico Docente Dr. León Cuervo Rubio de Pinar del Río en el período de tiempo de junio 2019 a junio 2020.Métodos: se desarrolló una investigación educativa con la asunción del método dialéctico materialista que posibilitó la integración de métodos de los niveles teórico y empírico y método estadístico. El universo conformado por 35 especialistas en Medicina Interna profesores, la muestra fue seleccionada de forma intencional y quedo conformada por 25 especialistas en Medicina interna profesores. La variable estudiada el proceso de superación profesional sobre NACResultados: insuficientes acciones de superación sobre NAC dirigidas a los profesores. Deficiente integración de los contenidos de la NAC en la educación en el trabajo a través del empleo del método clínico. Aún no se organizan acciones de superación con enfoque secuenciado de los objetivos y contenidos de la NAC. Debilidades en la preparación pedagógica de los profesores para incidir en el mejoramiento del desempeño sobre manejo de la NAC. Conclusiones: se constatan limitadas acciones para el perfeccionamiento del manejo de la NAC, el proceso de superación profesional planificado no posibilita la actualización y profundización en los contenidos de la NAC, necesarios para elevar el desempeño   de los docentes que transforme el estado de salud de los pacientes ingresados y mejorar su calidad de vida.  Introduction: the improvement of health professionals is a necessity due to the continuous scientific and technical development. Objective: to identify the regularities of the professional improvement process on Community Acquired Pneumonia (CAP) for specialists of Internal Medicine teachers at the Dr. León Cuervo Rubio Teaching Clinical Surgical Hospital in Pinar del Río, in the period of time from June 2019 to June 2020. Methods: an educational research was developed with the assumption of the dialectical materialist method that made possible the integration of methods of the theoretical and empirical levels and statistical method. The universe consisted of 35 specialists in Internal Medicine teachers, the sample was selected intentionally and consisted of 25 specialists in Internal Medicine teachers. The variable studied was the professional training process on NACResults: insufficient training actions on NAC directed to teachers. Deficient integration of NAC contents in on-the-job education through the use of the clinical method. Improvement actions with a sequential approach to the objectives and contents of NAC have not yet been organized. Weaknesses in the pedagogical preparation of teachers to have an impact on the improvement of NAC management performance.Conclusions: The planned professional development process does not allow the updating and deepening of the contents of CAP, which are necessary to improve the performance of teachers in order to transform the health status of admitted patients and improve their quality of life.

Resistance to immune checkpoint inhibitors secondary to myeloid-derived suppressor cells: A new therapeutic targeting of haematological malignancies

Producción CientíficaMyeloid-derived suppressor cells (MDSCs) are a set of immature myeloid lineage cells that include macrophages, granulocytes, and dendritic cell precursors. This subpopulation has been described in relation to the tumour processes at different levels, including resistance to immunotherapy, such as immune checkpoint inhibitors (ICIs). Currently, multiple studies at the preclinical and clinical levels seek to use this cell population for the treatment of different haematological neoplasms, together with ICIs. This review addresses the different points in ongoing studies of MDSCs and ICIs in haematological malignancies and their future significance in routine clinical practice

Regularities of professional development on Community Acquired Pneumonia for teachers of Internal Medicine

Introduction: the improvement of health professionals is a necessity due to the continuous scientific and technical development. Objective: to identify the regularities of the professional training process on Community Acquired Pneumonia for Internal Medicine teachers at León Cuervo Rubio Hospital in Pinar del Río. Method: educational research was developed based on the dialectical-materialistic method, integrating qualitative and quantitative aspects, using theoretical and empirical methods such as the historical-logical, systemic-structural, documentary review, surveys, interview and observation, respectively. In addition, descriptive and inferential statistical methods, the sample used was 25 intentionally selected specialists in Internal Medicine teachers. The variable studied was the professional improvement process on Community Acquired Pneumonia. Results: Insufficient training actions on CAP directed to teachers. Deficient integration of CAP contents in on-the-job education through the use of the clinical method. Training actions with a sequential approach to the objectives and contents of NAC have not yet been organized. Weaknesses in the pedagogical preparation of teachers to have an impact on the improvement of performance on management of Community Acquired Pneumonia. Conclusions: There are limited actions for the improvement of the management of CAP, the planned professional development process does not make possible the updating and deepening of the contents of CAP, necessary to improve the performance of teachers in order to transform the health status of patients admitted and improve their quality of life

