Live cell imaging reveals focal adhesions mechanoresponses in mammary epithelial cells under sustained equibiaxial stress

Mechanical stimuli play a key role in many cell functions such as proliferation, differentiation and migration. In the mammary gland, mechanical signals such as the distension of mammary epithelial cells due to udder filling are proposed to be directly involved during lactation and involution. However, the evolution of focal adhesions -specialized multiprotein complexes that mechanically connect cells with the extracellular matrix- during the mammary gland development, as well as the influence of the mechanical stimuli involved, remains unclear. Here we present the use of an equibiaxial stretching device for exerting a sustained normal strain to mammary epithelial cells while quantitatively assessing cell responses by fluorescence imaging techniques. Using this approach, we explored changes in focal adhesion dynamics in HC11 mammary cells in response to a mechanical sustained stress, which resembles the physiological stimuli. We studied the relationship between a global stress and focal adhesion assembly/disassembly, observing an enhanced persistency of focal adhesions under strain as well as an increase in their size. At a molecular level, we evaluated the mechanoresponses of vinculin and zyxin, two focal adhesion proteins postulated as mechanosensors, observing an increment in vinculin molecular tension and a slower zyxin dynamics while increasing the applied normal strain.Fil: Sigaut, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Von Bilderling, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Bianchi, Micaela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Burdisso, Juan Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; ArgentinaFil: Gastaldi, Laura Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; ArgentinaFil: Pietrasanta, Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentin

Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.Fil: Sigaut, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Bianchi, Micaela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Von Bilderling, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Pietrasanta, Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Antiproliferative effects of oxytocin and desmopressin on canine mammary cáncer cells

Neoplasms of the mammary gland represent the most frequent tumor type in the femaledog, and according to the histologic criteria, approximately 50% of them are malignant.In the most aggressive cases of mammary cancer, surgery is not enough to warrant afavorable outcome, and adjuvant therapies are needed to improve the patient?s overallsurvival. The aim of the present study was to evaluate the effects of two peptides onproliferation of a canine mammary cancer cell line derived from a simple carcinoma. Thecell line CMT-U27 was grown in 96-well plates, at two cell densities (4 × 103 and 8 × 103cells/well). Cultures were treated with oxytocin (OT) or desmopressin at five concentrations(10, 50, 100, 500, and 1000 nM). After 72 h of incubation, cell proliferation wasdetermined by the MTT assay. Results showed that with 4 × 103 cells/well, OT at 50,500, and 1000 nM was growth inhibitory for the cells, being statistically significant at1000 nM. On the contrary, no antiproliferative effect was observed with 10 or 100 nM.At 8 × 103 cells/well, OT showed a significant antiproliferative effect only with the highestconcentration (1000 nM). Desmopressin at 4 × 103 cells/well decreased cell viability atconcentrations of 50, 100, 500, and 1000 nM (statistically significant with the highestconcentration), while no effect was observed with 10 nM. With 8 × 103 cells/well, thispeptide reduced cell growth at 100, 500, and 1000 nM. In conclusion, we suggest thatthese peptides may be potential and promising compounds for the treatment of dogswith simple carcinomas of the mammary gland. In vivo studies are required to confirmthis hypothesis.Fil: Benavente, Micaela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Bianchi, Carolina Paula. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Imperiale, Fernanda Andrea. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Aba, Marcelo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; Argentin

Retroperitoneoscopic single site renal biopsy surgery: right indicatioBMC Urology ns for the right technique.

Bacground: Laparoendoscopic single-site surgery (LESS) has been developed in an attempt to further reduce the morbidity and scarring associated with laparoscopic surgery. In patients in whom there are indications to perform a laparoscopic renal biopsy, LESS surgery is a valid alternative to mini invasive surgery and is becoming more common. We report our experience on 14 renal biopsy procedures performed in a retroperitoneal LESS. Methods: LESS renal biopsy was performed in 14 patients 18 to 80 years old (mean age 58.3 years) during a 36 month period. All procedures were performed by a single operator. The patient was in a standard flank position. The procedure was performed using a 2.5 cm, single incision via a retroperitoneal access at the Petit ’ s triangle. A 5 mm biopsy forceps was used to collect the specimen under direct vision, and haemostasis was obtained with an Argon beam probe and the application of oxidized regenerated cellulose gauze. Results: Biopsy was performed successfully in all cases. Mean operative time was 52.64 min, blood loss was minimal, and the hospital stay ranged from 12 to 24 hours. None of the patients required narcotics or additional analgesia in the postoperative period. No postoperative complications occurred. Conclusions: The LESS technique is safe, reliable (100% success), easy to learn, and offers subjective cosmetic benefits to the patient. Minimal hospitalization requirement following retroperitoneal LESS biopsy is an additional timely advantage over laparoscopic renal biopsy. We think that with the right indications (marked obesity, failure of previous percutaneous biopsy attempts, a solitary kidney and coagulopathy) LESS renal biopsy is a good alternative to laparoscopy. Our next step will be a randomized prospective study of LESS compared with laparoscopy for renal biopsy to support our findings

