Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments

Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin.     

Long-term Changes and Seasonal Development of Phytoplankton in a Strongly Stratified,Hypertrophic Lake

Changes in the phytoplankton community of the hypertrophic, sharply stratified Lake Verevi have been studied over eight decades. Due to irregular discharge of urban wastewater, the trophic state of the lake has changed from moderately eutrophic to hypertrophic. We found that the trophic state in summer increased in the 1980s and remained at a hypertrophic level since then. Planktothrix agardhii was recorded first in the 1950s and became the dominant species in the 1980s, forming biomass maxima under the ice and in the metalimnion during the vegetation period. In summer 1989, P. agardhii contributed almost 100% of the phytoplankton biomass. Generally, the highest biomass values occurred in the metalimnion. In spring, when P. agardhii was less numerous, diatoms and cryptophytes prevailed. In springs 2000 and 2001 different diatoms dominated – Synedra acus var. angustissima (18.6 g m⁻³) and Cyclostephanos dubius (9.2 g m⁻³), respectively. In recent years, the spring overturn has been absent. In the conditions of strong thermal stratification sharp vertical gradients of light and nutrients caused a large number of vertically narrow niches in the water column. During a typical summer stage, the epilimnion, dominated by small flagellated chrysophytes, is nearly mesotrophic, and water transparency may reach 4 m. The lower part of the water column is hypertrophic with different species of cryptophytes and euglenophytes. A characteristic feature is the higher diversity of Chlorococcales. Often, species could form their peaks of biomass in very narrow layers, e.g. in August 2001 Ceratium hirundinella (18.6 g m⁻³) was found at a depth of 5 m (the lower part of the metalimnion with hypoxic conditions), Cryptomonas spp. (56 g m⁻³) at 6 m (with traces of oxygen and a relatively high content of dissolved organic matter) and euglenophytes (0.6 g m⁻³) at 7 m and deeper (without oxygen and a high content of dissolved organic matter).     

Selection of sporophytic and gametophytic self‐incompatibility in the absence of a superlocus

Self‐incompatibility (SI) is a complex trait that enforces outcrossing in plant populations. SI generally involves tight linkage of genes coding for the proteins that underlie self‐pollen detection and pollen identity specification. Here, we develop two‐locus genetic models to address the question of whether sporophytic SI (SSI) and gametophytic SI (GSI) can invade populations of self‐compatible plants when there is no linkage or weak linkage of the underlying pollen detection and identity genes (i.e., no S‐locus supergene). The models assume that SI evolves as a result of exaptation of genes formerly involved in functions other than SI. Model analysis reveals that SSI and GSI can invade populations even when the underlying genes are loosely linked, provided that inbreeding depression and selfing rate are sufficiently high. Reducing recombination between these genes makes conditions for invasion more lenient. These results can help account for multiple, independent evolution of SI systems as seems to have occurred in the angiosperms.     

The mechanism of extracellular stimulation of nerve cells on an electrolyte-oxide-semiconductor capacitor

Extracellular excitation of neurons is applied in studies of cultured networks and brain tissue, as well as in neuroprosthetics. We elucidate its mechanism in an electrophysiological approach by comparing voltage-clamp and current-clamp recordings of individual neurons on an insulated planar electrode. Noninvasive stimulation of neurons from pedal ganglia of Lymnaea stagnalis is achieved by defined voltage ramps applied to an electrolyte/HfO₂/silicon capacitor. Effects on the smaller attached cell membrane and the larger free membrane are distinguished in a two-domain-stimulation model. Under current-clamp, we study the polarization that is induced for closed ion channels. Under voltage-clamp, we determine the capacitive gating of ion channels in the attached membrane by falling voltage ramps and for comparison also the gating of all channels by conventional variation of the intracellular voltage. Neuronal excitation is elicited under current-clamp by two mechanisms: Rising voltage ramps depolarize the free membrane such that an action potential is triggered. Falling voltage ramps depolarize the attached membrane such that local ion currents are activated that depolarize the free membrane and trigger an action potential. The electrophysiological analysis of extracellular stimulation in the simple model system is a basis for its systematic optimization in neuronal networks and brain tissue.     

