A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context

BACKGROUND: In silico analyses based on sequence similarities with animal channels have identified a large number of plant genes likely to encode ion channels. The attempts made to characterise such putative plant channels at the functional level have most often relied on electrophysiological analyses in classical expression systems, such as Xenopus oocytes or mammalian cells. In a number of cases, these expression systems have failed so far to provide functional data and one can speculate that using a plant expression system instead of an animal one might provide a more efficient way towards functional characterisation of plant channels, and a more realistic context to investigate regulation of plant channels. RESULTS: With the aim of developing a plant expression system readily amenable to electrophysiological analyses, we optimised experimental conditions for preparation and transformation of tobacco mesophyll protoplasts and engineered expression plasmids, that were designed to allow subcellular localisation and functional characterisation of ion channels eventually in presence of their putative (possibly over-expressed) regulatory partners. Two inward K(+ )channels from the Shaker family were functionally expressed in this system: not only the compliant KAT1 but also the recalcitrant AKT1 channel, which remains electrically silent when expressed in Xenopus oocytes or in mammalian cells. CONCLUSION: The level of endogenous currents in control protoplasts seems compatible with the use of the described experimental procedures for the characterisation of plant ion channels, by studying for instance their subcellular localisation, functional properties, structure-function relationships, interacting partners and regulation, very likely in a more realistic context than the classically used animal systems

A Simple and Practical Approach to Unit Testing: The JML and JUnit Way

Writing unit test code is labor-intensive, hence it is often not done as an integral part of programming. However, unit testing is a practical approach to increasing the correctness and quality of software; for example, the Extreme Programming approach relies on frequent unit testing. In this paper we present a new approach that makes writing unit tests easier. It uses a formal specification language\u27s runtime assertion checker to decide whether methods are working correctly, thus automating the writing of unit test oracles. These oracles can be easily combined with hand-written test data. Instead of writing testing code, the programmer writes formal specifications (e.g., pre- and postconditions). This makes the programmer\u27s task easier, because specifications are more concise and abstract than the equivalent test code, and hence more readable and maintainable. Furthermore, by using specifications in testing, specification errors are quickly discovered, so the specifications are more likely to provide useful documentation and inputs to other tools. We have implemented this idea using the Java Modeling Language (JML) and the JUnit testing framework, but the approach could be easily implemented with other combinations of formal specification languages and unit test tools

A renewable energy-centred research agenda for planning and financing Nexus development objectives in rural sub-Saharan Africa

In rural sub-Saharan Africa – the global poverty hotspot – the vast majority of cropland is rainfed only, resulting in reduced and unstable yields. Smallholder farmers account for 80% of agricultural production but they have limited access to relevant services to support both commercial operations and their livelihoods: more than two-thirds of rural dwellers have no access to electricity (crucial for crop irrigation, processing, and storage) and about 40% have no access to clean water. Previous research has analysed integrated technological and resource management approaches to tackle these overlapping development gaps. To finance and implement such transformations in resource-constrained settings, it is now crucial to understand the business and investment implications, also considering the strong regional population growth and the increasing frequency and intensity of climate extremes. Here, we lay out a research agenda that promotes the integration of multi-scale modelling excellence along the climate-water-renewable energy-agriculture-development Nexus and the creation of robust business models for private companies that can sustainably support private smallholder farmers of SSAin their effort to eradicate poverty and inequality. The proposed agenda is a cornerstone of the EC-H2020 project LEAP-RE RE4AFAGRI (“Renewable Energy for African Agriculture: Integrating Modelling Excellence and Robust Business Models”). In proposing the agenda, we highlight the importance of integrating energy access into the Nexus framework from both research and investment perspectives