Estradiol-17β Injection Induces Ovulation in Llamas

This study aimed to investigate the effect of three different doses of estradiol-17b onovulation and subsequent luteal development and function in llamas. Twenty-three llamaswere examined daily by transrectal ultrasonography until the detection of an ovulatoryfollicle (≥8mm). Thereafter, animals were divided into five groups: Control (n = 3; treatedwith 1.6ml of saline solution), GnRH group (n = 6, treated with an intravenous injectionof 8.4 μg Buserelin), and estradiol groups that received 0.6mg (E1, n = 4), 1mg (E2,n = 4), or 1.6mg (E3, n = 6) of estradiol-17b intravenously. Detection of ovulationwas based on ultrasonographic visualization of disappearance of the largest follicleand subsequent presence of a newly formed corpus luteum (CL) and progesteroneconcentration exceeding 1 ng ml−1. Daily blood samples were collected to determineplasma progesterone concentration. Ovulation rate was 0% for control and E1 groups,25% for E2 group, and 100% for GnRH and E3 groups. Differences in the meanCL diameter between GnRH and E3 groups were not statistically significant. Plasmaprogesterone concentration was similar between groups during the different days inovulated animals. However, the day that the plasma progesterone concentration wasabove 1 ng ml−1 and the day that the highest plasma progesterone concentration wasachieved differed among E3 and GnRH groups, occurring later in females treated withestradiol. In conclusion, an injection of estradiol-17b is capable of inducing ovulation inllamas and the response depends on the dose used. Most of the animals required thehighest tested dose (1.6mg) to induce the ovulatory process. Although the CL diameterin females induced to ovulate with estradiol was similar to that in llamas induced toovulate with a GnRH analog, the rise in plasma progesterone concentration above 1 ngml−1 and the peak progesterone concentration were attained 1 day later in the estradioltreated females.Fil: Bianchi, Carolina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Benavente, Micaela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Viviani, Florencia. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Gallelli, Maria Florencia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aba, Marcelo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentin

Development of corpus luteum susceptibility to an analog of prostaglandin F 2α, throughout the luteal phase in llamas (Lama glama)

The aim of the present study was to evaluate the susceptibility of the corpus luteum to d-cloprostenol (synthetic analog of PGF 2α) throughout the luteal phase in llamas. Female llamas (n=43) were induced to ovulate by GnRH injection in the presence of an ovulatory follicle and randomly assigned into one of six groups: control and treated with an injection of d-cloprostenol on Day 3, 4, 5, 6 or 8 post GnRH. Blood samples were collected to determine plasma progesterone concentrations. There was no effect of treatment on animals injected on Day 3 or 4 post-GnRH. In animals treated on Day 5, different responses were observed. No effect of treatment was recorded in 27% of the animals whereas 55% of the llamas showed a transitory decrease followed by a recovery in plasma progesterone concentrations after d-cloprostenol injection, indicative of a resurgence of the corpus luteum, extending the luteal phase a day more than in control animals. In the remaining 18% of the animals injected on Day 5, (corresponding to those exhibiting the greatest plasma progesterone concentrations at the day of injection), complete luteolysis was observed. Plasma progesterone concentrations decreased to below 1ngml -1 24h after d-cloprostenol in llamas injected on Day 6 or 8 post-GnRH. In conclusion, the corpus luteum of llamas is completely refractory to PGF 2α until Day 4 after induction of ovulation, being partially sensitive by Day 5 and fully responsive to PGF 2α, by Day 6 after induction of ovulation.Fil: Bianchi, Carolina Paula. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; ArgentinaFil: Cavilla, María Verónica. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; ArgentinaFil: Jorgensen, E.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Benavente, Micaela Andrea. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; ArgentinaFil: Aba, Marcelo Alfredo. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; Argentin

Una experiencia innovadora en el curso de Fisiología del sistema nervioso de la carrera de Medicina Veterinaria

El curso de Fisiología del sistema nervioso y muscular de la Facultad de Ciencias Veterinarias, UNCPBA tiene como principio considerar al estudiante como sujeto de conocimiento, buscando que el estudiante vivencie en su experiencia de aprendizaje la construcción de los saberes disciplinares en su estrecha relación con el futuro ejercicio profesional evitando el aprendizaje descontextualizados y centrado en la memorización. Por ello, el equipo docente propone una estrategia de evaluación que se integre al proceso de enseñanza - aprendizaje, que dé cuenta del mismo y que exprese coherencia con los objetivos del curso. Así, se propone incorporar como herramienta de evaluación y acreditación, la realización de un Trabajo Práctico Integrador de Fisiología del Sistema Nervioso (TPIN) como actividad integradora de los contenidos, que promueva la construcción de los mismos en el análisis de una situación real contextualizada, que requiere del desarrollo de capacidades y habilidades de pensamiento complejas que se desplieguen a lo largo del proceso hasta culminar en la producción de un trabajo académico escrito y grupal. Esta propuesta resulta innovadora porque considera al estudiante como protagonista de su propio aprendizaje y permite una evaluación basada en la reflexión, el intercambio y en la adquisición de un aprendizaje significativo.Especialización en Docencia Universitari

Layer-by-Layer Assembled Microgels Can Combine Conflicting Properties: Switchable Stiffness and Wettability without Affecting Permeability

Responsive interfacial architectures of practical interest commonly require the combination of conflicting properties in terms of their demand upon material structure. Switchable stiffness, wettability, and permeability, key features for tissue engineering applications, are in fact known to be exclusively interdependent. Here, we present a nanoarchitectonic approach that decouples these divergent properties by the use of thermoresponsive microgels as building blocks for the construction of three-dimensional arrays of interconnected pores. Layer-by-layer assembled poly(N-isopropylacrylamide-co-methacrylic acid) microgel films were found to exhibit an increase in hydrophobicity, stiffness, and adhesion properties upon switching the temperature from below to above the lower critical solution temperature, whereas the permeability of redox probes through the film remained unchanged. Our findings indicate that the switch in hydrophilicity and nanomechanical properties undergone by the microgels does not compromise the porosity of the film, thus allowing the free diffusion of redox probes through the polymer-free volume of the submicrometer pores. This novel approach for decoupling conflicting properties provides a strategic route for creating tailorable scaffolds with unforeseen functionalities.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada