The PEN CSR, using external molecular programs to control directed evolution of enzymes

Selection-based strategies used in directed evolution methods proved to be very efficient but rely on the possibility of finding a case-specific selection tool that links the enzyme activity to its gene survival. Among all available directed evolution methods, the Compartmentalized Self-Replication (CSR) of Ghadessy and al. (1) and its derivatives allow to perform high-throughput in vitro selection tests thanks to microdroplets compartmentalization. Because engineered enzymes are often intended for use in unnatural environments, this kind of methods are particularly interesting. Yet, they can only be applied to polymerases replicating their own genetic sequence in the droplets. Here, we used an external DNA-based artificial network to create a feedback loop linking the activity of a nicking enzyme to the replication of its own gene. Molecular networks, such as the one using the PEN DNA toolbox (2), are designed to produce short DNA strands interacting within each other’s thanks to a set of enzymes. Taking short oligonucleotides at the input, they can generate short oligonucleotides of arbitrary sequence at the output. Enzyme activity can be assessed by these molecular networks thus allowing to produce a correlated amount of primers at the output. These primers can then be used to run the PCR of the enzyme’s gene. Compared to the Compartmentalized Partnered Replication (CPR) (3), our method keeps the ability to perform in vitro assays of the target enzyme and does not necessitate any gene circuitry optimization. Practically, bacteria carrying and expressing the mutants are co-encapsulated with the molecular program in individual droplets using microfluidics. The isothermal primers amplification (IPA) by the network is next initiated by raising the temperature to 45°C. After running the program for some time, each droplet contains an amount of the enzyme’s gene forward and reverse primers corresponding to the activity of the enzyme expressed by the bacterium. A PCR is then launched in the droplets. Its yield in each droplet will then depend on the amount of primers, therefore on the enzyme activity. Up to 107 parallel self-selections can be run simultaneously on individual copy of the gene. After emulsion breakage, we retrieve a library enriched in the best mutant genes that can be used for a new cycle. Two different selection pressures were applied by playing either on the IPA duration time to select for faster enzymes or by adding a heat shock at 65°C to select for higher thermostability. Next generation sequencing using MinION allowed us to follow the evolution process. This work is the first demonstration of the Programmable External Network based CSR (PEN CSR) method. Programs detecting other types of activity can be envisioned and would allow not only to greatly expand the scope of the CSR but also to implement smart selection functions. (1) F. J. Ghadessy and al. Proc. Natl. Acad. Sci. U.S.A., vol. 98, Apr. 2001. (2) K. Montagne and al. Mol. Syst Biol., vol. 7, Feb. 2011. (3) J.W. Ellefson and al. Nat. Biotechnol., vol. 32, Jan. 2014

La tutoría y su relación con el nivel de autoestima de los estudiantes del 6TO grado de primaria de la institución educativa N°20334 “Don José de San Martín” del distrito de Huaura, 2013

El propósito de este estudio fue determinar la relación que existe entre La tutoria y El nível de autoestima de los Estudiantes Del 6° grado de primaria de la Institución Educativa N° 20334 Don José de San Martín del distrito de Huaura en el período escolar 2013. El diseño de la investigación utilizado fue descriptivo-correlacional. La muestra estuvo constituida por 42 sujetos a quienes se le aplico un cuestionario para evaluar la labor de la tutoría en el aula y la aplicación de un inventario de autoestima para medir la variable dependiente. Los hallazgos indicaron que existe relación significativa entre la tutoría y la autoestima (p = 0.000 = 0,05). Así también existe relación entre los acompañamientos socioafectivo y cognitivo con el nivel de autoestima pues el estadístico Chi cuadrado devolvió un valor de p= 0.001, por lo que se puede afirmar contundentemente que la relación entre el acompañamiento pedagógico y la autoestima es baja así lo demuestra la correlación de Pearson con un valor de p= 0,389

