Protocol to detect senescence-associated ß-galactosidase and immunoperoxidase activity in fresh-frozen murine tissues.

Double labeling to identify different markers in the same tissue section represents a useful tool either for in situ diagnosis or characterization of molecular associations. Here, we present a protocol to detect senescence-associated ß-galactosidase (SA-ßGal) and immunoperoxidase (IPO) activity in fresh-frozen murine tissues. We describe steps for tissue collection, solution preparation, SA-ßGal staining, IPO staining, hematoxylin counterstaining, microscopic observation, and signal quantification. This protocol can be used to detect in situ proteins alongside SA-ßGal activity. For complete details on the use and execution of this protocol, please refer to Pacheco-Rivera et al.1.

Nucleoredoxin Redox Interactions Are Sensitized by Aging and Potentiated by Chronic Alcohol Consumption in the Mouse Liver.

Aging is characterized by increased reactive species, leading to redox imbalance, oxidative damage, and senescence. The adverse effects of alcohol consumption potentiate aging-associated alterations, promoting several diseases, including liver diseases. Nucleoredoxin (NXN) is a redox-sensitive enzyme that targets reactive oxygen species and regulates key cellular processes through redox protein-protein interactions. Here, we determine the effect of chronic alcohol consumption on NXN-dependent redox interactions in the liver of aged mice. We found that chronic alcohol consumption preferentially promotes the localization of NXN either into or alongside senescent cells, declines its interacting capability, and worsens the altered interaction ratio of NXN with FLII, MYD88, CAMK2A, and PFK1 proteins induced by aging. In addition, carbonylated protein and cell proliferation increased, and the ratios of collagen I and collagen III were inverted. Thus, we demonstrate an emerging phenomenon associated with altered redox homeostasis during aging, as shown by the declining capability of NXN to interact with partner proteins, which is enhanced by chronic alcohol consumption in the mouse liver. This evidence opens an attractive window to elucidate the consequences of both aging and chronic alcohol consumption on the downstream signaling pathways regulated by NXN-dependent redox-sensitive interactions.

Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery.

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.

Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing.

Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques. We discover a highly potent, cross-species compatible variant (AAV.cc47) that shows improved attributes benchmarked against AAV serotype 9 as evidenced by robust reporter and therapeutic gene expression, Cre recombination and CRISPR genome editing in normal and diseased mouse models. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. We envision that ccAAV vectors may not only improve predictive modeling in preclinical studies, but also clinical translatability by broadening the therapeutic window of AAV based gene therapies.

Loss of Dystroglycan Drives Cellular Senescence via Defective Mitosis-Mediated Genomic Instability.

Nuclear ß-dystroglycan (ß-DG) is involved in the maintenance of nuclear architecture and function. Nonetheless, its relevance in defined nuclear processes remains to be determined. In this study we generated a C2C12 cell-based DG-null model using CRISPR-Cas9 technology to provide insights into the role of ß-DG on nuclear processes. Since DG-null cells exhibited decreased levels of lamin B1, we aimed to elucidate the contribution of DG to senescence, owing to the central role of lamin B1 in this pathway. Remarkably, the lack of DG enables C2C12 cells to acquire senescent features, including cell-cycle arrest, increased senescence-associated-ß-galactosidase activity, heterochromatin loss, aberrant nuclear morphology and nucleolar disruption. We demonstrated that genomic instability is one driving cause of the senescent phenotype in DG-null cells via the activation of a DNA-damage response associated with mitotic failure, as shown by the presence of multipolar mitotic spindles, which in turn induced the formation of micronuclei and γH2AX foci (DNA-damage marker), telomere shortening and p53/p21 upregulation. Altogether, these events might ultimately lead to premature senescence, impeding the replication of the damaged genome. In summary, we present evidence supporting a role for DG in protecting against senescence, through the maintenance of proper lamin B1 expression/localization and proper mitotic spindle organization.

Diverse Escherichia coli lineages from domestic animals carrying colistin resistance gene mcr-1 in an Ecuadorian household.

