c-Abl Kinase Is Required for Satellite Cell Function Through Pax7 Regulation.

Satellite cells (SCs) are tissue-specific stem cells responsible for adult skeletal muscle regeneration and maintenance. SCs function is critically dependent on two families of transcription factors: the paired box (Pax) involved in specification and maintenance and the Muscle Regulatory Factors (MRFs), which orchestrate myogenic commitment and differentiation. In turn, signaling events triggered by extrinsic and intrinsic stimuli control their function via post-translational modifications, including ubiquitination and phosphorylation. In this context, the Abelson non-receptor tyrosine kinase (c-Abl) mediates the activation of the p38 α/ß MAPK pathway, promoting myogenesis. c-Abl also regulates the activity of the transcription factor MyoD during DNA-damage stress response, pausing differentiation. However, it is not clear if c-Abl modulates other key transcription factors controlling SC function. This work aims to determine the role of c-Abl in SCs myogenic capacity via loss of function approaches in vitro and in vivo. Here we show that c-Abl inhibition or deletion results in a down-regulation of Pax7 mRNA and protein levels, accompanied by decreased Pax7 transcriptional activity, without a significant effect on MRF expression. Additionally, we provide data indicating that Pax7 is directly phosphorylated by c-Abl. Finally, SC-specific c-Abl ablation impairs muscle regeneration upon acute injury. Our results indicate that c-Abl regulates myogenic progression in activated SCs by controlling Pax7 function and expression.

USP7-dependent control of myogenin stability is required for terminal differentiation in skeletal muscle progenitors.

Satellite cells (SCs) are myogenic progenitors responsible for skeletal muscle regeneration and maintenance. Upon activation, SCs enter a phase of robust proliferation followed by terminal differentiation. Underlying this myogenic progression, the sequential expression of muscle regulatory transcription factors (MRFs) and the downregulation of transcription factor paired box gene 7 (Pax7) are key steps regulating SC fate. In addition to transcriptional regulation, post-translational control of Pax7 and the MRFs provides another layer of spatiotemporal control to the myogenic process. In this context, previous work showed that Pax7 is ubiquitinated by the E3 ligase neural precursor cell-expressed developmentally downregulated protein 4 and interacts with several proteins related to the ubiquitin-proteasome system, including the deubiquitinase ubiquitin-specific protease 7 (USP7). Although USP7 functions in diverse cellular contexts, its role(s) during myogenesis remains poorly explored. Here, we show that USP7 is transiently expressed in adult muscle progenitors, correlating with the onset of myogenin expression, while it is downregulated in newly formed myotubes/myofibers. Acute inhibition of USP7 activity upon muscle injury results in persistent expression of early regeneration markers and a significant reduction in the diameter of regenerating myofibers. At the molecular level, USP7 downregulation or pharmacological inhibition impairs muscle differentiation by affecting myogenin stability. Co-immunoprecipitation and in vitro activity assays indicate that myogenin is a novel USP7 target for deubiquitination. These results suggest that USP7 regulates SC myogenic progression by enhancing myogenin stability.

Detección y tipificación de virus del papiloma humano en biopsias de carcinoma ductal infiltrante y lesiones benignas de mama en mujeres venezolanas

Objetivo: Realizar la detección y tipificación del virus del papiloma humano (VPH) en muestras de biopsias de tejido mamario con carcinoma ductal infiltrante. Métodos: Estudio descriptivo de corte transversal de 57 biopsias de carcinoma ductal infiltrante, y 41 biopsias de lesiones benignas de mama de pacientes venezolanas, estas fueron evaluadas utilizando la técnica PCR-RFLP en busca de la presencia del genoma del virus de papiloma humano. El riesgo OR fue evaluado mediante análisis estadístico con el paquete SPSS 12.0. Resultados: Treinta y tres (57,9 %) de las muestras de carcinoma ductal infiltrante tuvieron un resultado positivo para virus de papiloma humano, 19 de ellas pudieron ser tipificadas como: VPH-6b 15,15 %; VPH-11 3,03 %; VPH-18 12,12 %; VPH-33 27,27 %; VPH-45 3,03 % y VPH-58 3,03 %; de este grupo el 42,4 % fueron positivas no determinadas para la presencia de ADN del virus. Seis biopsias de lesiones benignas (14,6 %), presentaron infección por virus de papiloma humano, determinándose para ellas los tipos VPH-6b 33,33 %, VPH-11 16,67%, VPH-33 16,67% y 33,33 % positivas no determinadas. Se determinó estadísticamente que la presencia de virus de papiloma humano en tejido mamario aumenta 10,77 veces la posibilidad de desarrollar carcinoma ductal infiltrante. Conclusiones: Los hallazgos corroboran los resultados de otros investigadores, colocando al virus de papiloma humano como posible agente involucrado en la inmortalización de las células epiteliales de la mama.