Microbial production of natural raspberry ketone

Raspberry ketone is an important compound for the flavour industry. It is frequently used in products such as soft drinks, sweets, puddings and ice creams. The compound can be produced by organic synthesis. Demand for "natural" raspberry ketone is growing considerably. However, this product is extremely expensive. Consequently, there is a remaining desire to better understand how raspberry ketone is synthesized in vivo, and which genes and enzymes are involved. With this information we will then be in a better position to design alternative production strategies such as microbial fermentation. This article focuses on the identification and application of genes potentially linked to raspberry ketone synthesis. We have isolated candidate genes from both raspberry and other plants, and these have been introduced into bacterial and yeast expression systems. Conditions have been determined that result in significant levels of raspberry ketone, up to 5 mg/L. These results therefore lay a strong foundation for a potentially renewable source of "natural" flavour compounds making use of plant genes.     

Relaxed selection and the evolution of the chasmogamous flower of Impatiens capensis (Balsaminaceae)

Evolutionary Ecology - We exploited ecotypic variation in Impatiens capensis (Balsaminaceae) to test the hypothesis that mutation accumulation accompanying relaxed selection on the chasmogamous...     

A suction pump sampler for invertebrate drift detects exceptionally high concentrations of small invertebrates that drift nets miss

Hydrobiologia - Invertebrate drift is a key process in riverine ecosystems controlling aquatic invertebrate distribution and availability to fish as prey. However, accurately quantifying drifting...     

A Biotic Game Design Project for Integrated Life Science and Engineering Education

Engaging, hands-on design experiences are key for formal and informal Science, Technology, Engineering, and Mathematics (STEM) education. Robotic and video game design challenges have been particularly effective in stimulating student interest, but equivalent experiences for the life sciences are not as developed. Here we present the concept of a "biotic game design project" to motivate student learning at the interface of life sciences and device engineering (as part of a cornerstone bioengineering devices course). We provide all course material and also present efforts in adapting the project''s complexity to serve other time frames, age groups, learning focuses, and budgets. Students self-reported that they found the biotic game project fun and motivating, resulting in increased effort. Hence this type of design project could generate excitement and educational impact similar to robotics and video games.

This Education article is part of the Education Series.

