Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.Spanish ISCIII Health Research FundEuropean Union (EU) PI15/02015 PI18/00337 PI18/00330CECEyU of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) 2016000073391-TRA 2016000073332-TRA PI-57069 CARTPI-0001-201 PAIDI-Bio326 PI-0014-2016CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) 2016000073391-TRA 2016000073332-TRA PI-57069 CARTPI-0001-201 PAIDI-Bio326 PI-0014-2016Nicolas Monardes regional Ministry of Health contract 0006/2018Fundacion Poco FrecuenteSpanish Government FPU17/04327 FPU17/02268ISCIII through a PFIS fellowship FI19/00163SMSI PEJ-2018-001760-

Genome-edited adult stem cells: Next-generation advanced therapy medicinal products

Over recent decades, gene therapy, which has enabled the treatment of several incurable diseases, has undergone a veritable revolution. Cell therapy has also seen major advances in the treatment of various diseases, particularly through the use of adult stem cells (ASCs). The combination of gene and cell therapy (GCT) has opened up new opportunities to improve advanced therapy medicinal products for the treatment of several diseases. Despite the considerable potential of GCT, the use of retroviral vectors has major limitations with regard to oncogene transactivation and the lack of physiological expression. Recently, gene therapists have focused on genome editing (GE) technologies as an alternative strategy. In this review, we discuss the potential benefits of using GE technologies to improve GCT approaches based on ASCs. We will begin with a brief summary of different GE platforms and techniques and will then focus on key therapeutic approaches that have been successfully used to treat diseases in animal models. Finally, we discuss whether ASC GE could become a real alternative to retroviral vectors in a GCT setting.European Regional Development Fund (FEDER), Grant/Award Numbers: PI18/01610, PI18/00330, PI18/00337, grants PI12/01097; Spanish ISCIII Health Research Fun

Efeito da nutrição enteral precoce na mortalidade em doentes críticos com COVID-19

Background: Early enteral nutrition (EEN) has shown favorable clinical outcomes, such as lower risk of death, fewer frequency of infection and lower healthcare costs. Different societies recommend the provision of enteral nutrition within the first 24 to 48 hours of admission to the Intensive Care Unit in patients with COVID-19.  Methods: Retrospective cohort study including adult patients with severe COVID-19 and orotracheal intubation. Demographic and clinical characteristics, as well as use of drugs with nutritional relevance, such as vasopressors and steroids, as well as biochemical results (serum electrolytes) were registered. EEN was defined as the provision of enteral feeding within the first 24-48 hours of invasive mechanical ventilation (IMV). The primary outcome was in-hospital all-cause mortality.  Results: Overall, 404 patients were included in the study. EEN was achieved in 74% of all patients. EEN significantly reduced mortality in the bivariate model (RR 0.88, 95% CI 0.80 - 0.95) and in the multivariate model (adjusted OR 0.42, 95% CI 0.19 – 0.90). No differences in hospital length of stay and days on IMV in survivors were found.  Conclusions: EEN was associated with a lower risk of death in critically ill patients with COVID-19. Additional studies are necessary to further clarify the effects of early enteral feeding on patient outcomes. Antecedentes: la nutrición enteral temprana ha demostrado resultados clínicos favorables, tales como menor riesgo de mortalidad, menor frecuencia de infecciones y menores costos en salud. Diferentes sociedades recomiendan la provisión de nutrición enteral dentro de las primeras 24 a 48 horas del ingreso a la unidad de cuidados intensivos (UCI) en pacientes con enfermedad por coronavirus de 2019 (COVID-19).  Métodos: estudio de cohorte retrospectiva en pacientes adultos con COVID-19 grave e intubación orotraqueal. Se registraron las características demográficas y clínicas, así como el uso de fármacos con relevancia nutricional, como vasopresores y corticoides, y los resultados bioquímicos (electrolitos séricos). La nutrición enteral temprana se definió como la provisión de alimentación enteral en las primeras 24-48 horas de ventilación mecánica invasiva. El resultado primario fue la mortalidad hospitalaria por todas las causas.  Resultados: se incluyeron en el análisis a 404 pacientes. El 74 % de los casos recibió nutrición enteral temprana. La nutrición enteral temprana se asoció con una reducción estadísticamente significativa en la mortalidad por todas las causas en el modelo bivariado (riesgo relativo [RR]: 0,88; intervalo de confianza [IC] del 95 %: 0,80 a 0,95) y en el modelo multivariado ajustado (odds ratio [OR] ajustado: 0,42; IC 95 %: 0,19 a 0,90). No se encontraron diferencias significativas en la duración de la estancia hospitalaria ni en los días de VMI en los supervivientes.  Conclusiones: la nutrición enteral temprana se asocia con menor mortalidad por todas las causas en pacientes críticos con COVID-19. Son necesarios estudios adicionales para aclarar los efectos de la nutrición enteral temprana en los resultados de los pacientes. Introdução: a nutrição enteral precoce (NEP) tem mostrado resultados clínicos favoráveis, como menor risco de morte, menor frequência de infecção e menores custos de saúde. Diferentes sociedades recomendam o fornecimento de nutrição enteral nas primeiras 24 a 48 horas após a admissão na Unidade de Terapia Intensiva em pacientes com COVID-19.  Métodos: estudo de coorte retrospectivo incluindo pacientes adultos com COVID-19 grave e intubação orotraqueal. Foram registradas as características demográficas e clínicas, bem como o uso de medicamentos com relevância nutricional, como vasopressores e esteróides, e os resultados bioquímicos (eletrólitos séricos). A NEP foi definida como o fornecimento de alimentação enteral nas primeiras 24-48 horas de ventilação mecânica invasiva (VMI). O desfecho primário foi mortalidade intra-hospitalar por todas as causas.  Resultados: quatrocentos e quatro pacientes foram incluídos no estudo. A NEP foi alcançada em 74% de todos os pacientes. A NEP reduziu significativamente a mortalidade no modelo bivariado (RR 0,88, 95% IC 0,80 a 0,95) e no modelo multivariado (OR ajustado 0,42, 95% IC 0,19 – 0,90). Não foram encontradas diferenças no tempo de internação e nos dias de VMI nos sobreviventes. Conclusões: a NEP foi associada a menor risco de morte em pacientes gravemente doentes com COVID-19. Estudos adicionais são necessários para esclarecer melhor os efeitos da alimentação enteral precoce nos resultados dos pacientes.

