Peptidomimetic Polo-Box-Targeted Inhibitors that Engage PLK1 in Tumor Cells and Are Selective against the PLK3 Tumor Suppressor.

The polo-box domain (PBD) of PLK1 determines mitotic substrate recognition and subcellular localization. Compounds that target PLK1 selectively are required due to the tumor-suppressor roles of PLK3. A structure-activity analysis of the PBD phosphopeptide binding motif has identified potent peptides that delineate the determinants required for mimicry by nonpeptidic inhibitors and provide insights into the structural basis for the selectivity of inhibitors for the PLK1 PBD. Fragment-ligated inhibitory peptides (FLIPs) obtained through REPLACE have been optimized to enhance in vitro binding and a systematic analysis of selectivity for PLK1 vs PLK3 has been carried out for peptides and peptidomimetics. Furthermore, these more drug-like non-ATP-competitive inhibitors had on-target engagement in a cellular context, as evidenced by stabilization of PLK1 in a thermal-shift assay and by inhibition of the phosphorylation of TCTP, a target of PLK1. Investigation in cells expressing a mutant PLK1 showed that these cells are sensitive to PBD inhibitors but dramatically resistant to clinically investigated ATP-competitive compounds. These results further validate targeting the PBD binding site in the move towards PLK1 inhibitors that are active against tumors resistant to ATP inhibitors.

Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ.

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.

b-Annulated 1,4-dihydropyridines as Notch inhibitors.

The Notch signaling pathway is involved in cell proliferation and differentiation, and has been recognized as an active pathway in regenerating tissue and cancerous cells. Notch signaling inhibition is considered a viable approach to the treatment of a variety of conditions including colorectal cancer, pancreatic cancer, breast cancer and metastatic melanoma. The discovery that the b-annulated dihydropyridine FLI-06 (1) is an inhibitor of the Notch pathway with an EC50 ≈ 2.5 µM prompted us to screen a library of related analogs. After structure activity studies were conducted, racemic compound 7 was identified with an EC50 = 0.36 µM. Synthesis of individual enantiomers provided (+)-7 enantiomer with an EC50 = 0.13 µM, or about 20-fold the potency of 1.

Masa selar, no siempre un adenoma: presentación de 3 casos clínicos / A sellar mass, not always an adenoma: A report of 3 cases

El absceso hipofisario es un proceso infeccioso dentro de la silla turca, infrecuente, grave y de difícil diagnóstico debido a sintomatología variada e inespecífica. Presentamos 3 casos clínicos de abscesos hipofisarios, teniendo en común la presencia de lesiones preexistentes. Todos fueron intervenidos quirúrgicamente, 2 pacientes resultaron ser abscesos asépticos y en un paciente se aisló Aspergillus capsulatum. Presentaron buena evolución con el tratamiento médico pero con secuelas de hipopituitarismo. Es muy importante tener en cuenta el absceso hipofisario entre los diagnósticos diferenciales de las masas que se localicen en esa región debido a que el diagnóstico oportuno y el tratamiento correcto son relevantes para el pronóstico de estos pacientes.

Pituitary abscess is due to a severe and uncommon infection in the sella. It is difficult to diagnose due to varied and non-specific symptoms. A report is presented of 3 cases of pituitary abscess, which had the presence of pre-existing injuries in common. All were subjected to surgery, with aseptic abscesses found in 2 patients, and Aspergillus capsulatum was isolated in 1 patient. They showed good progress with medical treatment, but with sequelae of hypopituitarism. It is very important to consider the pituitary abscess in the differential diagnosis of the masses that are located in that region, as a timely diagnosis and proper treatment can be important for the prognosis of these patients.