OBJECTIVE: The aim of this study was to detect potential animal reservoirs of Escherichia coli carrying the mcr-1 gene in an Ecuadorian household. METHODS: The mobile colistin-resistance gene, mcr-1, was first detected in Ecuador in a commensal E. coli isolate from a boy. A cross-sectional study was performed to detect the possible source of colistin-resistant E. coli in the boy's household. Faecal swabs and soil faecal samples were collected from companion animals. Samples were plated on selective media to isolate colistin-resistant E. coli and isolates were submitted to PCR detection of mcr-1, pulsed field gel electrophoresis (PFGE), and multi-locus sequences typing (MLST). Moreover, the genomes of all the isolates were sequenced. RESULTS: Three different colistin-resistant E. coli sequence types (ST3941, 1630 and 2170), corresponding to three PFGE patterns, were obtained from a chicken and two dogs; these isolates were different from the human isolate (ST609). By whole-genome sequencing, the mcr-1.1 gene was found on IncI2 plasmids with very high nucleotide identity. CONCLUSIONS: Our results indicate a polyclonal dissemination of mcr-1.1 in the environment surrounding the first MCR-producing E. coli strain reported in Ecuador. Our findings support the idea of lateral dissemination of mcr-1.1 gene between unrelated E. coli isolates.

Conservative management in a patient with cervical ectopic pregnancy in Nariño, Colombia: Case report and review of the literature / Manejo conservador en una paciente con embarazo ectópico cervical en nariño, colombia: reporte de caso y revisión de la literatura

RESUMEN Objetivos: reportar el caso de un embarazo cervical (EC) que recibió manejo conservador exitoso y realizar una revisión de la literatura sobre el tratamiento médico y quirúrgico conservador. Materiales y métodos: se presenta el caso de una paciente con embarazo cervical, quien recibió manejo farmacológico con metotrexate (MTX) y posterior legrado con evolución clínica satisfactoria. Se realizó una búsqueda de artículos en Medline vía PubMed, LILACS, SciElo y Google académico con los términos: "cervical ectopic pregnancy", "conservative treatment", "curettage", "methotrexate", "uterine artery embolization" "hysteroscopy". Se seleccionaron reportes y series de caso, pacientes con embarazo cervical diagnosticado por ultrasonido, de cualquier edad gestacional, sometidas tratamiento médico o quirúrgico conservador. Resultados: se incluyeron 22 estudios; se identificaron 95 pacientes con EC tratados con MTX, con tratamiento exitoso en 93. La complicación más frecuente fue la hemorragia en 12 %; el 26 % requirió tratamiento quirúrgico complementario. Cada vez más, la embolización de arterias uterinas (EAU) se realiza de manera preventiva (7 casos) antes del legrado o del tratamiento con MTX. La histeroscopia es otra alternativa reciente (20 casos). En 2 casos se requirió histerectomía abdominal, uno de los cuales fue un embarazo ístmico cervical. Conclusiones: el tratamiento con MTX sigue siendo el más frecuentemente utilizado. La dilatación y el curetaje con taponamiento endocervical puede ser una opción por considerar en el manejo de urgencia del EC en instituciones de atención primaria. En instituciones donde se dispone de tecnologías de alta complejidad, la embolización de arterias uterinas previa a los procedimientos quirúrgicos y la histeroscopia son opciones que se deben considerar. Dado que actualmente es posible el diagnóstico temprano del EC, se requieren estudios multicéntricos que comparen las diferentes alternativas de manejo para una mejor evaluación de su seguridad y efectividad. Conclusiones: el tratamiento con MTX sigue siendo el más frecuentemente utilizado. La dilatación y el curetaje con taponamiento endocervical puede ser una opción por considerar en el manejo de urgencia del EC en instituciones de atención primaria. En instituciones donde se dispone de tecnologías de alta complejidad, la embolización de arterias uterinas previa a los procedimientos quirúrgicos y la histeroscopia son opciones que se deben considerar. Dado que actualmente es posible el diagnóstico temprano del EC, se requieren estudios multicéntricos que comparen las diferentes alternativas de manejo para una mejor evaluación de su seguridad y efectividad

