A rapid introduction to neurological biochemistry using <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Short, cost-effective teaching activities are a useful way of providing an integrated view on biological processes. Here we describe a brief, hands-on workshop that allows pre-university students to explore their understanding of a neurological pathway from its chemical bases to phenotype. The workshop effectively introduces the students to data collection and analysis in an enjoyable way and at an appropriate level, determined by an end of session feedback survey. The design of the workshop can be adapted and scaled to generate diverse sessions such as university teaching practicals or summer school training workshops.     

Enhancing Elementary Students’ Experiences Learning about Circuits Using an Exploration–Explanation Instructional Sequence

Using an exploration–explanation sequence of science instruction helps teachers unveil students’ prior knowledge about circuits and engage them in minds-on science learning. In these lessons, fourth grade students make predictions and test their ideas about circuits in series through hands-on investigations. The teacher helps students make connections between their hands-on experiences collecting data and new terms. This lesson shows how teachers can incorporate formative assessments such as checkpoints, self tests, and exit slips into the explanation phase of instruction so students can evaluate and self-monitor their understanding of circuits in series. These activities meet the National Science Education Standards for active, student-center learning environments that cultivate the critical thinking skills necessary to learn science.     

“Sharks in Your Hands”—A Case Study on Effects of Teaching Strategies to Change Knowledge and Attitudes Towards Sharks

This study was designed to exemplify how hands-on based teaching strategies enhanced students’ knowledge and positive attitudes towards sharks. Hands-on activities for sharks’ biological and morphological features were carried out. Eleven elementary school students from a remote area in Taiwan were recruited and assigned to the hands-on condition. They were encouraged to draw pictures of sharks before and after the instruction as the main data for pre- and post-test comparison. Two years later, the retention test and attitude inventory towards sharks were implemented. The results revealed that large effect size emerged for both the post-test and retention test. In regards to attitude inventory, students involved in hands-on activities also significantly outperformed the baseline group. Many of them have taken notice of television programs and books about sharks or marine ecology since the hands-on activities, indicating the instruction had a beneficial impact on their extracurricular lives. Empirical findings of this study suggest hands-on instruction is a powerful strategy for learning, both for immediate and prolonged effects on improving students’ knowledge and attitudes toward sharks.     

Elementary education majors experience hands-on learning in introductory biology

Faculty members from the University of South Dakota attended the Curriculum Reform Institute offered by the University of Wisconsin at Oshkosh, WI, during the summer of 2002 to design a course sequence for elementary education majors that better meets their needs for both content and pedagogy based on the science education standards. The special section of introductory biology that resulted from this workshop is designed to use laboratories and activities that either help students learn major concepts in the life sciences or model how to teach these concepts to their future K-8 students. This study describes how the active, hands-on learning opportunity for preservice teachers with its emphasis on both content and performance-based assessment was implemented in an introductory biology course for elementary education majors during the spring of 2004. During the initial offering of this course, student perceptions about what helped them to learn in the special section was compared with their nonscience major peers in the large lecture-intensive class that they would have taken. Each group of students completed early and late web-based surveys to assess their perceptions about learning during the courses. After the completion of the course, students in the special section appreciated how the relevance of science and conducting their own scientific experimentation helped them learn, enjoyed working and studying in small groups, valued diverse class time with very little lecture, were more confident in their abilities in science, and were more interested in discussing science with others. This course format is recommended for science classes for preservice teachers.     