Size and structure of the sequence space of repeat proteins

The coding space of protein sequences is shaped by evolutionary constraints set by requirements of function and stability. We show that the coding space of a given protein family-the total number of sequences in that family-can be estimated using models of maximum entropy trained on multiple sequence alignments of naturally occuring amino acid sequences. We analyzed and calculated the size of three abundant repeat proteins families, whose members are large proteins made of many repetitions of conserved portions of *30 amino acids. While amino acid conservation at each position of the alignment explains most of the reduction of diversity relative to completely random sequences, we found that correlations between amino acid usage at different positions significantly impact that diversity. We quantified the impact of different types of correlations, functional and evolutionary, on sequence diversity. Analysis of the detailed structure of the coding space of the families revealed a rugged landscape, with many local energy minima of varying sizes with a hierarchical structure, reminiscent of fustrated energy landscapes of spin glass in physics. This clustered structure indicates a multiplicity of subtypes within each family, and suggests new strategies for protein design.Fil: Marchi, Jacopo. Ecole Normale Supérieure; FranciaFil: Galpern, Ezequiel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Espada, Rocio. PSL University; FranciaFil: Ferreiro, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Walczak, Aleksandra M.. Ecole Normale Supérieure; FranciaFil: Mora, Thierry. Ecole Normale Supérieure; Franci

Advances in the management and translocation methodology of the endangered mollusk Patella ferruginea in artificial habitats of port infrastructures: implications for its conservation

The limpet (Patella ferruginea), endemic to the western Mediterranean, is the most endangered intertidal mollusk listed in the Habitats Directive (EU). The translocation methodology of this species is a scientific challenge faced by those port infrastructures – where there are established breeding subpopulations - that may be subject to emergency works, restoration, or extension of breakwaters. This study presents the results of an innovative two-phase pilot project (P1A/B and P2), involving the legally authorized translocation of individuals of this species obtained in three relocation experiments in the Marina of Puerto José Banús (Málaga) in July 2021 (phase 1: P1A and B) and May 2022 (phase 2: P2) (study period: July 2021-October 2022). The specimens translocated in both phases were from the same port except for those of P1B, which came from the Marbella Marina as an exceptional case. These translocations were undertaken on concrete breakwater tetrapod units. We show that the somewhat forced, slow and unregulated extraction of specimens to be translocated during the first experiment of the P1A pilot project was the main cause of mortality, as it generated an important exhaustion of the fixation musculature, which resulted in a low survival (66%) after the first month of transfer, whereas during the same period of time. In the subsequent experiments (where the problem was methodologically corrected, a fast extraction technique -less than 5 seconds-, regulated and without injuries), in P1B was 100%, in P2 with cage was 93.3%, and, in P2 without cage it was 83.3%, which reflected an improvement in translocation methodology. The results of this research are compared according to the descriptors of success and failure of each protocol in order to observe the differences and thus enable their replicability

First Latin American clinical practice guidelines for the treatment of systemic lupus erythematosus: Latin American Group for the Study of Lupus (GLADEL, Grupo Latino Americano de Estudio del Lupus)-Pan-American League of Associations of Rheumatology (PANLAR)