ABSTRACT Objectives: To report a case of cervical pregnancy (CP) treated successfully with a conservative approach, and to conduct a review of the literature regarding conservative medical and surgical treatment. Materials and Methods: Patient with cervical pregnancy treated pharmacologically with methotrexate (MTX) followed by dilation and curettage, with a satisfactory clinical course. A search of articles was conducted in Medline via PubMed, LILACS, SciElo and Google Scholar using the terms "cervical ectopic pregnancy," "conservative treatment," "curettage,¼ "methotrexate," "uterine artery embolization," "hysteroscopy." Reports and case series were selected of patients with cervical pregnancy diagnosed on ultrasound at any gestational age, subjected to conservative medical or surgical treatment. Results: A total of 22 studies were included; 95 patients with CP treated with MTX were identified, 93 of them successfully treated. The most frequent complication was bleeding in 12%; 26% required complementary surgical treatment. Increasingly, uterine artery embolization (UAE) is carried out preventatively (7 cases) before curettage or treatment with MTX. The hysteroscopy is another recent alternative (20 cases). Abdominal hysterectomy was required in two cases, one of which was a cervico-isthmic pregnancy. Conclusions: Treatment with MTX continues to be the most frequent strategy. Dilation and curettage with endocervical plugging may be an option to consider in the emergency management of EP in primary care institutions. In institutions equipped with high complexity technology, uterine artery embolization before the surgical procedure and histeroscopy are options to be considered. Considering that early diagnosis of EP is now possible, multi-center studies comparing different management options are needed for better assessment of their safety and effectiveness.

Conocimiento y factores de finalización de la lactancia materna en mujeres de una comunidad en Veracruz, México / Knowledge and factors to stop breastfeeding in women of a community in Veracruz, Mexico / Connaissances et facteurs d'achèvement de l'allaitement maternel chez les femmes d'une communauté à Veracruz, au Mexique / Conhecimento e fatores de conclusâo da amamentaçâo em mulheres de uma comunidade em Veracruz, México

Resumen: Objetivo: Identificar el déficit de conocimiento y los factores de finalización de la lactancia materna en mujeres que acuden al Centro de Salud de Arroyo Blanco en Xalapa, Veracruz. Material y Métodos: Investigación descriptiva, transversal, muestreo no probabilístico a conveniencia en una muestra integrada por 31 mujeres embarazadas. Resultados: El 96.8% menciona tener conocimiento sobre la LM, sin embargo el 12.9% desconoce la técnica adecuada para lactar a su bebé y el 22.6% desconoce la posición correcta de amamantar. Algunos factores de finalización son: poca producción de leche materna (19.4%) y la incorporación a su trabajo (9.7%). Conclusiones: Aunque la mayoría de las mujeres menciona tener el conocimiento sobre la LM, una proporción considerada manifestó tener déficit de conocimiento, ya que desconocen la técnica y posición adecuada para lactar. La información insuficiente sobre la LM, coincide con lo encontrado por Ulunque y cols., los cuales señalan que el conocimiento en las mujeres de su estudio era insuficiente e inadecuado.

Abstract: Objetive: Identify the lack of knowledge and completion factors of breastfeeding in women who come to the Health Center of Arroyo Blanco in Xalapa, Veracruz. Materials and methods: Descriptive, cross-sectional study, non-probabilistic sampling at convenience in a sample composing of 31 pregnant women. Results: Results: 96.8% mentioned having knowledge about breastfeeding. However, 12.9% do not know the proper technique for breastfeeding their baby and 22.6% do not know the correct position to perform it. Some factors of completion are: low breast milk production (19.4%) and incorporation into their work (9.7%). Conclusions: Although most women mentioned having knowledge about BF, a considerate proportion reported having lack of knowledge about it, because they do not know the technique and proper position to breastfeed. Insufficient information on breastfeeding, agrees with what was found by Ulunque et al, who point out that the knowledge about breastfeeding in their study by women was insufficient and inadequate.