Hands-on robotic and video game design projects and competitions are widespread and have proven particularly effective at sparking interest and teaching K–12 and college students in mechatronics, computer science, and Science, Technology, Engineering, and Mathematics (STEM). Furthermore, these projects foster teamwork, self-learning, design, and presentation skills [1,2]. Such playful and interactive media that provide fun, creative, open-ended learning experiences for all ages are arguably underdeveloped in the life sciences. Most hands-on education occurs in traditionally structured laboratory courses with a few exceptions like the International Genetically Engineered Machine (iGEM) competition [3]. Furthermore, there is an increasing need to bring the traditional engineering and life science disciplines together. In order to fill these gaps, we present the concept of a biotic game design project to foster student development in a broad set of engineering and life science skills in an integrated manner (Fig. 1). Though we primarily discuss our specific implementation as a cornerstone project-based class [4], alternative implementations are possible to motivate a variety of learning goals under various constraints such as student age and cost (see supplements for all course material).Open in a separate windowFig 1We developed a bioengineering devices course that employed biotic game design as a motivating project scheme. A: Biotic games enable human players to interact with cells. B: Conceptual overview of a biotic game setup. C: Students built and played biotic games. Image credits: A C64 joystick by Speed-link, 1984 (http://commons.wikimedia.org/wiki/File:Joystick_black_red_petri_01.svg); Euglena viridis by C. G. Ehrenberg, 1838; C Photo, N. J. C.Biotic games are games that operate on biological processes (Fig. 1) [5]. The biotic games we present here involve the single-celled phototactic eukaryote, Euglena gracilis. These microscopic organisms are housed in a microfluidic chip and are displayed in a magnified image on a video screen. Players interact with these cells by modulating the intensity and direction of light perpendicular to the microfluidic chip via a joystick, thereby influencing the cells’ phototactic motion. Software tracks the position of individual euglena with respect to virtual objects overlaid on the screen, creating myriad opportunities for creative game design and play. For example, in a simple game, points might be scored when a cell hits a virtual box (see S1 Video).The biotic game design project we developed was intended to motivate all the broad categories of theoretical and hands-on skills for creating any integrated instrument intended to house and to interface with biological materials, i.e., optics, electronics, sensing, actuation, microfluidics, fabrication, image processing, programming, and creative design. We termed the synthesis of these skills “biotics” in analogy to mechatronics. Our intended audience for this course was bioengineering undergraduate students at Stanford University who already had some programming experience but little to no experience in device design, fabrication, and integration. We also incorporated bioethics into the curriculum to emphasize the social responsibility of every engineer and demonstrate the potential for the biotic game project to motivate multiple fields. The course we taught spanned ten weeks, divided roughly equally into a set of technical units and the biotic game project, with two 4-hour lab sections and a single 1.5-hour lecture each week. For details and all course documents, please refer to the supplemental material.The technical section of the course focused on developing hands-on skills and theoretical understanding related to devices in a conventionally structured laboratory setting. We introduced students to fundamental electronics concepts and components such as voltage, current, resistors, capacitors, LEDs, filters, operational amplifiers, motors, microcontrollers (Arduino Uno), and breadboards. We followed a similar traditional approach in introducing optics, presenting the thin lens equation, ray tracing, conjugate planes, basic optical system design, and Köhler illumination. We covered additional topics in less detail: MATLAB programming, particle tracking, computer-aided design (CAD), fabrication, and microfluidics (learning objectives are provided at the beginning of each unit in the supplemental material).During the project-based section, students built their own biotic games. We left specific choices of implementation, architecture, and design to the students to encourage creativity and exploration but required students to revisit the technical skills they learned in the first section by integrating some specific requirements into their games (Fig. 2). Students built a bright field microscope with Köhler illumination and projected their images onto a webcam (optics). Glass and polydimethylsiloxane (PDMS) components comprised the microfluidic chip (microfluidics) and housed the euglena (microbiology). The holder for the chip and euglena-steering LEDs was designed in Solidworks (CAD) and 3-D printed (fabrication). The students constructed a polycarbonate housing for the game controller using a band saw and drill press (fabrication). The students revisited electronic breadboarding and soldering when creating the electronic circuits to communicate between the LEDs, joystick, microcontroller, and computer. Finally, they used MATLAB to program the microcontroller, implement real time image recognition, and provide the user interface for the game experience (image processing and programming).Open in a separate windowFig 2Biotic game-based courses encourage students to integrate a versatile set of relevant STEM topics.Image credits: Taken by N. J. C. (credit for the work and artifacts to the students who took the course).We challenged students to consider the ethical implications [6] of manipulating life in a game context before building their projects. Although phototaxis experiments with euglena are commonplace in education, and have hitherto raised no ethical concerns, the equivalent manipulation in the form of a game warrants its own ethical analysis as provided by Harvey et al. [7]. The students read and discussed this paper, then wrote a 200-word essay on whether they found it permissible or not to make and play biotic games. Students had the choice to switch to a nongame project of equivalent complexity. All students found euglena-based games permissible, pointing out that “they are nonsentient and cannot feel pain,” followed by a diverse range of considerations such as “the euglena are still free to act as they please,” “there needs to be an educational intention,” or “a pet…provides a way…to work on responsibility and caring.” Based on further student-initiated discussions that spontaneously emerged throughout the course, we believe that biotic games are effective in providing a stimulating, student-relevant, in-class context for bioethics.We motivated the game design project to the students as having educational potential at two levels, i.e., learning by building and learning by playing; we lectured them about the needs and opportunities for new approaches to K–12 STEM education [8,9]. The students were then asked to consider building a game that had educational value for the player. Educational value has many aspects, which was reflected in students’ statements regarding their intended educational outcomes for their games on their course project websites. These ranged from more factual learning objectives (“learn about…” “…inner working,” “…structural detail,” “… light responses,” “…euglena behavior”) to objectives affecting attitude (“spark interest,” “generate fascination,” “encourage to explore,” “respect for life”). We also had a game designer give a guest lecture to the students. For pragmatic reasons, we requested the students keep games very simple (ideally having just a single in-game objective) and cap game duration at one minute. Before, during, and after their projects, students received feedback from instructors as well as from their peers on their games from technical and user perspectives.The games that the students ultimately produced were diverse and creative (Fig. 2 and S1 Video), including single and multiplayer scenarios, games where euglena hit virtual targets, and games where euglena pushed virtual objects. Games that involved pushing objects across the screen (relying on collective motion of many organisms) were generally more consistent at correlating player strategy to scored points than those that involved hitting target objects. The quality and robustness of these integrated projects naturally varied, and individual groups placed more or less emphasis on different aspects based on personal preferences and learning goals (for example, fabricating a more elaborate housing for the game controller versus programming more complex game mechanics). A key point was that the students did not rely on prepared materials or platforms to develop their games but rather had to design, build, and test their game setups from scratch, thereby revisiting and deepening the primary learning goals of the course with some freedom to follow their own learning aspirations (Fig. 2). The final project deliverables were a two-minute project demonstration video, a website describing the elements of the project, and a game that all instructors and students played on the final day (Fig. 1B), which led to lots of laughter as well as in-depth discussions on technical details.Many students self-reported that they enjoyed the project and that it led to increased motivation and effort during the course. In response to the question “Do you think you were motivated to try harder or had more fun (and thereby learned more) during your final project because you were making a game (rather than just building a technical instrument, for example)? If so—please give some examples:” 15 out of 17 students responded “Very/definitely” on a five point scale. As examples, students listed: “wanted to make the best game,” “want to make it clever and cool in the eyes of classmates who are play testing,” “motivated during final push,” “willing to put in more time,” “was fun”/”made it fun,” “create a game that actually works,” “reinforced what was learned before,” and “provided room for creativity.” These comments reflect the overall excitement we saw for the biotic game project. While these responses do not constitute rigorous proof regarding course effectiveness (which will require more detailed and controlled assessments in the future), we consider this course a success based on our teaching experiences.45 students have now taken this class over the past three years, with 18 students in our most recent offering. We used each year to iterate and improve our implementation. For example, we changed the organism and stimulus from Paramecia galvanotaxis [5] to Euglena phototaxis, which gave more reliable long-term responses. We also added a simple microfluidics unit enabling students to build more robust organism housing chambers. We changed the microscope structure from LEGO to Thorlabs parts (essentially trading the emphasis on 3-D structural design, flexibility, and cost for a more in-depth focus on high-end optics and their alignment). Finally, we explicitly asked the students to design and fabricate a housing for the game controller to better incorporate fabrication skills like using a band saw and tapping screw threads. So far, we primarily used MATLAB as the programming component given its widespread use in education and research and the available Arduino interface. However, MATLAB is not particularly well-suited to support game design and is also not free, making translation into lower resource settings challenging. For the future, we are considering moving to smartphone-based control (such as Android) given that these mobile environments are very flexible and increasingly used for control of scientific and consumer instruments and are becoming more widespread in education. We also see the opportunity to better emphasize and teach the approach of iterative design; for example, by letting students prototype and test their game ideas on paper [10] and simple programming environments like Scratch [11] first, before attempting the full implementation. It would likely also be very rewarding for the students to be able to take their project home at the end of the course. In summary, many different course design decisions can be made based on specific intended educational outcomes. Not all of these can be fit into one course at the same time, and clear decisions should be made on how to balance covering a breadth of topics with depth on a selected few.As a preliminary test of another age range, time frame, and budget, we taught a greatly simplified 3-hour workshop where high school and middle school students assembled a low-cost microscope and microfluidics chamber, attached it to a smartphone, and stimulated euglena using a preprogrammed Arduino-based controller (see supplements). We had no game interface implemented yet on the phone, but the students could observe the euglena responses to the light stimuli. All students were able to complete the project and take their microscopes home. Over half of our undergraduate student teams also volunteered to present their game projects for this outreach event which took place multiple weeks after their class had ended. This separate experience suggests that the biotic game concept holds promise for reaching a wider age range in a shortened timespan and at a greatly reduced budget, and that completed games can be used in outreach activities. We are currently developing a kit modeled after this unit.In conclusion, we consider biotic games promising in motivating integrated, hands-on learning at the interface of life science and engineering. Our efforts so far indicate that this concept could be adapted to various age groups and learning goals with the potential for wider future impacts on education. We draw upon the analogy to robotics, where microcontrollers went from initially unfathomable as an educational tool to the vision of Papert and collaborators and their use of programmable robotics with children [12], eventually leading to multiple commercial realizations (LEGO mindstorm, Arduino, etc.), a large public following, and a major role in education both in the classroom and through competitions such as First Robotics [1]. We also see additional potential for integrating more creative and artistic aspects into STEM, i.e., leading to generalized Science, Technology, Engineering, Arts, and Mathematics (STEAM) disciplines [13]. We invite others to join us in these endeavors—all instructional materials are available in the appendix for further adaptations and educational use.