Analysis of the Interaction of the Nucleotide Base with Myosin and the Effect on Substrate Efficacy

A wide variety of purine- and pyrimidine-based nucleotides can serve as a substrate for actomyosin mechanics, but with varying effectiveness. To understand the myosin-ATP interaction and in particular, the interactions with the base, we have used molecular dynamics simulations to model the interactions of myosin with ATP, CTP, UTP, aza-ATP, ITP, and GTP (in decreasing order of effectiveness as a substrate for the generation of motility) docked at the active site. The simulations with ATP, and x-ray structures, show a triad of conserved amino acids lining the nucleotide site that form a cyclical chain of nucleotide-protein hydrogen bonding interactions: ATP → Y135 → Y116 → N188 → ATP. Mechanical efficacy of a substrate correlates with its ability to maintain this coordination. Simulations modeling the active site of other myosin isoforms with different amino acids in the triad likewise imply that the amino acid composition at the nucleotide site could modulate function. The modeling has predictive power. In silico mutation experiments suggest mutations that would enhance GTP as a substrate for myosin while simultaneously making ATP a less effective substrate.     

Lessons Learned from Implementing a Wet Laboratory Molecular Training Workshop for Beach Water Quality Monitoring

Rapid molecular testing methods are poised to replace many of the conventional, culture-based tests currently used in fields such as water quality and food science. Rapid qPCR methods have the benefit of being faster than conventional methods and provide a means to more accurately protect public health. However, many scientists and technicians in water and food quality microbiology laboratories have limited experience using these molecular tests. To ensure that practitioners can use and implement qPCR techniques successfully, we developed a week long workshop to provide hands-on training and exposure to rapid molecular methods for water quality management. This workshop trained academic professors, government employees, private industry representatives, and graduate students in rapid qPCR methods for monitoring recreational water quality. Attendees were immersed in these new methods with hands-on laboratory sessions, lectures, and one-on-one training. Upon completion, the attendees gained sufficient knowledge and practice to teach and share these new molecular techniques with colleagues at their respective laboratories. Key findings from this workshop demonstrated: 1) participants with no prior experience could be effectively trained to conduct highly repeatable qPCR analysis in one week; 2) participants with different desirable outcomes required exposure to a range of different platforms and sample processing approaches; and 3) the collaborative interaction amongst newly trained practitioners, workshop leaders, and members of the water quality community helped foster a cohesive cohort of individuals which can advocate powerful cohort for proper implementation of molecular methods.     

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.     

Estimation of passive and active properties in the human heart using 3D tagged MRI

Advances in medical imaging and image processing are paving the way for personalised cardiac biomechanical modelling. Models provide the capacity to relate kinematics to dynamics and—through patient-specific modelling—derived material parameters to underlying cardiac muscle pathologies. However, for clinical utility to be achieved, model-based analyses mandate robust model selection and parameterisation. In this paper, we introduce a patient-specific biomechanical model for the left ventricle aiming to balance model fidelity with parameter identifiability. Using non-invasive data and common clinical surrogates, we illustrate unique identifiability of passive and active parameters over the full cardiac cycle. Identifiability and accuracy of the estimates in the presence of controlled noise are verified with a number of in silico datasets. Unique parametrisation is then obtained for three datasets acquired in vivo. The model predictions show good agreement with the data extracted from the images providing a pipeline for personalised biomechanical analysis.     

Opportunities to replace the use of animals in sepsis research. The report and recommendations of a Focus on Alternatives workshop

Sepsis and multiple organ failure are common causes of death in patients admitted to intensive care units. The incidence of sepsis and associated mortalities has been steadily increasing over the past 20 years. Sepsis is a complex inflammatory condition, the precise causes of which are still poorly understood. Animal models of sepsis have the potential to cause substantial suffering, and many of them have been poorly representative of the human syndrome. However, a number of non-animal approaches, including in vitro, in silico and clinical studies, show promise for addressing this situation. This report is based on discussions held at an expert workshop convened by Focus on Alternatives and held in 2004 at the Wellcome Trust, London. It provides an overview of some non-animal approaches to sepsis research, including their strengths and weaknesses, and argues that they should be prioritised for further development.     