Résumé: Objectif: Identifier le manque de connaissances et les facteurs d'achèvement de l'allaitement chez les femmes qui vont au centre de santé d'Arroyo Blanco à Xalapa, Veracruz. Matériel et méthodes: Recherche descriptive, transversale échantillonnage de commodité non-probabilité dans un échantillon constitué de 31 femmes enceintes. Résultats: 96,8% ont déclaré avoir des connaissances en matière d'allaitement. Cependant, 12,9% ne connaissent pas la technique appropriée pour allaiter leur bébé et 22,6% ne connaissent pas la bonne position pour l'exécuter. Certains facteurs d'achèvement sont: une faible production de lait maternel (19,4%) et l'intégration dans leur travail (9,7%). Conclusions: Bien que la plupart des femmes mentionnent avoir des connaissances en matière d'allaitement, une proportion considérée ont déclaré avoir un manque de connaissances à ce sujet, car ils ne connaissent ni la technique ni la position pour allaiter. L'Information insuffisant sur l'allaitement, est d'accord avec les conclusions d'Ulunque et al, qui soulignent que les connaissances sur l'allaitement des femmes dans leur étude étaient insuffisantes et inadéquate.

Resumo: Objetivo: Identificar o déficit de conhecimento e os fatores de completude da amamentaçâo em mulheres atendidas no Centro de Saúde Arroyo Blanco, em Xalapa, Veracruz. Materiais e métodos: Pesquisa descritiva, transversal, amostragem nao probabilística, por conveniência, em uma amostra composta por 31 gestantes. Resultados: 96,8% referiram ter conhecimento sobre a MM, porém 12,9% desconhecem a técnica adequada para amamentar e 22,6% desconhecem a posiçâo correta da amamentaçâo. Alguns fatores de conclusao sao: baixa produçâo de leite materno (19,4%) e incorporaçâo em seu trabalho (9,7%). Conclusao: Embora a maioria das mulheres mencionasse ter conhecimento sobre o AM, uma proporçâo considerou ter déficit de conhecimento, uma vez que desconhecem a técnica e a posiçâo para amamentar. A informaçâo insuficiente sobre o ML, concorda com a encontrada por Ulunque et al., Que indicam que o conhecimento nas mulheres de seu estudo era insuficiente e inadequado.

Cambios en prevalencias de conocimientos, actitudes y prácticas de sexualidad en adolescentes escolarizados, Bogotá, 2011-2015 / Changes in prevalence of knowledge, attitudes and practices of sexuality in teenage students, Bogotá, 2011-2015 / Mudanças na prevalência de conhecimentos, atitudes e práticas de sexualidade em adolescentes escolares, Bogotá, 2011-2015

RESUMEN Objetivo Presentar los cambios en las prevalencias de conocimientos, actitudes y prácticas en adolescentes escolarizados en torno a sexualidad luego de un proceso de intervención. Metodología Estudio descriptivo observacional. Se aplicó un cuestionario a 319 adolescentes, pertenecientes a tres colegios públicos, durante cinco años consecutivos a medida que avanzaban en los grados escolares (séptimo a undécimo). Se realizaron talleres de promoción de la salud. Se midieron los cambios de prevalencias de identificación del ciclo menstrual, de la anticoncepción de emergencia y de la protección del condón, así como de la aceptación de los padres a la planificación, de haber tenido relaciones sexuales, razones para hacerlo y si usó protección en la primera y última relación. Se compararon los resultados por género. Resultados La identificación de la anticoncepción de emergencia se incrementó del 52% en séptimo al 70% en undécimo; igualmente la certeza de protección del condón (del 45% al 62%, respectivamente). El promedio de la prevalencia de relaciones sexuales fue del 24%, el de protección en la primera relación fue del 52% y, en la última, el 81%; no se encontraron diferencias estadísticas de estas variables al compararlas por género. La percepción de la aceptación de la planificación por parte de los padres se incrementó del 45% al 79%. Conclusión El programa puso en evidencia la necesidad de fortalecer el proceso educativo a través de las prevalencias encontradas.(AU)