Desarrollo de una metodología voltamperométrica para la determinación de Aflatoxina B1 usando un electrodo de carbón vítreo modificado con una película de bismuto y nanopartículas de oro

La Aflatoxina B1 es una micotoxina altamente cancerígena que se encuentra en una gran variedad de alimentos y piensos, por lo tanto, su cuantificación es importante para la industria de los alimentos. En este trabajo de investigación se describe el desarrollo de una metodología electroanalítica utilizando un electrodo de carbón vítreo modificado superficialmente con una película de bismuto y nanopartículas de oro para la cuantificación de Aflatoxina B1, por las ventajas que presenta frente a otras metodologías que ya se han estudiado y validado para la cuantificación de esta sustancia. Los límites de detección y de cuantificación que se obtuvieron después de la de la optimización de la Voltamperometría de Onda Cuadrada mediante un diseño Box-Behnken fueron 11.19 y 37.31 ng L-¹, respectivamente. Estos parámetros nos indican que es posible cuantificar la aflatoxina B1 en un intervalo de concentraciones a nivel traza, tal como se encuentra en alimentos.feeds, therefore, its quantification is important for the food industry. This research paper describes the development of an electroanalytical methodology using a glassy carbon electrode modified with a bismuth film and gold nanoparticles for quantification of Aflatoxin B1, due to the advantages it presents over other methodologies that have already been studied and validated for the quantification of this substance. The detection and quantification limits obtained after optimization of Square Wave Voltammetry using a Box-Behnken design were 11.19 and 37.31 ng L-¹, respectively. These parameters indicated that it is possible to quantify the Aflatoxin B1 in a wide concentration range at trace levels, as expected in food

Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies

Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation.Fundacion Poco Frecuente (Almeria)Asociacion Espanola de Enfermos de Glucogenosis (AEEG)Asociacion Espanola de Enfermos de Pompe (AEEP

