Marker-free co-selection for successive rounds of prime editing in human cells.

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.

p.G12C KRAS mutation prevalence in non-small cell lung cancer: Contribution from interregional variability and population substructures among Hispanics.

BACKGROUND: The KRAS exon 2 p. G12C mutation in patients with lung adenocarcinoma has been increasing in relevance due to the development and effectiveness of new treatment medications. Studies around different populations indicate that regional variability between ethnic groups and ancestries could play an essential role in developing this molecular alteration within lung cancer. METHODS: In a prospective and retrospective cohort study on samples from lung adenocarcinoma from 1000 patients from different administrative regions in Colombia were tested for the KRAS p.G12C mutation. An analysis of STR populations markers was conducted to identify substructure contributions to mutation prevalence. RESULTS: Included were 979 patients with a national mean frequency for the KRAS exon 2 p.G12C mutation of 7.97% (95%CI 6.27-9.66%). Variation between regions was also identified with Antioquia reaching a positivity value of 12.7% (95%CI 9.1-16.3%) in contrast to other regions such as Bogota DC (Capital region) with 5.4% (2.7-8.2%) and Bolivar with 2.4% (95%CI 0-7.2%) (p-value = 0.00262). Furthermore, Short tandem repeat population substructures were found for eight markers that strongly yielded association with KRAS exon 2 p.G12C frequency reaching an adjusted R2 of 0.945 and a p-value of < 0.0001. CONCLUSIONS: Widespread identification of KRAS exon 2 p.G12C mutations, especially in cases where NGS is not easily achieved is feasible at a population based level that can characterize regional and national patterns of mutation status. Furthermore, this type of mutation prevalence follows a population substructure pattern that can be easily determined by population and ancestral markers such as STR.

EGFR Inhibitors Plus Bevacizumab are Superior Than EGFR Inhibitors Alone as First-Line Setting in Advanced NSCLC With EGFR Mutations and BIM Deletion Polymorphisms (BIM-CLICaP).

PURPOSE: BIM activation is essential for epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)-triggered apoptosis in EGFR-mutant non-small-cell lung cancer (NSCLC). A deletion in the intron two of the BIM gene results in generation of alternatively spliced isoforms that impairs their apoptotic response to TKIs, conferring the NSCLC cells intrinsic resistance to these medications. Patients with both alterations have poor clinical evolution. The current study aimed to investigate the clinical efficacy and tolerability of EGFR-TKIs plus bevacizumab (Bev) versus EGFR-TKIs alone as first-line treatment in advanced NSCLC patients with EGFR mutations and BIM deletions (BIMdel). MATERIALS AND METHODS: A retrospective analysis was conducted. BIMdel was detected using polymerase chain reaction analysis and direct sequencing of DNA. BIM protein expression was investigated by immunohistochemistry, and BIM mRNA levels by reverse transcriptase-polymerase chain reaction. Clinical characteristics, overall survival, progression-free survival (PFS), overall response rate (ORR), and treatment-related adverse events were compared between both groups. RESULTS: Thirty-three patients were included; 15 received EGFR-TKIs, and 18 received EGFR-TKIs plus Bev. The median age was 63 years, with a majority of recruited female patients. All included individuals had an Eastern Cooperative Oncology Group performance score of 2 or less. The addition of Bev resulted in a significantly higher ORR (94.4% v 40%, P > .001). Median PFS was longer with the use of the combination therapy (11.12 v 7.87 months; P = .001). Median overall survival tended to be longer in the EGFR-TKIs plus Bev (30.9 v 25.4 months; P = .06) but failed to reach statistical significance. Response in terms of both partial and complete as well as overall favorably affected PFS. CONCLUSION: EGFR-TKIs plus Bev conferred a significantly higher ORR and PFS in advanced NSCLC patients with EGFR mutation and BIMdel. Further prospective studies are needed to validate these findings.

Persistent High-Risk HPV Infection and Molecular Changes Related to the Development of Cervical Cancer.