Proceso de admisión al residentado médico en el Perú: características, preferencias y propuestas de los médicos que se preparan para rendir el examen de admisión 2011 / Admission competition for medical residency in Peru: characteristics, preferences, and proposals from physicians who are preparing for taking the national medical residency examination in 2011

Objetivo: Describir las características, preferencias y propuestas de los médicos que se preparan para rendir el examen, respecto al proceso de admisión vigente al residentado médico en el Perú. Material y método: Se realizó un estudio descriptivo, considerando una población referencial de 4 328 postulantes, según el examen de residentado médico 2010, utilizando el programa estadístico Epidat 3,1, con un nivel de confianza del 95, y precisión del 5, se obtuvo 353 como valor muestral. Utilizando un cuestionario autoaplicable y validado previamente en 20 médicos postulantes. Resultados: De los 416 médicos encuestados (mediana de 27 años) la mayoría procede de universidades del interior del país (52,6). El 42,5 trabaja actualmente, de éstos, el 79,7 en el sector privado y el 79,2 bajo la modalidad de locación de servicios. El 48,6 realizó el Servicio Rural Urbano Marginal en Salud (SERUMS) en establecimientos correspondientes al Quintil I (extrema pobreza). El 54,3 ha tenido una experiencia previa del proceso de admisión al residentado médico. El 48,6 está en desacuerdo con el proceso actual del residentado médico, 67,5 está a favor del examen único nacional, y 39,2 prefiere el concurso por especialidades. El 38,9 considera que la evaluación del examen debe ser realizada por el CONAREME y cada universidad. El 54,1 está en desacuerdo al incremento del número de vacantes cautivas y 78,1 expresó estar en desacuerdo ante la propuesta del Ministerio de Salud de implementar tiempo de servicio obligatorio adicional en el residentado médico en regiones de menor desarrollo del país. Conclusiones: La mayoría de médicos encuestados procede de universidades del interior del país, sin carga familiar importante, menos de la mitad trabaja y lo hace en su mayoría bajo la modalidad de locación de servicios.

Objetive: To describe features, preferences, and proposals from physicians that are preparing themselves to take the 2011 National Medical Residency Examination in Peru. Material and ethods: This is a descriptive study, considering a referential population including 4328 candidates, according to the 2010 National Medical Residency Examination. The Epidat 3.1 statistical software was used, with a 95 confidence limit, and a 5 precision value, and the simple size was established in 353 physicians. A self-applicable and previously validated questionnaire was previously used as a pilot in 20 candidates. Results: Of the 416 surveyed physicians (median age, 27 years), most of them come from provinces outside Lime (52.6). Nearly half of all physicians (42.5) are currently working, 79.5 of them in the private sector and 79.2 on an outsourcing basis. Nearly half of all candidates (48.6) performed their rural healthcare service (SERUMS, according to its Spanish initials) in extremely poor areas in Peru (Quintile I). More than half of the candidates (54.3) had already taken the Medical Residency Examination in the past. Nearly half of the candidates (48,6) do not agree with the current status for the National Medical Residency Examination, 67.5 prefer a single national examination, and 39,2 prefer that the contest is separated for each specialty. More than one third (38.9) considered that the examination should be led by CONAREME (National Commission for Medical Residency) and each Medical School. More than half of the candidates do not agree with the increase of the so-called captive places, and 78.1 disagreed with the proposal by the Ministry of Health to establish an additional mandatory period of work for medical specialists in the less developed areas in the country.

Reduced expression of the Na+/Ca2+ exchanger in adult cardiomyocytes via adenovirally delivered shRNA results in resistance to simulated ischemic injury.

The Na(+)/Ca(2+) exchanger (NCX) is proposed to be an important protein in the regulation of Ca(2+) movements in the heart. This Ca(2+) regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca(2+) overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to approximately 60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. Ca(2+) homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic Ca(2+) and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes.