Physiological lentiviral vectors for the generation of improved CAR-T cells

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms.However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50%of the treated patients.MostCAR-Tcells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of theirmoderate andTCR-like expression profile. Compared with CAR-T cells generated with human elongation factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo.Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing ofAW-CAR-T cells in good manufacturing practice (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.Spanish ISCIII Health Research FundEuropean Commission PI15/02015 PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA 2016000073332-TRA PI-57069 PA IDI-Bio326 CARTPI-0001-201 PECART-0031-2020 Red RANTECAR CAR-T 2019 00400200101918 PLEC2021-008094 PI-0014-2016 PEER-0286-2019Spanish Government PLEC2021-008094 00123009/SNEO-20191072Nicolas Monardes contracts from regional Ministry of Health 0006/2018 C2-0002-2019German Research Foundation (DFG) FPU16/05467 FPU17/02268 FPU17/04327 MCI DIN2018-010180Fundacion Andaluza Progreso y SaludGerman Research Foundation (DFG) PEJ-2018-001760-AJunta de Andalucia PE-0223-2018Biomedicine Programme of the University of Granada (Spain

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 30 long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivatorfree, all-one-one system capable of generating Dox-inducible ATMPs.Spanish ISCIII Health Research FundEuropean Union (EU) PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672Red TerAvJunta de Andalucia FEDER/European Cohesion Fund (FSE) for AndalusiaSpanish Government PI18/00337 PI21/00298European Union-NextGenerationEU - Maria Zambrano Senior Program RD21/0017/0004 PI18/00330 PI17/00672Ministry of Health 2016000073332-TRA PI-57069 CARTPI-0001-201 PE-CART-0031-2020 PI-0014-2016 PECART-0027-2020 ProyExcel_00875 PEER-0286-2019European Cooperation in Science and Technology (COST) 00123009/SNEO-20191072MINECO - European Regional Development Fund PLEC2021-008094Spanish Government 0006/2018FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades CA21113Spanish Government SAF2015-71589-PMCI RYC-2016-21395German Research Foundation (DFG) PY20_00619 y A-CTS-28_UGR20Biomedicine Program of the University of Granada (Spain) FPU16/05467 FPU17/02268 FPU17/04327 DIN2018-010180 DIN2020-011550 PEJ-2018-001760-

Correlation between clinical parameters characterising peri-implant and periodontal health : a practice-based research in Spain in a series of patients with implants installed 4-5 years ago

Objectives: To explore peri-implant health (and relation with periodontal status) 4-5 years after implant insertion. Study D esign: A practice-based dental research network multicentre study was performed in 11 Spanish centres. The first patient/month with implant insertion in 2004 was considered. Per patient four teeth (one per quadrant) showing the highest bone loss in the 2004 panoramic X-ray were selected for periodontal status assessment. Bone losses in implants were calculated as the differences between 2004 and 2009 bone levels in radiographs. Results: A total of 117 patients were included. Of the 408 teeth considered, 73 (17.9%) were lost in 2009 (losing risk: >50% for bone losses ?7mm). A total of 295 implants were reviewed. Eight of 117 (6.8%) patients had lost implants (13 of 295 implants installed; 4.4%). Implant loss rate (quadrant status) was 1.4% (edentulous), 3.6% (preserved teeth), and 11.1% (lost teeth) (p=0.037). The percentage of implant loss significantly (p<0.001) increased when the medial/distal bone loss was ?3 mm. The highest (p?0.001) pocket depths were found in teeth with ?5mm and implants with ?3mm bone losses, with similar mean values (?4mm), associated with higher rates of plaque index and bleeding by probing. Conclusions: The significant bi-directional relation between plaque and bone loss, and between each of these two parameters/signs and pocket depths or bleeding (both in teeth and implants, and between them) together with the higher percentage of implants lost when the bone loss of the associated teeth was ?3 mm suggest that the patient?s periodontal status is a critical issue in predicting implant health/lesion

Genetic diversity of HLA system in four populations from Baja California, Mexico: Mexicali, La Paz, Tijuana and rural Baja California

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 250 Mexicans from the states of Baja California Norte and Baja California Sur living in Mexicali (N = 100), La Paz (N = 75), Tijuana (N = 25) and rural communities (N = 50) to obtain information regarding allelic and haplotypic frequencies. The most frequent haplotypes for the Baja California region include nine Native American and five European haplotypes. Admixture estimates revealed that the main genetic components are European (50.45 ± 1.84% by ML; 42.03% of European haplotypes) and Native American (43.72 ± 2.36% by ML; 40.24% of Native American haplotypes), while the African genetic component was less apparent (5.83 ± 0.98% by ML; 9.36% of African haplotypes)