Systemic lupus erythematosus (SLE), a complex and heterogeneous autoimmune disease, represents a significant challenge for both diagnosis and treatment. Patients with SLE in Latin America face special problems that should be considered when therapeutic guidelines are developed. The objective of the study is to develop clinical practice guidelines for Latin American patients with lupus. Two independent teams (rheumatologists with experience in lupus management and methodologists) had an initial meeting in Panama City, Panama, in April 2016. They selected a list of questions for the clinical problems most commonly seen in Latin American patients with SLE. These were addressed with the best available evidence and summarised in a standardised format following the Grading of Recommendations Assessment, Development and Evaluation approach. All preliminary findings were discussed in a second face-to-face meeting in Washington, DC, in November 2016. As a result, nine organ/system sections are presented with the main findings; an 'overarching' treatment approach was added. Special emphasis was made on regional implementation issues. Best pharmacologic options were examined for musculoskeletal, mucocutaneous, kidney, cardiac, pulmonary, neuropsychiatric, haematological manifestations and the antiphospholipid syndrome. The roles of main therapeutic options (ie, glucocorticoids, antimalarials, immunosuppressant agents, therapeutic plasma exchange, belimumab, rituximab, abatacept, low-dose aspirin and anticoagulants) were summarised in each section. In all cases, benefits and harms, certainty of the evidence, values and preferences, feasibility, acceptability and equity issues were considered to produce a recommendation with special focus on ethnic and socioeconomic aspects. Guidelines for Latin American patients with lupus have been developed and could be used in similar settings.Fil: Pons Estel, Bernardo A.. Centro Regional de Enfermedades Autoinmunes y Reumáticas; ArgentinaFil: Bonfa, Eloisa. Universidade de Sao Paulo; BrasilFil: Soriano, Enrique R.. Instituto Universitario Hospital Italiano de Buenos Aires. Rectorado.; ArgentinaFil: Cardiel, Mario H.. Centro de Investigación Clínica de Morelia; MéxicoFil: Izcovich, Ariel. Hospital Alemán; ArgentinaFil: Popoff, Federico. Hospital Aleman; ArgentinaFil: Criniti, Juan M.. Hospital Alemán; ArgentinaFil: Vásquez, Gloria. Universidad de Antioquia; ColombiaFil: Massardo, Loreto. Universidad San Sebastián; ChileFil: Duarte, Margarita. Hospital de Clínicas; ParaguayFil: Barile Fabris, Leonor A.. Hospital Angeles del Pedregal; MéxicoFil: García, Mercedes A.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Amigo, Mary Carmen. Centro Médico Abc; MéxicoFil: Espada, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Catoggio, Luis J.. Hospital Italiano. Instituto Universitario. Escuela de Medicina; ArgentinaFil: Sato, Emilia Inoue. Universidade Federal de Sao Paulo; BrasilFil: Levy, Roger A.. Universidade do Estado de Rio do Janeiro; BrasilFil: Acevedo Vásquez, Eduardo M.. Universidad Nacional Mayor de San Marcos; PerúFil: Chacón Díaz, Rosa. Policlínica Méndez Gimón; VenezuelaFil: Galarza Maldonado, Claudio M.. Corporación Médica Monte Sinaí; EcuadorFil: Iglesias Gamarra, Antonio J.. Universidad Nacional de Colombia; ColombiaFil: Molina, José Fernando. Centro Integral de Reumatología; ColombiaFil: Neira, Oscar. Universidad de Chile; ChileFil: Silva, Clóvis A.. Universidade de Sao Paulo; BrasilFil: Vargas Peña, Andrea. Hospital Pasteur Montevideo; UruguayFil: Gómez Puerta, José A.. Hospital Clinic Barcelona; EspañaFil: Scolnik, Marina. Instituto Universitario Hospital Italiano de Buenos Aires. Rectorado.; ArgentinaFil: Pons Estel, Guillermo J.. Centro Regional de Enfermedades Autoinmunes y Reumáticas; Argentina. Hospital Provincial de Rosario; ArgentinaFil: Ugolini Lopes, Michelle R.. Universidade de Sao Paulo; BrasilFil: Savio, Verónica. Instituto Universitario Hospital Italiano de Buenos Aires. Rectorado.; ArgentinaFil: Drenkard, Cristina. University of Emory; Estados UnidosFil: Alvarellos, Alejandro J.. Hospital Privado Universitario de Córdoba; ArgentinaFil: Ugarte Gil, Manuel F.. Universidad Cientifica del Sur; Perú. Hospital Nacional Guillermo Almenara Irigoyen; PerúFil: Babini, Alejandra. Instituto Universitario Hospital Italiano de Buenos Aires. Rectorado.; ArgentinaFil: Cavalcanti, André. Universidade Federal de Pernambuco; BrasilFil: Cardoso Linhares, Fernanda Athayde. Hospital Pasteur Montevideo; UruguayFil: Haye Salinas, Maria Jezabel. Hospital Privado Universitario de Córdoba; ArgentinaFil: Fuentes