Investigación de metapneumovirus humano en pacientes hospitalizados: estudio multicéntrico / Research of human metapneumovirus in hospitalized patients: a multicenter study

Introducción: las infecciones respiratorias agudas bajas (IRAB) son la primer causa de hospitalización a lo largo del año. La etiología viral es la más frecuente. El Metapneumovirus humano (MNVh) ha sido vinculado a las IRAB con aspirado negativo para Virus respiratorio sincicial (VRS) y Adenovirus (AD). Objetivo: determinar la prevalencia, epidemiología, clínica y severidad de las infecciones por MNVh, e intentar detectar un patrón radiológico relacionado con el mismo. Métodos: se estudiaron niños entre 0 y 2 años de edad internados por IRAB en el Centro Hospitalario Pereira Rossell, Hospital Central de las Fuerzas Armadas, Hospital Policial, Hospital Británico y Asociación Española Primera de Socorros Mutuos en el período 1 de abril al 30 de noviembre de 2006. Los aspirados nasofaríngeos fueron analizados en la Sección Virología de la Facultad de Ciencias. Resultados: se estudiaron 185 pacientes, obteniéndose 17 resultados positivos para MNVh (9,2%), con 9 coinfecciones con VRS. La mayoría de los pacientes tenían 6 meses o menos de edad. Las manifestaciones clínicas principales fueron polipnea, tirajes y sibilancias. Los principales hallazgos radiológicos fueron infiltrado intersticial difuso e hiperinsuflación. Ningún paciente requirió internación en unidad de cuidados intensivos y no hubo casos fatales. Conclusiones: la prevalencia, características clínicas y evolutivas de las infecciones por MNVh no mostraron diferencias frente a las producidas por el VRS.

A comparison of adenovirally delivered molecular methods to inhibit Na+/Ca2+ exchange.

The ability to use molecular biology tools to down-regulate Na+/Ca2+ exchanger (NCX) expression will allow us to better understand the regulation of Ca(i)2+ and contractility in heart. Three different techniques to deplete NCX expression were compared: short hairpin RNA (shRNA), antisense RNA and exchanger inhibitory peptide expression via adenoviral transfection. Our results demonstrate that the most efficient method to deplete NCX expression and activity from cardiomyocytes is shRNA. It is also possible to replace the endogenous NCX with alternative isoforms or mutant forms of the NCX. Adenovirally delivered shRNA is an efficient tool for the study of the NCX and could be adapted for many other cardiac proteins.

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload.

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.

Adenovirally delivered shRNA strongly inhibits Na+-Ca2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes.

The cardiac Na(+)-Ca(2+) exchanger (NCX1) is the main mechanism for Ca(2+) efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na(+)-Ca(2+) exchange activity. Exchanger expression was inhibited by up to approximately 94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca(2+) transient amplitude, a depressed rate of Ca(2+) rise and decline, elevated diastolic [Ca(2+)], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca(2+) pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.

Chlamydia pneumoniae stimulates proliferation of vascular smooth muscle cells through induction of endogenous heat shock protein 60.

Chlamydia pneumoniae infection has been linked with atherosclerosis. However, the mechanism responsible for the atherogenic effects of C pneumoniae remains unclear. Heat shock proteins (HSPs) have been found in atherosclerotic lesions. HSPs of HSP70 and HSP90 families are involved in the regulation of cell cycle progression and cell proliferation. We assessed the hypothesis that HSP60 is induced in vascular cells infected with C pneumoniae and stimulates cell proliferation. Rabbit vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were infected with C pneumoniae. Western blot analysis demonstrated the induction of endogenous HSP60 expression in C pneumoniae-infected VSMCs. C pneumoniae infection significantly increased the number of VSMCs, and the mitogenic effect correlated with the expression level of endogenous HSP60. In contrast to VSMCs, C pneumoniae infection had no effect on the expression level of HSP60 and did not stimulate cell proliferation in HUVECs. Exogenous addition of recombinant chlamydial HSP60 had no mitogenic effect on VSMCs and HUVECs. However, overexpression of HSP60 within VSMCs by infection with adenovirus encoding human HSP60 resulted in a significant increase in cell numbers compared with uninfected VSMCs. These results suggest that overexpression of endogenous HSP60 may be a central intracellular event responsible for the mitogenic effects induced by C pneumoniae infection. In addition to C pneumoniae, other infectious agents and atherogenic risk factors may also stimulate VSMC proliferation and contribute to the lesion formation through the induction of HSP60.