POR UNA CULTURA DE PAZ: UNA MIRADA DESDE LAS CIENCIAS DE LA CONDUCTA

En
 virtud
 de
 lo
 anterior,
 los
 estudiosos
 de
 las
 ciencias
 de
 la
 conducta
 
de
 la
 Universidad
Autónoma 
del
 Estado 
de 
México,

ante 
la
persistencia
 y 
proliferación
 de
 estos 
hechos
 en
 diversas
 partes
 del
Mundo
 y
 de
 nuestro 
país 
en 
particular, se
 convocó
 a
 los
 estudiosos
 interesados
 y
 a
 la
 sociedad
 en
 general
 a
 presentar
 trabajos
 para
 analizar,
 debatir
 y
 proponer
 estrategias
 de
 acción
 y
 dirección,
 que
 fortalezcan
 una
 convivencia y bienestar con sentido humanista para una cultura de paz. El
 presente
 texto
 es
 producto
 de 
esta convocatoria 
que
 recoge 
los
trabajos 
de 

los
 interesados 
en 
la
 temática,

 de
 diferentes 
países
(España,
Argentina,
Cuba,
Brasil,
Costa
 Rica
 y
 México)
 retomando
 con
 ello
 sus
 experiencias
 relativas
 al
 estudio,
 análisis,
 comprensión
 e
 instrumentación
 de
 la
 cultura
 de
 paz
 en
 los
 distintos
 ámbitos
 institucionales
 en
 los
 que
 participan:
 educativo,
 salud,
 penitenciario,
 social,
laboral,
familia,
alimentario,
psicológico,
por 
mencionar 
algunos.
 El
 presente
 libro,
 propicia
 un
 espacio
 de
 reflexión,
 diálogo
 y
 posicionamiento
 de
 las 
ciencias 
de 
la 
conducta
 para 
la 
apropiación,
análisis,
debate
 y 
propuestas 
que
 fortalezcan 
una
 cultura
 de 
paz
 a
través
 de 
la
 convivencia 
y
 el 
bienestar
 social 
con
 sentido 
humanista.
El
 sistema 
económico
 neoliberal
 y 
el 
proceso
 de 
globalización 
han
 contribuido
al
 logro
 de
 avances
 significativos
 en
 la
 ciencia
 y
 la
 tecnología,
 pero
 también
 han
 propiciado
 la
 polarización
 de
 las
 sociedades
 lo
 que
 ha
 impactado
 de
 manera
 negativa
 a
 la
 sociedad
 en
 su
 conjunto,
 pero
 en
 mayor
 medida
 a
 los grupos
 vulnerables. Dicha
 polarización
 ha
 traído
 consigo
 un
 desarrollo
 desigual
 del
 mundo
 que
 se
 expresa
 de
 diferentes
 maneras
 tanto
 en
 países
 desarrollados
 como
 en
 los
 llamados
 del
 tercer
 mundo,
 en
 donde
 no
 están
 satisfechas
 las
 necesidades
 humanas 
elementales
 de
 todos 
los
sectores 
de 
la 
población,
siempre 
falta 
algo. 
Si 
a
 esto 
le
 sumamos 
los
conflictos
 internacionales por
 diferentes
 motivos
 que
 enfrentan
 algunas
 naciones,
 una
 insuficiente
 cobertura
 educativa
 y
 de
 salud,

 desempleo
 y
 pobreza 
extrema,
 entre 
otras
 cosas; 
estamos
 frente
 a
retos 
de
 gran
 envergadura
 para
 los
 gobiernos,
 para
 los
 estudiosos
 y
 para
 la
 sociedad
 civil
 en
 general. Uno 
de 
los
 intentos
 para
 frenar 
y prevenir 
la
 agudización
 de 
estas 
problemáticas
 es
 la
 cultura 
de 
paz,
cuyo
 estudio
y propuestas 
han 
ido 
avanzando 
en 
diferentes
 sentidos 
y 
de 
manera 
favorable,
el 
tema 
está 
presente 
en 
diferentes 
Organismos
 Internacionales
 como
 la
 ONU,
 la
 UNESCO,
 la
 OCDE,
 El
 Banco
 Mundial,
 entre
 otros.
 Pero
 falta 
mucho 
por 
hacer.Universidad Autónoma del Estado de Méxic