Shp2 Knockdown and Noonan/LEOPARD Mutant Shp2–Induced Gastrulation Defects

Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown–induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome

Sheep-Specific Immunohistochemical Panel for the Evaluation of Regenerative and Inflammatory Processes in Tissue-Engineered Heart Valves

The creation of living heart valve replacements via tissue engineering is actively being pursued by many research groups. Numerous strategies have been described, aimed either at culturing autologous living valves in a bioreactor (in vitro) or inducing endogenous regeneration by the host via resorbable scaffolds (in situ). Whereas a lot of effort is being invested in the optimization of heart valve scaffold parameters and culturing conditions, the pathophysiological in vivo remodeling processes to which tissue-engineered heart valves are subjected upon implantation have been largely under-investigated. This is partly due to the unavailability of suitable immunohistochemical tools specific to sheep, which serves as the gold standard animal model in translational research on heart valve replacements. Therefore, the goal of this study was to comprise and validate a comprehensive sheep-specific panel of antibodies for the immunohistochemical analysis of tissue-engineered heart valve explants. For the selection of our panel we took inspiration from previous histopathological studies describing the morphology, extracellular matrix composition and cellular composition of native human heart valves throughout development and adult stages. Moreover, we included a range of immunological markers, which are particularly relevant to assess the host inflammatory response evoked by the implanted heart valve. The markers specifically identifying extracellular matrix components and cell phenotypes were tested on formalin-fixed paraffin-embedded sections of native sheep aortic valves. Markers for inflammation and apoptosis were tested on ovine spleen and kidney tissues. Taken together, this panel of antibodies could serve as a tool to study the spatiotemporal expression of proteins in remodeling tissue-engineered heart valves after implantation in a sheep model, thereby contributing to our understanding of the in vivo processes which ultimately determine long-term success or failure of tissue-engineered heart valves

La trigonometría como herramienta para medir nuestro entorno

En esta experiencia de aula se presenta el trabajo de un grupo de estudiantes de grado décimo que realizaron una actividad en la clase de trigonometría en la que aplicaron conceptos trigonométricos para calcular las medidas de las instalaciones de la institución educativa a la cual pertenecen. El objetivo es mostrar un ejemplo de cómo se puede generar un ambiente de aprendizaje en el que los estudiantes puedan elaborar significados de objetos matemáticos como lo son las razones trigonométricas mediante una labor que permita la aplicación fundamental de la trigonometría realizando mediciones indirectas