ABSTRACT Objective To present the changes in the prevalence of knowledge, attitudes and practices in high school adolescents in sexuality after an intervention process. Methodology Observational descriptive study. It was administered a questionnaire to 319 adolescents, attending three public schools, for five consecutive years as they progressed in school years (seventh to eleventh). Health promotion workshops were held. Changes in the prevalence were measured (identification of the menstrual cycle, emergency contraception and, condom protection, as well as parents' acceptance of contraception methods, having sex, reasons for do it and, whether they used protection in the first and last relationship). The results were compared by gender. Results The identification of emergency contraception was increased from 52% in seventh to 70% in eleven; also, the certainty of the condom protection (from 45% to 62%, respectively). The average of the prevalence of sexual intercourse was 24%, protection in the first relationship 52% and, in the last one was 81%; no statistical differences of these variables were found when comparing them by gender. Parents' perception of acceptance of planning increased from 45% to 79%. Conclusion The program results highlighted the need to strengthen the educational processes.(AU)

RESUMO: Objetivo Apresentar as mudanças na prevalência de conhecimentos, atitudes e práticas em adolescentes escolarizados em torno da sexualidade após um processo de intervenção. Metodologia Estudo observacional descritivo. Um questionário foi aplicado a 319 adolescentes, pertencentes a três escolas públicas, por cinco anos consecutivos, à medida que avançavam nas séries escolares (sétima a décima primeira). Foram realizadas oficinas de promoção da saúde. As mudanças na prevalência da identificação do ciclo menstrual, contracepção de emergência e proteção do preservativo foram medidas, bem como a aceitação do planejamento pelos pais, tendo tido relações sexuais, razões para fazê-lo e se ele usou proteção em o primeiro e o último relacionamento. Os resultados por sexo foram comparados. Resultados A identificação da contracepção de emergência aumentou de 52% no sétimo para 70% no décimo primeiro; também a certeza da proteção do preservativo (de 45% a 62%, respectivamente). A prevalência média de relações sexuais foi de 24%, a de proteção na primeira relação foi de 52% e, na última, de 81%; Não foram encontradas diferenças estatísticas para essas variáveis quando comparadas por gênero. A percepção dos pais quanto à aceitação do planejamento aumentou de 45% para 79%. Conclusão O programa destacou a necessidade de fortalecer o processo educacional por meio das prevalências encontradas.(AU)

Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1-6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.

[Changes in prevalence of knowledge, attitudes and practices of sexuality in teenage students, Bogotá, 2011-2015]. / Cambios en prevalencias de conocimientos, actitudes y prácticas de sexualidad en adolescentes escolarizados, Bogotá, 2011-2015.

OBJECTIVE: To present the changes in the prevalence of knowledge, attitudes and practices in high school adolescents in sexuality after an intervention process. METHODOLOGY: Observational descriptive study. It was administered a questionnaire to 319 adolescents, attending three public schools, for five consecutive years as they progressed in school years (seventh to eleventh). Health promotion workshops were held. Changes in the prevalence were measured (identification of the menstrual cycle, emergency contraception and, condom protection, as well as parents' acceptance of contraception methods, having sex, reasons for do it and, whether they used protection in the first and last relationship). The results were compared by gender. RESULTS: The identification of emergency contraception was increased from 52% in seventh to 70% in eleven; also, the certainty of the condom protection (from 45% to 62%, respectively). The average of the prevalence of sexual intercourse was 24%, protection in the first relationship 52% and, in the last one was 81%; no statistical differences of these variables were found when comparing them by gender. Parents' perception of acceptance of planning increased from 45% to 79%. CONCLUSION: The program results highlighted the need to strengthen the educational processes.