Silva, Yurilis J.. Universidad de Oriente - Núcleo Bolívar; VenezuelaFil: Montandon De Oliveira E Silva, Ana Carolina. Universidade Federal de Goiás; BrasilFil: Eraso Garnica, Ruth M.. Universidad de Antioquia; ColombiaFil: Herrera Uribe, Sebastián. Hospital General de Medellin Luz Castro de Gutiérrez; ColombiaFil: Gómez Martín, DIana. Instituto Nacional de la Nutrición Salvador Zubiran; MéxicoFil: Robaina Sevrini, Ricardo. Universidad de la República; UruguayFil: Quintana, Rosana M.. Hospital Provincial de Rosario; Argentina. Centro Regional de Enfermedades Autoinmunes y Reumáticas; ArgentinaFil: Gordon, Sergio. Hospital Interzonal General de Agudos Dr Oscar Alende. Unidad de Reumatología y Enfermedades Autoinmunes Sistémicas; ArgentinaFil: Fragoso Loyo, Hilda. Instituto Nacional de la Nutrición Salvador Zubiran; MéxicoFil: Rosario, Violeta. Hospital Docente Padre Billini; República DominicanaFil: Saurit, Verónica. Hospital Privado Universitario de Córdoba; ArgentinaFil: Appenzeller, Simone. Universidade Estadual de Campinas; BrasilFil: Dos Reis Neto, Edgard Torres. Universidade Federal de Sao Paulo; BrasilFil: Cieza, Jorge. Hospital Nacional Edgardo Rebagliati Martins; PerúFil: González Naranjo, Luis A.. Universidad de Antioquia; ColombiaFil: González Bello, Yelitza C.. Ceibac; MéxicoFil: Collado, María Victoria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Sarano, Judith. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Retamozo, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Sattler, María E.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Gamboa Cárdenas, Rocio V.. Hospital Nacional Guillermo Almenara Irigoyen; PerúFil: Cairoli, Ernesto. Universidad de la República; UruguayFil: Conti, Silvana M.. Hospital Provincial de Rosario; ArgentinaFil: Amezcua Guerra, Luis M.. Instituto Nacional de Cardiologia Ignacio Chavez; MéxicoFil: Silveira, Luis H.. Instituto Nacional de Cardiologia Ignacio Chavez; MéxicoFil: Borba, Eduardo F.. Universidade de Sao Paulo; BrasilFil: Pera, Mariana A.. Hospital Interzonal General de Agudos General San Martín; ArgentinaFil: Alba Moreyra, Paula B.. Universidad Nacional de Córdoba. Facultad de Medicina; ArgentinaFil: Arturi, Valeria. Hospital Interzonal General de Agudos General San Martín; ArgentinaFil: Berbotto, Guillermo A.. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Gerling, Cristian. Hospital Interzonal General de Agudos Dr Oscar Alende. Unidad de Reumatología y Enfermedades Autoinmunes Sistémicas; ArgentinaFil: Gobbi, Carla Andrea. Universidad Nacional de Córdoba. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gervasoni, Viviana L.. Hospital Provincial de Rosario; ArgentinaFil: Scherbarth, Hugo R.. Hospital Interzonal General de Agudos Dr Oscar Alende. Unidad de Reumatología y Enfermedades Autoinmunes Sistémicas; ArgentinaFil: Brenol, João C. Tavares. Hospital de Clinicas de Porto Alegre; BrasilFil: Cavalcanti, Fernando. Universidade Federal de Pernambuco; BrasilFil: Costallat, Lilian T. Lavras. Universidade Estadual de Campinas; BrasilFil: Da Silva, Nilzio A.. Universidade Federal de Goiás; BrasilFil: Monticielo, Odirlei A.. Hospital de Clinicas de Porto Alegre; BrasilFil: Seguro, Luciana Parente Costa. Universidade de Sao Paulo; BrasilFil: Xavier, Ricardo M.. Hospital de Clinicas de Porto Alegre; BrasilFil: Llanos, Carolina. Universidad Católica de Chile; ChileFil: Montúfar Guardado, Rubén A.. Instituto Salvadoreño de la Seguridad Social; El SalvadorFil: Garcia De La Torre, Ignacio. Hospital General de Occidente; MéxicoFil: Pineda, Carlos. Instituto Nacional de Rehabilitación; MéxicoFil: Portela Hernández, Margarita. Umae Hospital de Especialidades Centro Medico Nacional Siglo Xxi; MéxicoFil: Danza, Alvaro. Hospital Pasteur Montevideo; UruguayFil: Guibert Toledano, Marlene. Medical-surgical Research Center; CubaFil: Reyes, Gil Llerena. Medical-surgical Research Center; CubaFil: Acosta Colman, Maria Isabel. Hospital de Clínicas; ParaguayFil: Aquino, Alicia M.. Hospital de Clínicas; ParaguayFil: Mora Trujillo, Claudia S.. Hospital Nacional Edgardo Rebagliati Martins; PerúFil: Muñoz Louis, Roberto. Hospital Docente Padre Billini; República DominicanaFil: García Valladares, Ignacio. Centro de Estudios de Investigación Básica y Clínica; MéxicoFil: Orozco, María Celeste. Instituto de Rehabilitación Psicofísica; ArgentinaFil: Burgos, Paula I.. Pontificia Universidad Católica de Chile; ChileFil: Betancur, Graciela V.. Instituto de Rehabilitación Psicofísica; ArgentinaFil: Alarcón, Graciela S.. Universidad Peruana Cayetano Heredia; Perú. University of Alabama at Birmingahm; Estados Unido