Tissue properties and collagen remodeling in heart valve tissue engineering

Valvular heart disease is a major health problem worldwide causing morbidity and mortality. Heart valve replacement is frequently applied to avoid serious cardiac, pulmonary, or systemic problems. However, the current replacements do not consist of living tissue and, consequently, cannot grow, repair, or remodel in response to changing functional demands. Heart valve tissue engineering (HVTE) seeks to overcome the shortcomings of the existing valve replacements by creating living autologous heart valves. One of the main challenges of HVTE is to control tissue formation, collagen remodeling and consequent tissue mechanical properties during the in vitro culture phase. Additionally, it is important to define benchmarks based on the target native heart valve tissues to compare with the tissue structure and mechanical properties of tissue-engineered (TE) heart valves. The aim of this thesis is to define benchmarks, understand and optimize tissue development and resulting tissue mechanical properties of TE heart valves, with special emphasis on collagen remodeling. In order to provide insights into the evolution and maturation of the extracellular matrix and mechanical properties and to provide benchmarks for TE heart valves, matrix composition, maturation and mechanical properties of native human aortic and pulmonary heart valves were studied. It was observed that the matrix composition and the mechanical properties change with age and that a significant part of the mechanical behaviour of the human native heart valve leaflets is defined by the composition and maturation of the matrix. Tissue (mechanical) properties of TE heart valves should be optimized towards the provided benchmarks during the in vitro culture phase. To this end, possible indicators of in vitro tissue outcome were determined to enable prediction of the properties of the autologous tissues cultured for individual patients. It was found that a-Smooth muscle actin (aSMA) might be such an indicator. In addition, interspecies differences in tissue (mechanical) properties were evaluated to determine whether ovine TE heart valves are representative of human TE heart valves as the ovine model is the prescribed animal model to evaluate heart valve replacements. This study suggested that the culture process of ovine tissue can be controlled, whereas the mechanical properties, and hence functionality, of tissues cultured with human cells are more difficult to predict, indicating once more the importance of early markers to predict tissue outcome. As a further step towards clinical application and to circumvent the use of animal-derived medium components in the culture protocol, fetal bovine serum was replaced by human platelet lysate for the culture of autologous TE heart valve constructs. Although tissue composition and maturation were similar, mechanical properties were much lower for the tissues cultured in platelet lysate, most likely due to an increased production of matrix-degrading enzymes leading to an altered collagen architecture. Thus, collagen architecture, rather than collagen content alone, is dominant in defining the tissue mechanical properties. To stimulate tissue formation and maturation towards the right collagen architecture for in vivo mechanical functionality, mechanical conditioning of the engineered tissue is commonly pursued. Previous studies indicated that intermittent conditioning, in which cyclic and static strain are alternated, is favoured to obtain mature tissues in a short time period. To unravel the underlying mechanism of intermittent conditioning, the effects of cyclic strain and static strain after cyclic strain were examined at gene expression level. This study indicated that a period of static strain is required for collagen synthesis and remodeling, while continuous cyclic strain shifts this balance towards collagen remodeling and maturation. These results imply that the mechanical conditioning protocol should change over time from intermittent conditioning to continuous cyclic strain to improve collagen maturation after its synthesis and, therewith, the mechanical properties of TE heart valves. In summary, the results from this thesis suggest that in addition to collagen content and maturation, collagen organization is particularly important in defining the tissue mechanical properties. Thus, optimization of culture protocols should focus on obtaining the proper collagen architecture for creating mechanically functioning TE heart valves. Autologous culture of TE heart valves using human platelet lysate is not preferred, since it prevents the formation of a load-bearing organized collagen network. Mechanical conditioning protocols should start with intermittent conditioning, followed by continuous cyclic strain to enhance collagen maturation after its synthesis. Considering the interpatient variability in tissue outcome of tissues cultured with similar protocols, it must be noted that further refinement, or even personalization, of culture protocols might be necessary. To this end, markers of tissue outcome, such as aSMA, are necessary to predict and adapt culture protocols and, therewith, individual tissue outcome at an early stage during culture. Although these suggestions require additional (in vivo) study, the results of this thesis provide substantial insight on how to improve in vitro HVTE strategies to control tissue properties and collagen remodeling for optimization of TE heart valves towards their native counterparts

COVID-19 in challenge-based learning at TU/e

Challenge-based learning (CBL) forms the core of the educational vision of the Eindhoven University of Technology (TU/e) for 2030. It concerns an innovative type of learning where students work on real-life open-ended challenges that directly impact our world (e.g. related to United Nations’ Sustainable Development Goals), and where students take ownership of their learning. Besides deepening disciplinary knowledge and skills in context, students learn to collaborate with different disciplines and stakeholders, and deal with complex, open-ended processes. TU/e aims to implement CBL as an educational concept, a learning framework for all programs. TU/e innovation Space is the center of expertise for CBL and student entrepreneurship at TU/e, a learning hub for education innovation, and an open community where students, researchers, industry, and societal organizations can exchange knowledge and develop responsible solutions for real world challenges.In order to specifically target the challenges of our world today, the ‘TU/e against COVID-19’ brokering platform is set up. TU/e innovation Space is the broker for this platform with the purpose to match and to bundle initiatives, brainpower and services through a platform of supply and demand. As such, connecting students, researches and external parties, such as hospitals, companies and service providers.Since the launch of the platform end of March, a stream of initiatives has arisen. Amongst these are the development of a chatbot, which a hospital can use to simplify their internal question and answer process towards employees. This allows busy physicians to focus on primary care. Another example is a social distancing app, which aims to stimulate the distance standard of the 1.5-meter economy in a more natural way during and after the peak of the epidemic. Furthermore, a young TU/e student start-up has developed an application to determine the identity of who is behind a computer based on the pattern of strokes on the keyboard. This is an effective way to detect exam fraud in a period of massive digital education. A final example is a project in which students and researchers are working on an effective way to administer aerosol treatment to small children, without infecting nurses.Although this initiative is relatively new, the formula of TU/e innovation Space is already proving its strength. Apart from the applications that result from it, it has also showed to be an excellent way to stay connected as students and TU/e staff in these difficult times of COVID-19