OBJETIVO: Presentar los cambios en las prevalencias de conocimientos, actitudes y prácticas en adolescentes escolarizados en torno a sexualidad luego de un proceso de intervención. METODOLOGÍA: Estudio descriptivo observacional. Se aplicó un cuestionario a 319 adolescentes, pertenecientes a tres colegios públicos, durante cinco años consecutivos a medida que avanzaban en los grados escolares (séptimo a undécimo). Se realizaron talleres de promoción de la salud. Se midieron los cambios de prevalencias de identificación del ciclo menstrual, de la anticoncepción de emergencia y de la protección del condón, así como de la aceptación de los padres a la planificación, de haber tenido relaciones sexuales, razones para hacerlo y si usó protección en la primera y última relación. Se compararon los resultados por género. RESULTADOS: La identificación de la anticoncepción de emergencia se incrementó del 52% en séptimo al 70% en undécimo; igualmente la certeza de protección del condón (del 45% al 62%, respectivamente). El promedio de la prevalencia de relaciones sexuales fue del 24%, el de protección en la primera relación fue del 52% y, en la última, el 81%; no se encontraron diferencias estadísticas de estas variables al compararlas por género. La percepción de la aceptación de la planificación por parte de los padres se incrementó del 45% al 79%. CONCLUSIÓN: El programa puso en evidencia la necesidad de fortalecer el proceso educativo a través de las prevalencias encontradas.

Conservative management in a patient with cervical ectopic pregnancy in Nariño, Colombia: Case report and review of the literature / Manejo conservador en una paciente con embarazo ectópico cervical en Nariño, Colombia: reporte de caso y revisión de la literatura

Objectives: To report a case of cervical pregnancy (CP) treated successfully with a conservative approach, and to conduct a review of the literature regarding conservative medical and surgical treatment. Materials and methods: Patient with cervical pregnancy treated pharmacologically with methotrexate (MTX) followed by dilation and curettage, with a satisfactory clinical course. A search of articles was conducted in Medline via PubMed, LILACS, SciElo and Google Scholar using the terms "cervical ectopic pregnancy," "conservative treatment," "curettage,¼ "methotrexate," "uterine artery embolization," "hysteroscopy." Reports and case series were selected of patients with cervical pregnancy diagnosed on ultrasound at any gestational age, subjected to conservative medical or surgical treatment. Results: A total of 22 studies were included; 95 patients with CP treated with MTX were identified, 93 of them successfully treated. The most frequent complication was bleeding in 12%; 26% required complementary surgical treatment. Increasingly, uterine artery embolization (UAE) is carried out preventatively (7 cases) before curettage or treatment with MTX. The hysteroscopy is another recent alternative (20 cases). Abdominal hysterectomy was required in two cases, one of which was a cervico-isthmic pregnancy. Conclusions: Treatment with MTX continues to be the most frequent strategy. Dilation and curettage with endocervical plugging may be an option to consider in the emergency management of EP in primary care institutions. In institutions equipped with high complexity technology, uterine artery embolization before the surgical procedure and histeroscopy are options to be considered. Considering that early diagnosis of EP is now possible, multi-center studies comparing different management options are needed for better assessment of their safety and effectiveness.