American College of Rheumatology Provisional Criteria for Clinically Relevant Improvement in Children and Adolescents With Childhood-Onset Systemic Lupus Erythematosus

10.1002/acr.23834ARTHRITIS CARE & RESEARCH715579-59

Table_1_Advances in the management and translocation methodology of the endangered mollusk Patella ferruginea in artificial habitats of port infrastructures: implications for its conservation.docx

The limpet (Patella ferruginea), endemic to the western Mediterranean, is the most endangered intertidal mollusk listed in the Habitats Directive (EU). The translocation methodology of this species is a scientific challenge faced by those port infrastructures – where there are established breeding subpopulations - that may be subject to emergency works, restoration, or extension of breakwaters. This study presents the results of an innovative two-phase pilot project (P1A/B and P2), involving the legally authorized translocation of individuals of this species obtained in three relocation experiments in the Marina of Puerto José Banús (Málaga) in July 2021 (phase 1: P1A and B) and May 2022 (phase 2: P2) (study period: July 2021-October 2022). The specimens translocated in both phases were from the same port except for those of P1B, which came from the Marbella Marina as an exceptional case. These translocations were undertaken on concrete breakwater tetrapod units. We show that the somewhat forced, slow and unregulated extraction of specimens to be translocated during the first experiment of the P1A pilot project was the main cause of mortality, as it generated an important exhaustion of the fixation musculature, which resulted in a low survival (66%) after the first month of transfer, whereas during the same period of time. In the subsequent experiments (where the problem was methodologically corrected, a fast extraction technique -less than 5 seconds-, regulated and without injuries), in P1B was 100%, in P2 with cage was 93.3%, and, in P2 without cage it was 83.3%, which reflected an improvement in translocation methodology. The results of this research are compared according to the descriptors of success and failure of each protocol in order to observe the differences and thus enable their replicability.</p

Hemodialysis-Associated Immune Dysregulation in SARS-CoV-2-Infected End-Stage Renal Disease Patients

Patients on hemodialysis show dysregulated immunity, basal hyperinflammation and a marked vulnerability to COVID-19. We evaluated the immune profile in COVID-19 hemodialysis patients and the changes associated with clinical deterioration after the hemodialysis session. Recruited patients included eight hemodialysis subjects with active, PCR-confirmed SARS-CoV-2 infection, five uninfected hemodialysis patients and five healthy controls. In SARS-CoV-2-infected hemodialysis patients TNF-α, IL-6 and IL-8 were particularly increased. Lymphopenia was mostly due to reduction in CD4+ T, B and central memory CD8+ T cells. There was a predominance of classical and intermediate monocytes with reduced HLA-DR expression and enhanced production of pro-inflammatory molecules. Immune parameters were analysed pre- and post-hemodialysis in three patients with COVID-19 symptoms worsening after the hemodialysis session. There was a higher than 2.5-fold increase in GM-CSF, IFN-γ, IL-1β, IL-2, IL-6, IL-17A and IL-21 in serum, and augmentation of monocytes-derived TNF-α, IL-1β and IL-8 and CXCL10 (p < 0.05). In conclusion, COVID-19 in hemodialysis patients associates with alteration of lymphocyte subsets, increasing of pro-inflammatory cytokines and monocyte activation. The observed worsening during the hemodialysis session in some patients was accompanied by augmentation of particular inflammatory cytokines, which might suggest biomarkers and therapeutic targets to prevent or mitigate the hemodialysis-related deterioration during SARS-CoV-2 infection

TIC as a information tool of scientific methodology

Diseño, elaboración y procesado de vídeos en los que se incluye la metodología empleada en una investigación avanzada.Design, development and processing of videos which includes the methodology used in advanced research.Depto. de Química InorgánicaFac. de Ciencias QuímicasFALSEsubmitte