Objective: To perform the detection and typing of human papilloma (HPV) virus in biopsy samples of breast tissue invasive ductal cancer. Methods: Cross-sectional study of 57 biopsies of invasive ductal carcinoma, and 41 biopsies of benign breast lesions of Venezuelan patients were evaluated using the PCR-RFLP technique for the presence of the human papillomavirus genome. The OR risk was evaluated by statistical analysis using SPSS package. Results: Thirty-three (57.9%) of invasive ductal carcinoma samples had a positive result for human papillomavirus, 19 of them could be classified as: HPV-6b 15.15%; HPV-11 3.03%; HPV-18 12.12%; HPV-33 27.27%; HPV-45 3.03% and HPV-58 3.03%. This group 42.4% were positive not determined for the presence of virus DNA. Six biopsies of benign lesions (14.6%) had human papillomavirus infection, determining for themselves the types HPV-6b 33.33%, 16.67% HPV-11, HPV-33 16.67% and 33.33% not determined positive. It is statistically determined that the presence of human papillomavirus in breast tissue 10.77 times increases the possibility of developing invasive ductal carcinoma. Conclusions: These findings corroborate the results of other researchers, placing human papillomavirus as a possible agent involved in the immortalization of epithelial cells of the breast.

Estandarización de un sistema PCR multiplex para la determinación molecular de infertilidad masculina idiopática

Objetivo: Diseñar y optimizar sistemas de reacción en cadena de polimerasa individuales y multiplex para la detección de microdeleciones de genes asociados a infertilidad masculina en el cromosoma Y. Métodos: Se estandarizaron sistemas de reacción en cadena de polimerasa multiplex utilizando oligonucleótidos STS (Sequence Target Site) específicos asociados a infertilidad masculina, con previa estandarización de cada par de oligos en reacciones individuales. Resultados: Se logró estandarizar 7 sistemas individuales y 2 sistemas multiplex de alta sensibilidad y especificidad que pueden indicar la presencia o ausencia de un gen, en este caso, son utilizados para indicar la mutación por microdeleción de algún fragmento específico de Yq que conlleva a la inactivación de un gen. Conclusiones: Se pudo realizar la estandarización de dos sistemas multiplex para el análisis de microdeleciones del cromosoma Y asociados a infertilidad masculina como una herramienta molecular para el diagnóstico rápido y preciso de esta patología. El amplificado del marcador RBM1 no pudo ser incluido en ninguna de los dos multiplex estandarizados, no obstante, se sugiere el estudio de otros marcadores de infertilidad masculina que puedan ser incluidos en la estandarización de nuevos multiplex.

Objective: To design and optimize individual systems and multiplex polymerase chain reaction for detection of microdeletions of male infertility associated genes on the Y chromosome. Methods: multiplex polymerase chain reaction systems were standardized using oligonucleotides STS (Sequence Target Site) specific to male infertility associated with prior standardization of each pair of oligos in individual reactions. Results: It was possible to standardize 7 individual systems and two multiplex systems high sensitivity and specificity that may indicate the presence or absence of a gene, in this case, are used to indicate the mutation microdeletion of a specific fragment Yq leading to the inactivation of a gene. Conclusions: standardization could make two multiplex systems for the analysis of microdeletions of the Y chromosome associated with male infertility as a molecular tool for rapid and accurate diagnosis of this disease. The amplified RBM1 marker could not be included in either standard multiplex, despite the studies of other markers of male infertility is suggested to be included in new in the standardization of new multiplexes.

Electroforesis horizontal en geles de poliacrilamida en la detección y tipificación del virus de papiloma humano / Horizontal electrophoresis in polyacrylamide gels in the detection and typing of human papillomavirus

OBJETIVO: estandarizar el uso de electroforesis horizontal en poliacrilamida como un método rápido de fácil implementación y de alta sensibilidad para la detección y tipificación del virus del papiloma humano por reacción en cadena de la polimerasa-polimorfismos de longitud de fragmentos de restricción con la enzima HpyCH4V como única enzima de restricción. MÉTODOS: Se utilizó el ácido desoxirribonucleico de 17 tipos de virus del papiloma humano, clonados en la colección del Laboratorio de Biología y Medicina Experimental (LABIOMEX-ULA), para la amplificación de la región flanqueada por los oligonucleótidos MY09/ MY11, los amplificados obtenidos se sometieron a digestión enzimática con la enzima HpyCH4V. El producto se corrió en geles de poliacrilamida de rápida polimerización en equipos de electroforesis horizontales. El ácido desoxirribonucleico se tiñó con bromuro de etidio y fueron fotografiados con la utilización de un trans-iluminador de luz ultravioleta. RESULTADOS: Se corrieron muestras de 17 tipos diferentes de virus del papiloma humano en geles de poliacrilamida en electroforesis horizontal sudmarina. Se observaron patrones de digestión, con la enzima de restricción HpyCH4V, bien definidos y distintos para 15 tipos diferentes. Se presentó una coincidencia de patrón polimorfismos de longitud de fragmentos de restricción para los tipos 45 y 52 ambos de alto riesgo. Se obtuvo una excelente resolución en corridas de 2,5 cm de longitud para cualquier patrón polimorfismos de longitud de fragmentos de restricción de los 17 tipos de virus del papiloma humano analizados. CONCLUSIÓN: El análisis de las corridas permitió una eficiente caracterización de los 17 tipos virales. La comparación del índice de movilidad relativa con polimorfismos de longitud de fragmentos de restricción virtual de los fragmentos amplificados presentó diferencias mínimas, el análisis de la movilidad en agarosa y poliacrilamida se ajustaron perfectamente permitiendo la sustitución de la agarosa por la poliacrilamida.