A Simple and Practical Approach to Unit Testing: The JML and JUnit Way

Writing unit test code is labor-intensive, hence it is often not done as an integral part of programming. However, unit testing is a practical approach to increasing the correctness and quality of software; for example, the Extreme Programming approach relies on frequent unit testing. In this paper we present a new approach that makes writing unit tests easier. It uses a formal specification language\u27s runtime assertion checker to decide whether methods are working correctly, thus automating the writing of unit test oracles. These oracles can be easily combined with hand-written test data. Instead of writing testing code, the programmer writes formal specifications (e.g., pre- and postconditions). This makes the programmer\u27s task easier, because specifications are more concise and abstract than the equivalent test code, and hence more readable and maintainable. Furthermore, by using specifications in testing, specification errors are quickly discovered, so the specifications are more likely to provide useful documentation and inputs to other tools. We have implemented this idea using the Java Modeling Language (JML) and the JUnit testing framework, but the approach could be easily implemented with other combinations of formal specification languages and unit test tools

Muscle RING-finger 2 and 3 maintain striated-muscle structure and function

Background: The Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo. Methods: MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses. Results: DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts. Conclusions: The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo

Autobiography as unconventional history: Constructing the author

The experience of historians as autobiographers has led them to reconsider the nature of historical knowledge and the function of the historian as an intermediary between the past and present. In the new theoretical context of the social sciences and historiography, we can take this proposal further and consider autobiography as a valid form of history—or, at least, as ‘unconventional history’, understood as negotiations with history that transcend or subvert traditional chronological monographs, posit the ‘subjective’ as a useful form of knowledge, and engage the constructed nature of the text. Taking this hypothesis as a starting point, this article reads historians' autobiographical texts to explore if we can/should continue to defend the classic distinction between subject and object, historian scientist and historian author. In this article I compare the work of several historian autobiographers that permit us to identify different methodologies in approaching the story of the self that also reflects different theoretical conceptions of history. I argue that historians that may be considered ‘constructionist’, such as Fernand Braudel, Annie Kriegel, George Duby, and Eric Hobsbawm, design their autobiographies in the same way they articulate their historical texts: by foregrounding objectivity and establishing critical distance between the subject—the historian who narrates the story—and the object—one's own life. Unconventional or experimental approaches, such as those espoused by Robert Rosenstone, Dominick LaCapra, or Clifford Geertz, result in more self-conscious autobiographies, which are, paradoxically, often more realistic and more revealing of the epistemological nature of life writing. ----------------- La experiencia de los historiadores como autobiógrafos les ha llevado a reconsiderar la naturaleza del conocimiento histórico y la función del historiador como un intermediario entre el pasado y el presente. En el nuevo contexto teórico de las ciencias sociales y la historiografía podemos tomar esta propuesta más allá y considerar la autobiografía como una forma válida de historia-o, al menos, de historia ‘poco convencional’-, entendida como negociaciones con la historia que trascienden o subvierten las tradicionales monografías cronológicas, plantean lo "subjetivo" como una forma útil de conocimiento y participan de la naturaleza construida del texto. Tomando esta hipótesis como punto de partida, este artículo lee los textos autobiográficos de los historiadores para explorar si se puede / debe seguir defendiendo la clásica distinción entre sujeto y objeto, historiador científico e historiador escritor. En este artículo comparo el trabajo de varios historiadores autobiógrafos que nos permiten identificar las diferentes metodologías para acercarse a la historia del yo y que también reflejan las diferentes concepciones teóricas de la historia. Sostengo que los historiadores que pueden considerarse "constructivistas", como Fernand Braudel, Annie Kriegel, George Duby y Eric Hobsbawm, diseñan sus autobiografías de la misma forma que articulan sus textos históricos: poniendo en primer plano la objetividad y estableciendo una distancia crítica entre el sujeto -el historiador que narra la historia-y el objeto- la vida de cada uno. Enfoques no convencionales o experimentales, como los expuestos por Robert Rosenstone, Dominick LaCapra, o Clifford Geertz, resultan autobiografías más autoconscientes, que son, paradójicamente, a menudo más realistas y más reveladoras de la naturaleza epistemológica de la escritura de la vida