Objetivos: Reportar el caso de un embarazo cervical (EC) que recibió manejo conservador exitoso y realizar una revisión de la literatura sobre el tratamiento médico y quirúrgico conservador. Materiales y métodos: Se presenta el caso de una paciente con embarazo cervical, quien recibió manejo farmacológico con metotrexate (MTX) y posterior legrado con evolución clínica satisfactoria. Se realizó una búsqueda de artículos en Medline vía PubMed, LILACS, SciElo y Google académico con los términos: "cervical ectopic pregnancy", "conservative treatment", "curettage", "methotrexate", "uterine artery embolization" "hysteroscopy". Se seleccionaron reportes y series de caso, pacientes con embarazo cervical diagnosticado por ultra- sonido, de cualquier edad gestacional, sometidas tratamiento médico o quirúrgico conservador. Resultados: Se incluyeron 22 estudios; se identificaron 95 pacientes con EC tratados con MTX, con tratamiento exitoso en 93. La complicación más frecuente fue la hemorragia en 12 %; el 26 % requirió tratamiento quirúrgico complementario. Cada vez más, la embolización de arterias uterinas (EAU) se realiza de manera preventiva (7 casos) antes del legrado o del tratamiento con MTX. La histeroscopia es otra alternativa reciente (20 casos). En 2 casos se requirió histerectomía abdominal, uno de los cuales fue un embarazo ístmico cervical. Conclusiones: el tratamiento con MTX sigue siendo el más frecuentemente utilizado. La dilatación y el curetaje con taponamiento endocervical puede ser una opción por considerar en el manejo de urgencia del EC en instituciones de atención primaria. En instituciones donde se dispone de tecnologías de alta complejidad, la embolización de arterias uterinas previa a los procedimientos quirúrgicos y la histeroscopia son opciones que se deben considerar. Dado que actualmente es posible el diagnóstico temprano del EC, se requieren estudios multicéntricos que comparen las diferentes alternativas de manejo para una mejor evaluación de su seguridad y efectividad. Conclusiones: El tratamiento con MTX sigue siendo el más frecuentemente utilizado. La dilatación y el curetaje con taponamiento endocervical puede ser una opción por considerar en el manejo de urgencia del EC en instituciones de atención primaria. En instituciones donde se dispone de tecnologías de alta complejidad, la embolización de arterias uterinas previa a los procedimientos quirúrgicos y la histeroscopia son opciones que se deben considerar. Dado que actualmente es posible el diagnóstico temprano del EC, se requieren estudios multicéntricos que comparen las diferentes alternativas de manejo para una mejor evaluación de su seguridad y efectividad.

Representación social de la diabetes tipo 2 en mujeres bajo la teoría del núcleo central / Social Representation of the Diabetes Type 2 in Women according to the Central Core / Theory Representação social da diabete tipo 2 em mulheres sob a teoria do núcleo central

Introducción: La diabetes como enfermedad crónica es un evento catastrófico que origina comportamientos negativos hacia la adherencia terapéutica, lo que hace necesario usar teorías cognoscitivas y sociales para reorientar los cuidados de enfermería. Objetivo: Dar respuesta a las preguntas orientadoras de la investigación: ¿cuál es la representación social que la mujer tiene sobre la diabetes tipo 2? ¿Qué elementos integran el núcleo central y los sistemas periféricos de la representación social? Método: Revisión integrativa utilizando como guía el método prisma, de artículos cuyos descriptores primarios fueran "representaciones sociales", "diabetes tipo 2" y "mujeres" y secundarios "pacientes con diabetes tipo 2". La búsqueda se realizó en bases de datos informatizadas: Web of Science, Medline, PubMed, SciELO, LILACS, Bireme y CUIDEN. Resultados: Los hallazgos señalan que el fenómeno de la diabetes dio origen a una representación social irreversible. Este sistema de informaciones, actitudes y creencias se organizan y estructuran alrededor del padecimiento, alimentación, ejercicio y medicación, lo que dificulta la identidad social y, probablemente, la adherencia terapéutica a la enfermedad. Conclusión: El identificar la estructura de la representación social y elementos que integran el núcleo central les permite a los profesionales de la salud planear intervenciones que favorezcan la identidad social y la adherencia terapéutica de las mujeres en la diabetes tipo 2.

Introduction: As a chronic disease, diabetes is a disastrous event bringing about negative behaviors regarding the treatment adherence, which makes necessary the use of cognitive and social theories in order to redirect the nursing care. Objective: To answer the guiding research questions: What is the social representation that women have about Diabetes Type 2? What elements form the central core and the peripheral systems of the social representation? Method: This is a comprehensive review using the Prism Method as a guide. It includes articles whose primary descriptors are "social representations", "Diabetes Type 2" and "women"; and the secondary descriptor "type 2 diabetes patients". The search was carried out in systematized databases: Web of Science, Medline, PubMed, SciELO, LILACS, Bireme, and CUIDEN. Results: The findings indicate that diabetes phenomenon gave rise to an irreversible social representation. This system of data, attitudes and beliefs is organized and structured around the suffering, feeding, exercise and medication, which makes difficult to ascertain the social identity and, probably, the treatment adherence specific to this disease. Conclusion: To identify the structure of the social representation and elements forming the central core allows the health professionals to plan interventions intended to favor the social identity and treatment adherence among women with diabetes type 2.