This article is a preliminary investigational study that is aimed at giving hints about the interesting biomarkers involved in the transition process from low-grade cervix lesion to invasive cervical cancer. Our study focuses on the risk factors and tumour molecular changes in one patient. First in 1986, she was diagnosed a preinvasive cervix lesion. Then, 16 years later, she was diagnosed an invasive cervical cancer. The 2002 diagnosis was a squamous cell carcinoma of the cervix, stage IIIB (FIGO), whereas in 1986, she had been diagnosed a high-grade squamous intraepithelial cervical lesion. Retrospectively, the analysis of samples of preneoplastic lesions and invasive cervical cancer confirmed the histopathological diagnoses and detected the presence of HPV type and HPV-16 variants, as well as the overexpression of proteins such as hTERT, IGF1Rα, IGF1Rß, CAIX, and GLUT1. Finally, the Arg72Pro polymorphism was detected in TP53. The role of high-risk HPV and HPV-16 variants and of hTERT, IGF1Rα, IGF1Rß, CAIX, and GLUT1 variations seemed confirmed in the development and progression of cervical cancer. As a result, analyzing the molecular changes in one and same tumour that progresses from a low-grade cervix lesion to invasive cervical cancer could provide valuable information in order to improve detection, diagnosis, and treatment in the future.

hTERT Protein Expression in Cytoplasm and Nucleus and its Association With HPV Infection in Patients With Cervical Cancer.

BACKGROUND: Few studies have analyzed the association between human telomerase reverse transcriptase (hTERT) protein expression (nuclear and cytoplasmic localization), hTERT methylation status, and human papillomavirus (HPV) genotype infection in cervical cancer. PATIENTS AND METHODS: One hundred seventy-three patients with cervical cancer were analyzed. hTERT protein expression was detected by immunohistochemistry. hTERT DNA methylation analysis was performed using a PCR-RLB-hTERT assay, targeting two regions of the hTERT promoter. Type specific HPV infection was detected by using GP5+/GP6+PCR-RLB. RESULTS: hTERT protein expression was found in both cytoplasm and nucleus (78.0% of the samples showed a cytoplasmic localization and 79.8% had a nuclear localization). A statistically significant association was found between alpha 9 and 7 HPV species with a non-methylation pattern of the hTERT promoter and between these species and high expression of hTERT protein with nuclear localization. CONCLUSION: hTERT protein is found in both the nucleus and cytoplasm of patients with cervical cancer and confirm the relationship between the non-methylated status of hTERT promoter and some HPV species as well as the relationship between these species and hTERT protein expression.

Formación de un divertículo epifrénico como complicación de la cirugía antirreflujo: importancia de la fluoroscopia / Development of an epiphrenic diverticulum as a complication of antireflux surgery: importance of fluoroscopy

En este artículo se muestran los hallazgos radiológicos en el esofagograma del divertículo epifrénico en un paciente con disfagia para sólidos y pérdida de peso, con antecedente de cirugía antirreflujo. Además, se encuentra una plicatura muy apretada y, como consecuencia, la formación del divertículo epifrénico, entidad de rara presentación. Las técnicas de fluoroscopia siguen vigentes para la valoración anatómica y funcional del tracto gastrointestinal

This article demonstrates the radiological findings in the esophagogram of the epiphrenic diverticulum in a patient with solid dysphagia and weight loss with a history of antireflux surgery. In this case, a very tight plication was found and as a consequence the formation of the epiphrenic diverticulum, a rare entity Fluoroscopy techniques are still used for anatomical and functional evaluation of the gastrointestinal tract.

Embarazo abdominal calcificado de 40 años de evolución, hallazgo incidental: Presentación de caso / Calcified Abdominal Pregnancy 40 Years of Evolution, Incidental Finding: a Case Report

Debido a la baja incidencia de embarazos ectópicos abdominales y, más aún, de la formación de litopedion, se reporta el caso de una paciente de 84 años quien consulta por cuadro clínico de colelitiasis y pancreatitis de origen biliar, con hallazgo incidental de litopedion en cavidad abdominal de más de 40 años, documentado y diagnosticado por medio de radiografía convencional de abdomen y confirmado con tomografía computarizada multidetector (TCMD). Se realizó una revisión de la literatura mundial sobre los aspectos clínicos, radiológicos y patológicos del embarazo abdominal con presentación de litopedion

Due to the low incidence of abdominal ectopic pregnancy and, even more, of the formation of lithopedion, the case of an 84-years-old female patient is presented, who was admitted with clinical symptoms of cholelithiasis and biliary pancreatitis, with an incidental finding of lithopedion in the abdominal cavity, of more than 40 years. It was documented and diagnosed using conventional abdominal radiography, and confirmed with multidetector computed tomography (MDCT). The article presents a review of world literature on the clinical, radiological and pathological features of abdominal pregnancy with lithopedion.

Promoción de alimentación saludable en hogares comunitarios infantiles del municipio de Sopó (Cundinamarca. Colombia) bajo la estrategia de Atención Primaria en Salud / Promoting healthy eating in community children's day care centers in the municipality of Sopo (Cundinamarca, Colombia) under the Primary Health Care strategy

Objetivo: Fortalecer hábitos saludables alimenticios en los hogares infantiles comunitarios y sus familias en el municipio de Sopó (Cundinamarca, Colombia) desde la Atención Primaria en Salud. Metodología: Investigación-acción participativa realizada con 67 niños de 5 hogares comunitarios, sus madres comunitarias, padres de familia y administración municipal durante el segundo semestre de 2014, mediante el abordaje de 3 ejes: 1. Conociendo sobre alimentación saludable, 2. Motivando sobre alimentación saludable y 3. Articulando a la comunidad. Resultados: Las madres comunitarias conocen la importancia de la alimentación saludable y el número de frutas y verduras que se deben consumir diariamente. Los padres de familia reconocen a sus hijos como agentes de cambios para mejorar la motivación en el hogar para el consumo de una alimentación saludable. Los niños han mejorado su consumo de frutas y verduras, aunque persiste la preferencia por alimentación no saludable. Por último, se pudo articular la propuesta con el sector salud y educación del municipio y se reconoció la importancia de trabajar colaborativamente para mejorar la salud de los niños. Conclusiones: Los niños demostraron que son capaces de reconocer que las frutas y las verduras son componentes fundamentales en su dieta para estar sanos y fuertes; además es importante resaltar que los niños se convirtieron en agentes de cambio respecto a la alimentación saludable en sus hogares involucrando a los padres y motivando a las madres comunitarias.

Objetive: To strengthen healthy eating habits in community children's day care centers and their families in the municipality of Sopo (Colombia) from the Primary Health Care. Methodology: Participatory Action Research conducted with 67 children of 5 community children's day care centers, their community mothers (teachers), parents and local government during the second half of 2014, by addressing three areas: 1. Knowing about healthy eating, 2. Motivating on healthy eating and 3. Articulating the community. Results: The community mothers know the importance of healthy eating, and know the number of fruits and vegetables should be consumed daily. Parents recognize their children as agents of change to improve the motivation at home to eating a healthy diet. Children have improved their consumption of fruits and vegetables, although the preference for unhealthy diet persists. Finally it was possible to articulate the proposal with the health and education sectors of the municipality, recognizing the importance of working collaboratively to improve the health of children. Conclusions: Children showed that they are able to recognize that fruits and vegetables are key components in your diet to be healthy and strong, it is also important to note that children become agents of change regarding healthy eating at home, involving parents, and motivating the community mothers.