Sheep-Specific Immunohistochemical Panel for the Evaluation of Regenerative and Inflammatory Processes in Tissue-Engineered Heart Valves

The creation of living heart valve replacements via tissue engineering is actively being pursued by many research groups. Numerous strategies have been described, aimed either at culturing autologous living valves in a bioreactor (in vitro) or inducing endogenous regeneration by the host via resorbable scaffolds (in situ). Whereas a lot of effort is being invested in the optimization of heart valve scaffold parameters and culturing conditions, the pathophysiological in vivo remodeling processes to which tissue-engineered heart valves are subjected upon implantation have been largely under-investigated. This is partly due to the unavailability of suitable immunohistochemical tools specific to sheep, which serves as the gold standard animal model in translational research on heart valve replacements. Therefore, the goal of this study was to comprise and validate a comprehensive sheep-specific panel of antibodies for the immunohistochemical analysis of tissue-engineered heart valve explants. For the selection of our panel we took inspiration from previous histopathological studies describing the morphology, extracellular matrix composition and cellular composition of native human heart valves throughout development and adult stages. Moreover, we included a range of immunological markers, which are particularly relevant to assess the host inflammatory response evoked by the implanted heart valve. The markers specifically identifying extracellular matrix components and cell phenotypes were tested on formalin-fixed paraffin-embedded sections of native sheep aortic valves. Markers for inflammation and apoptosis were tested on ovine spleen and kidney tissues. Taken together, this panel of antibodies could serve as a tool to study the spatiotemporal expression of proteins in remodeling tissue-engineered heart valves after implantation in a sheep model, thereby contributing to our understanding of the in vivo processes which ultimately determine long-term success or failure of tissue-engineered heart valves

Sheep-specific immunohistochemical panel for the evaluation of regenerative and inflammatory processes in tissue-engineered heart valves

\u3cp\u3eThe creation of living heart valve replacements via tissue engineering is actively being pursued by many research groups. Numerous strategies have been described, aimed either at culturing autologous living valves in a bioreactor (in vitro) or inducing endogenous regeneration by the host via resorbable scaffolds (in situ). Whereas a lot of effort is being invested in the optimization of heart valve scaffold parameters and culturing conditions, the pathophysiological in vivo remodeling processes to which tissue-engineered heart valves are subjected upon implantation have been largely under-investigated. This is partly due to the unavailability of suitable immunohistochemical tools specific to sheep, which serves as the gold standard animal model in translational research on heart valve replacements. Therefore, the goal of this study was to comprise and validate a comprehensive sheep-specific panel of antibodies for the immunohistochemical analysis of tissue-engineered heart valve explants. For the selection of our panel we took inspiration from previous histopathological studies describing the morphology, extracellular matrix composition and cellular composition of native human heart valves throughout development and adult stages. Moreover, we included a range of immunological markers, which are particularly relevant to assess the host inflammatory response evoked by the implanted heart valve. The markers specifically identifying extracellular matrix components and cell phenotypes were tested on formalin-fixed paraffin-embedded sections of native sheep aortic valves. Markers for inflammation and apoptosis were tested on ovine spleen and kidney tissues. Taken together, this panel of antibodies could serve as a tool to study the spatiotemporal expression of proteins in remodeling tissue-engineered heart valves after implantation in a sheep model, thereby contributing to our understanding of the in vivo processes which ultimately determine long-term success or failure of tissue-engineered heart valves.\u3c/p\u3

Decreased mechanical properties of heart valve tissue constructs cultured in platelet lysate as compared to fetal bovine serum

In autologous heart valve tissue engineering, there is an ongoing search for alternatives of fetal bovine serum (FBS). Human platelet-lysate (PL) might be a promising substitute. In the present article, we aimed to examine the tissue formation, functionality, and mechanical properties of engineered three-dimensional tissue constructs cultured in PL as a substitute for FBS. Our results show that tissue constructs that were cultured in PL and FBS produce similar amounts of collagen, glycosoaminoglycans, and collagen crosslinks, and that the cellular phenotype remains unchanged. Nevertheless, mechanical testing showed that the ultimate tensile strength in PL constructs was on average approximately three times lower as compared to FBS (0.25 vs. 0.74¿MPa, respectively,