OBJECTIVE: to standardize the use of horizontal electrophoresis in polyacrylamide as a quick method of easy implementation and high sensitivity for the detection and typing of Human Papillomavirus by PCR-FRLP with HpyCH4V enzyme as unique restriction enzyme. METHODS: DNA from 17 Human Papillomavirus types, cloned in the collection of the Laboratory of Experimental Biology and Medicine, for amplification of the region flanked by the MY09/ MY11 oligonucleotides were used, the amplified obtained were subjected to enzymatic digestion with the enzyme HpyCH4V. The product was run on polyacrylamide gels rapid polymerization horizontal electrophoresis equipment. Stained with ethidium bromide and were photographed with the use of a trans-illuminating UV. RESULTS: Samples of 17 different Human Papillomavirus types polyacrylamide gel electrophoresis were run horizontally. Digestion patterns were observed with the restriction enzyme HpyCH4V, well-defined and different for different types 15. A pattern match for RFLP types 45 and 52 both high risk presented. Excellent resolution on runs of 2.5 cm in length for any RFLP pattern of the 17 Human Papillomavirus types analyzed was obtained. CONCLUSION: The analysis of the runs allows efficient characterization of the 17 Human Papillomavirus types. Comparing the relative mobility rate with the virtual RFLP of amplified fragments present minimal differences, analyzing mobility in agarose and polyacrylamide perfectly adjusted allowing for substitution of agarose by polyacrylamide.

Pneumotórax hipertensivo na sala de recuperação pós-anestésica: relato de caso / Tension pneumothorax in post-anesthetic care unit: case report

JUSTIFICATIVA E OBJETIVOS: Há 100 por cento de prevalência de pneumotórax nos casos de trauma penetrante torácico, sendo que o pneumotórax hipertensivo pode ser desencadeado, entre outras causas, por uma lesão pulmonar não diagnosticada inicialmente, ou associado à ventilação mecânica, apresentando alta taxa de mortalidade. O objetivo deste relato é apresentar um caso de pneumotórax hipertensivo diagnosticado na Sala de Recuperação Pós-Anestésica (SRPA). RELATO DO CASO: Paciente do sexo masculino, 34 anos, estado físico ASA I E, vítima de lesões por arma de fogo. Foi submetido a laparotomia exploradora e exploração de artéria e veia femorais à direita, sob anestesia geral balanceada com indução em seqüência rápida, com estabilidade hemodinâmica durante todo o procedimento cirúrgico. Na SRPA, apresentou instabilidade hemodinâmica, com insuficiência respiratória, sudorese, taquicardia e hipertensão arterial. A tomografia computadorizada de tórax evidenciou hemopneumotórax à direita, sendo imediatamente drenado. Foi transferido para o Centro de Terapia Intensiva, apresentando melhora progressiva do quadro, com alta hospitalar, sem seqüelas, após 22 dias. CONCLUSÕES: O pneumotórax hipertensivo é uma doença letal que pode ser facilmente reconhecida através dos exames clínico e radiológico; devendo ser sempre suspeitado na presença de traumatismo torácico, e neste caso, deve-se realizar imediatamente a drenagem de tórax antes da ventilação mecânica e de procedimentos cirúrgicos.

[Tension pneumothorax in post-anesthetic care unit: case report.]. / Pneumotórax hipertensivo na sala de recuperação pós-anestésica: relato de caso.

BACKGROUND AND OBJECTIVES: The incidence of pneumothorax after penetrating chest trauma is 100%. Tension pneumothorax, a high mortality rate condition, may be triggered, among other causes, by pulmonary injury not previously identified, and may also be associated to mechanical ventilation. This report presents a case of tension pneumothorax diagnosed in the Post-Anesthetic Care Unit (PACU). CASE REPORT: A 34-year-old black male patient, physical status ASA I E, victim of gunshot wound was submitted to explorative laparotomy and right femoral artery and vein exploration under general balanced anesthesia with rapid sequence induction. The patient kept hemodynamically stable throughout the procedure. However, in the PACU patient presented hemodynamic instability with respiratory failure, sweating, tachycardia and hypertension. Chest CT-scan revealed right hemopneumothorax, which was immediately drained. Patient was then transferred to the Intensive Care Unit, where he progressively improved to be discharged from the hospital 22 days later, without sequelae. CONCLUSIONS: Tension pneumothorax is a fatal condition which may be easily identified through clinical and radiological evaluations. It should be always suspected in the presence of chest trauma and, in this case, immediate chest drainage should be performed prior to mechanical ventilation or any surgical procedure.