Las proteínas B3 y PDI aisladas de plaquetas humanas se unen con el rotavirus ECwt in vitro / B3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

Antecedentes. En la actualidad, no se dispone de integrina b3 de manera comercial y la PDI comercial tiene costos muy altos, lo cual dificulta el acceso a estas dos proteínas para realizar estudios conducentes a establecer si b3 y PDI interactúan con cepas de rotavirus silvestres. Objetivo. Explorar una metodología que permitiese aislar las proteínas b3 y PDI a partir de plaquetas humanas para generar anticuerpos policlonales en conejo contra la integrina b3 y evaluar la interacción entre las proteínas PDI y b3 con el rotavirus ECwt. Material y métodos. Mediante la técnica de electroforesis preparativa en condiciones reductoras, se separaron las proteínas de un lisado de plaquetas humanas y posteriormente se electroeluyeron. Mediante las técnicas de coinmunoprecipitación, “Western blotting” y ELISA de captura se analizó la interacción del rotavirus ECwt con las proteínas b3, y PDI. Resultados. Las proteínas totales de un lisado de plaquetas humanas se separaron mediante electroforesis en condiciones reductoras, se identificaron las proteínas b3 y PDI en un segmento del gel, utilizando anticuerpos comerciales en “Western blotting” y luego se aislaron estas dos proteínas del resto del gel. Posteriormente las proteínas se electroeluyeron del segmento del gel y se analizó su pureza. Conclusión. Se logró purificar parcialmente a partir de plaquetas humanas, utilizando electroforesis preparativa, cantidades relativamente altas de proteína b3 y PDI. El aislamiento de estas proteínas nos permitió generar un anticuerpo policlonal contra b3 y establecer que b3 y PDI se unen in vitro, luego de incubar las proteínas aisladas con el rotavirus ECwt, e in vivo, después de incubar el rotavirus con las vellosidades aisladas del intestino delgado de ratón lactante de la cepa ICR.

Caracterización parcial de los genes codificantes para la proteína de choque térmico HSP70 de Trypanosoma rangeli / Partial characterization of coding genes for Trypanosoma rangeli HSP70

Objetivo: dada la relevancia inmunológica de la región amino terminal de la proteína HSP70 de la Tripanosoma cruzi, así como el hecho de que la inmunización de ratones con Tripanosoma rangeli protege a los animales contra la infección por T. cruzi, el presente trabajo se centró en el aislamiento y caracterización molecular del fragmento homólogo en T. rangeli. Materiales y métodos: la región amino terminal del gen codificante para la HSP70 de T. rangeli fue amplificada mediante PCR utilizando los oligonucleotidos TrHSP70f/R2, diseñados con base en la secuencia homóloga de T. cruzi. El fragmento amplificado fue purificado, clonado en el vector pGEM® –Teasy (Promega) y secuenciado en un 373 Automatic DNA sequencer (Applied Biosystems). La organización genómica de los genes HSP70 se determino mediante ensayos de “Southern blot” y PFGE. Resultados: los genes HSP70 de T. rangeli tienen un tamaño aproximado de 2.400 pb, se encuentran repetidos en tandem y se localizan en un cromosoma de 1.030 y 1.090 Kb en la cepa H14, KP1(+) y de 1.100 KB en la cepa Tre, KP1 (-). La secuencia de nucleótidos correspondiente a la región amino terminal de la proteína tiene una identidad del 99 por ciento entre las cepas H14 y Tre de T. rangeli y del 94 por ciento entre éstas y T. cruzi, preséntando además polimorfismos para diversas enzimas de restricción. La secuencia de amiácidos de dicha región entre ambos parásitos tiene una identidad del 95 por ciento. Conclusiones: el extremo amino terminal de las proteínas HSP70 de T. cruzi y T. rangeli guardan una elevada identidad de secuencia lo que abre un camino a la posible utilización de la HSP70 de T. rangeli en inmunoterapia. Asimismo y dado que en esta región se localizan epítopes B y T inductores de respuesta inmune en pacientes chagásicos, la HSP70 puede estar implicada en la reacción inmunológica cruzada entre estos parásitos