Bacillus subtilis RarA acts as a positive RecA accessory protein

Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly

The RecD2 helicase balances RecA activities

DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA–ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth

Analysis of autophagy gene polymorphisms in Spanish patients with head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth cancer on incidence worldwide. Tobacco and alcohol consumption are the most classical risk factors associated with its development. Autophagy process has a dual effect both in tumourigenesis and tumour suppressing activity. To investigate the importance of this pathway in HNSCC susceptibility, a risk factor matched case-control association study was performed with four candidate polymorphisms in autophagy genes (ATG2B, ATG5, ATG10, ATG16L1). We found an association between the variant in ATG10 rs1864183 and a higher susceptibility to develop laryngeal cancer, ATG2B rs3759601 and pharyngeal cancer and ATG16L1 rs2241880 and oral carcinoma. ATG5 rs2245214 SNP was not associated with any location. Overall, our results indicate the importance of the autophagy pathway in the susceptibility of head and neck squamous cell carcinoma and demonstrate the heterogeneity between its locations encompassed under a single terminology

Genetic Susceptibility in Head and Neck Squamous Cell Carcinoma in a Spanish Population

Despite classical environmental risk factors like tobacco, alcohol or viral infection, not all individuals develop head and neck cancer. Therefore, identification of the genetic susceptibility produced by single nucleotide polymorphisms (SNPs) is an important task. A total of 296 human papillomavirus negative head and neck cancer (HNC) patients (126 laryngeal, 100 pharyngeal and 70 oral cavity) were included in the study, involving 29 candidate SNPs in genes within important carcinogenic pathways (oncogenesis and tumour suppression, DNA repair, inflammation, oxidation and apoptosis). Genotyping was performed using TaqMan probes or restriction fragment length assays in peripheral blood DNA. In addition, 259 paired controls were also evaluated with the same risk factors for each specific location. Nine SNPs in DNA repair (ERCC1 rs11615, ERCC2 rs13181), inflammatory (IL2 rs2069762, IL6 rs1800795), oxidative (NFE2L2 rs13035806 and rs2706110) and apoptotic genes (TP53 rs1042522, MDM2 rs2279744, BCL2 rs2279115) were differently associated with HNSCC susceptibility by location. Some of these SNPs were not described before in this tumour type. In conclusion, we describe several SNPs associated with HNC in a Spanish population

Oncogenic driver mutations predict outcome in a cohort of head and neck squamous cell carcinoma (HNSCC) patients within a clinical trial

234 diagnostic formalin-fixed paraffin-embedded (FFPE) blocks from homogeneously treated patients with locally advanced head and neck squamous cell carcinoma (HNSCC) within a multicentre phase III clinical trial were characterised. The mutational spectrum was examined by next generation sequencing in the 26 most frequent oncogenic drivers in cancer and correlated with treatment response and survival. Human papillomavirus (HPV) status was measured by p16INK4a immunohistochemistry in oropharyngeal tumours. Clinicopathological features and response to treatment were measured and compared with the sequencing results. The results indicated TP53 as the most mutated gene in locally advanced HNSCC. HPV-positive oropharyngeal tumours were less mutated than HPV-negative tumours in TP53 (p<0.01). Mutational and HPV status influences patient survival, being mutated or HPV-negative tumours associated with poor overall survival (p<0.05). No association was found between mutations and clinicopathological features. This study confirmed and expanded previously published genomic characterization data in HNSCC. Survival analysis showed that non-mutated HNSCC tumours associated with better prognosis and lack of mutations can be identified as an important biomarker in HNSCC. Frequent alterations in PI3K pathway in HPV-positive HNSCC could define a promising pathway for pharmacological intervention in this group of tumours