The optimization of aminooxadiazoles as orally active inhibitors of Cdc7.

A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC50=0.71 µM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL=18 L/h/kg). Extensive optimization resulted in azaindole 26 (Cdc7 IC50=1.1 nM, pMCM2 IC50=32 nM) that demonstrated robust lowering of pMCM2 in a mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.

Inhibition of histone deacetylation does not block resilencing of p16 after 5-aza-2'-deoxycytidine treatment.

Epigenetic drugs are in use in clinical trials of various human cancers and are potent at reactivating genes silenced by DNA methylation and chromatin modifications. We report here the analysis of a set of normal fibroblast and cancer cell lines after combination treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA). Low doses of the drug combination caused cell cycle arrest, whereas high doses induced apoptosis in T24 bladder carcinoma cells. Both p16 (CDKN2A/INK4) and p21 (CIP1/SDI1/WAF1) expression were induced to similar levels in normal and cancer cells in a dose-dependent fashion after combination treatments. We detected a distinct increase of histone H3 acetylation at lysine 9/14 near the transcription start sites, in both LD419 normal fibroblasts and T24 bladder carcinoma cells, whereas the acetylation changes in the p21 locus were less apparent. Interestingly, the levels of trimethylation of histone H3 on lysine 9, which usually marks inactive chromatin regions and was associated with the p16 promoter in silenced T24 cells, did not change after drug treatments. Furthermore, we provide evidence that the remethylation of the p16 promoter CpG island in T24 cells after 5-aza-CdR treatment cannot be halted by subsequent continuous PBA treatment. The p16 gene is resilenced with kinetics similar to 5-aza-CdR only-treated cells, which is also marked by a localized loss of histone acetylation at the transcription start site. Altogether, our data provide new insights into the mechanism of epigenetic drugs and have important implications for epigenetic therapy.

Isolation and Characterization of a Distinct Subpopulation from the WM115 Cell Line That Resembles In Vitro Properties of Melanoma Cancer Stem Cells.

Despite key advances in cancer therapies, malignant tumors, such as melanoma, continue to be one of the leading causes of mortality. Recent debate on whether cancer can originate from a tumor-initiating subpopulation has permeated oncology and stem cell research. It has been well established that primary and immortalized tumor cells consist of heterogeneous cell populations. The profound effect of tumor heterogeneity on tumor growth and drug resistance remains elusive, but it is highly likely that subpopulations of cancer cells have different capabilities of self-renewal and drug resistance. Discrepancies between excellent in vitro potency and efficacy and poor patient response have been observed on multiple cancer therapeutics. Although this observation can be attributed to many factors, a better understanding of the contribution from subpopulations within a cancer will help bridge the gap between in vitro assay results and patient prognosis. To comprehend this impact, it is critical to isolate and characterize cancer subpopulations that possess higher growth and drug resistance properties so that novel therapeutics can be developed to eventually eradicate all cancer cells. In this article, we describe a method to enrich a subpopulation, CB4, from the melanoma cell line WM115. CB4 exhibited higher anchorage-independent growth, higher survival under serum starvation condition, and lower drug sensitivity to commonly used melanoma treatment compared with WM115. Details of functional properties and gene expression of CB4 compared with WM115 are reported. Our study demonstrates that it is feasible to isolate and enrich a subpopulation that exhibits higher growth capacity and treatment resistance from an immortalized tumor cell line.

A software framework enabling analysis of plate-based flow cytometry data for high-throughput screening.

Flow cytometry (FCM) is an important technology with a broad spectrum of applications ranging from basic research to clinical diagnostics. In a typical FCM experiment, thousands of cells are queried with respect to size, shape, and abundance of multiple cell surface antigens. Recent advances in FCM techniques and instrumentation have enabled researchers to raise the throughput of experimentation dramatically. However, data analysis has remained a time-consuming activity requiring significant manual intervention for gating as well as for overall data reduction and interpretation. Presented in this article is a novel, algorithmically flexible, internally developed, software framework for the analysis of plate-based FCM data for high-throughput screening (HTS). Utilizing a post-treatment pooling strategy, >87,000 individual wells representing over 240,000 compounds were automatically gated, percent of control (POC) calculated, results assembled, deconvolved, and sorted, allowing researchers to visually assess wells of interest in minutes.

Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival.

Previous studies have shown that DNA methyltransferase (Dnmt) 1 is required for maintenance of bulk DNA methylation and is essential for mouse development. However, somatic disruption of DNMT1 in the human cancer cell line HCT116 was not lethal and caused only minor decreases in methylation. Here, we report the identification of a truncated DNMT1 protein, which was generated by the disruption of DNMT1 in HCT116 cells. The truncated protein, which had parts of the regulatory N-terminal domain deleted but preserved the catalytic C-terminal domain, was present at different levels in all DNMT1 single-knockout and DNMT1/DNMT3b double-knockout cell lines tested and retained hemimethylase activity. DNMT1 RNAi resulted in decreased cell viability in WT and knockout cells and further loss of DNA methylation in DNMT1 knockout cells. Furthermore, we observed a delay in methylation after replication and an increase in hemimethylation of specific CpG sites in cells expressing the truncated protein. Remethylation studies after drug-induced hypomethylation suggest a putative role of DNMT1 in the de novo methylation of a subtelomeric repeat, D4Z4, which is lost in cells lacking full-length DNMT1. Our data suggest that DNMT1 might be essential for maintenance of DNA methylation, proliferation, and survival of cancer cells.

Cateterización venosa central a través de la vena yugular externa / Central venous Cateterización through the external juglar vein

Presenta un estudio prospectivo, descriptivo, transversal, recopila datos de cateterización venosa central a través de la vena yugular externa en 46 pacientes en estado crítico en quienes se realizó cirugías de emergencia y se estimo podrían ocurrir pérdidas sanguíneas importantes y por manejo hemodinámico e hidroelectrolítico de cuidado por patología asociada o camulación venosa difícil. Se planteó la yugular externa como alternativa a la colocación de un catéter en la vena yugular interna o vena subclavia, las mismas que de acuerdo a las estadísticas mundiales, trae consigo un alto porcentaje de complicaciones pospunción que en muchas ocasiones comprometen la vida del enfermo, así como el grado de dificultad que estas técnicas conllevan para personal no experimentado. A la luz de los resultados: porcentaje de certeza en el acceso al sistema venoso central del 91xciento, comprobado radiológicamente con ubicación correcta de la punta del catéter en un 92.8xciento; tiempo de ejecución promedio de 5.4 minutos; porcentaje de complicaciones del 2.1xciento, referido a la formación de hematoma autolimitado en el sitio de punción; hacen de la técnica una vía alternativa de gran utilidad en el manejo de pacientes en el perioperatorio.

Embarazo precoz y maternidad en solteras como variables de paternidad responsable y su relación con inadecuada educación sexual / Unmarried precocious pregnancy and maternity in like variables of responsible paternity and their relation with inadequate sexual education

Expone que mediante el presente estudio se establece y confirma una clara relación sexual entre la deficiente educación sexual y la alta incidencia de embarazo precoz y maternidad en solteras, dos de los principales problemas detectados en las poblaciones de Mira y la Libertad (Provincia del Carchi). Se demuestra que el nivel educativo es semejante en las dos poblaciones, existiendo diferencias significativas con las publicadas a nivel nacional.

Semiología pediátrica en recién nacidos y lactantes / Pediactric semiology in new born and feeding on milk

En la pediatría, así como en otras ramas de la medicina se ha descuidado la enseñanza del examen metódico de los pacientes por lo que es importante insistiren practicar la exploración y transmitir a los médicos en formación, experiencias y conocimientos adquiridos al estudio y la práctica diaria. Que este trabajo sea de utilidad al estudiante en formación, al residente y todas aquellas personas que diariamente se enfrentan al delicado manejo del recién nacido y lactante, es el objetivo deseado al realizar esta obra donde se combinan los hallazgos clínicos producto de la anamnesis y el examen físico lo que permitirá el diagnóstico y manejo adecuado de nuestros niños