Introdução: O diabete como urna doença crónica é um evento catastrófico que origina comportamentos negativos para a adesão terapêutica, o que faz preciso usar teorias cognoscitivas e sociais para reorientar os cuidados de enfermagem. Objetivo: Dar resposta às perguntas norteadoras da pesquisa: qual a representação social que a mulher possue sobre o diabete tipo 2? Quais elementos compõem o núcleo central e os sistemas periféricos da representação social? Método: Revisão integrativa utilizando como guia o método prisma, de artigos cujos descritores primarios foram "representações sociais", "diabete tipo 2" e "mulheres" e secundarios "pacientes com diabete tipo 2". A busca foi realizada em bases de dados informatizadas: Web of Science, Medline, PubMed, SciELO, LILACS, Birerne e CUIDEN. Resultados: Os achados apontam que o fenômeno da diabete deu origem a urna representação social irreversível. Esse sistema de informações, atitudes e crenças é organizado e estruturado em torno do padecimento, alimentação, exercício e medicação, o que dificulta a identidade social e, provavelmente, a adesão terapêutica à doença. Conclusão: Identificar a estrutura da representação social e elementos que integram o núcleo central permite os pro fissionais de a saúde planejarem intervenções favorecendo a identidade social e a adesão terapêutica das mulheres em diabetes tipo 2.

Aquaporin-4-dependent glymphatic solute transport in the rodent brain.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-ß. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures.

Tissue-Dependent Expression and Translation of Circular RNAs with Recombinant AAV Vectors In Vivo.

Circular RNAs (circRNAs) are long-lived, covalently closed RNAs that are abundantly expressed and evolutionarily conserved across eukaryotes. Possible functions ranging from microRNA (miRNA) and RNA binding protein sponges to regulators of transcription and translation have been proposed. Here we describe the design and characterization of recombinant adeno-associated viral (AAV) vectors packaging transgene cassettes containing intronic sequences that promote backsplicing to generate circularized RNA transcripts. Using a split GFP transgene, we demonstrate the capacity of vectors containing different flanking intronic sequences to efficiently drive persistent circRNA formation in vitro. Further, translation from circRNA is efficiently driven by an internal ribosomal entry site (IRES). Upon injecting AAV vectors encoding circRNA in mice, we observed robust transgene expression in the heart, but low transduction in the liver for the intronic elements tested. Expression in the murine brain was restricted to astrocytes following systemic or intracranial administration, while intravitreal injection in the eye yielded robust transgene expression across multiple retinal cell layers. These results highlight the potential for exploiting AAV-based circRNA expression to study circRNA function and tissue-specific regulation in animal models, as well as development of therapeutic platforms using this approach.

Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier.

Effective gene delivery to the CNS by intravenously administered adeno-associated virus (AAV) vectors requires crossing the blood-brain barrier (BBB). To achieve therapeutic CNS transgene expression, high systemic vector doses are often required, which poses challenges such as scale-up costs and dose-dependent hepatotoxicity. To improve the specificity and efficiency of CNS gene transfer, a better understanding of the structural features that enable AAV transit across the BBB is needed. We generated a combinatorial domain swap library using AAV1, a serotype that does not traverse the vasculature, and AAVrh.10, which crosses the BBB in mice. We then screened individual variants by phylogenetic and structural analyses and subsequently conducted systemic characterization in mice. Using this approach, we identified key clusters of residues on the AAVrh.10 capsid that enabled transport across the brain vasculature and widespread neuronal transduction in mice. Through rational design, we mapped a minimal footprint from AAVrh.10, which, when grafted onto AAV1, confers the aforementioned CNS phenotype while diminishing vascular and hepatic transduction through an unknown mechanism. Functional mapping of this capsid surface footprint provides a roadmap for engineering synthetic AAV capsids for efficient CNS gene transfer with an improved safety profile.

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion.

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.