A model of stem cell population dynamics: in silico analysis and in vivo validation

The proper renewal and maintenance of tissues by stem cell populations is simultaneously influenced by anatomical constraints, cell proliferation dynamics and cell fate specification. However, their relative influence is difficult to examine in vivo. To address this difficulty we built, as a test case, a cell-centered state-based computational model of key behaviors that govern germline development in C. elegans, and used it to drive simulations of cell population dynamics under a variety of perturbations. Our analysis provided unexpected possible explanations for laboratory observations, including certain 'all-or-none' phenotypes and complex differentiation patterns. The simulations also offered insights into niche-association dynamics and the interplay between cell cycle and cell fate. Subsequent experiments validated several predictions generated by the simulations. Notably, we found that early cell cycle defects influence later maintenance of the progenitor cell population. This general modeling approach is potentially applicable to other stem cell systems.     

Dynamical analysis of the calcium signaling pathway in cardiac myocytes based on logarithmic sensitivity analysis

Many cellular functions are regulated by the Ca(2+) signal which contains specific information in the form of frequency, amplitude, and duration of the oscillatory dynamics. Any alterations or dysfunctions of components in the calcium signaling pathway of cardiac myocytes may lead to a diverse range of cardiac diseases including hypertrophy and heart failure. In this study, we have investigated the hidden dynamics of the intracellular Ca(2+) signaling and the functional roles of its regulatory mechanism through in silico simulations and parameter sensitivity analysis based on an experimentally verified mathematical model. It was revealed that the Ca(2+) dynamics of cardiac myocytes are determined by the balance among various system parameters. Moreover, it was found through the parameter sensitivity analysis that the self-oscillatory Ca(2+) dynamics are most sensitive to the Ca(2+) leakage rate of the sarcolemmal membrane and the maximum rate of NCX, suggesting that these two components have dominant effects on circulating the cytosolic Ca(2+).     

Molecular Modeling on Inhibitor Complexes and Active-Site Dynamics of Cytochrome P450 C17, a Target for Prostate Cancer Therapy

A molecular model for the P450 enzyme cytochrome P450 C17 (CYP17) is presented based on sequence alignments of multiple template structures and homology modeling. This enzyme plays a central role in the biosynthesis of testosterone and is emerging as a major target in prostate cancer, with the recently developed inhibitor abiraterone currently in advanced clinical trials. The model is described in detail, together with its validation, by providing structural explanations to available site-directed mutagenesis data. The CYP17 molecule in this model is in the form of a triangular prism, with an edge of ∼ 55 Å and a thickness of ∼ 37 Å. It is predominantly helical, comprising 13 α helices interspersed by six 3₁₀ helices and 11 β-sheets. Multinanosecond molecular dynamics simulations in explicit solvent have been carried out, and principal components analysis has been used to reveal the details of dynamics around the active site. Coarse-grained methods have also been used to verify low-frequency motions, which have been correlated with active-site gating. The work also describes the results of docking synthetic inhibitors, including the drug abiraterone and the natural substrate pregnenolone, in the CYP17 active site together with molecular dynamics simulations on the complexes.     

Dependence of Protein Crystal Stability on Residue Charge States and Ion Content of Crystal Solvent

Protein crystallization is frequently induced by the addition of various precipitants, which directly affect protein solubility. In addition to organic cosolvents and long-chain polymers, salts belong to the most widely used precipitants in protein crystallography. However, despite such widespread usage, their mode of action at the atomistic level is still largely unknown. Here, we perform extensive molecular dynamics simulations of the villin headpiece crystal unit cell to examine its stability at different concentrations of sodium sulfate. We show that the inclusion of ions in crystal solvent at high concentration can prevent large rearrangements of the asymmetric units and a loss of symmetry of the unit cell without significantly affecting protein dynamics. Of importance, a similar result can be achieved by neutralizing several specific charged residues suggesting that they may play an active role in crystal destabilization due to unfavorable electrostatic interactions. Our results provide a microscopic picture behind salt-induced stabilization of a protein crystal and further suggest that adequate modeling of realistic crystallization conditions may be necessary for successful molecular dynamics simulations of protein crystals.     

Multiscale agent-based cancer modeling

Agent-based modeling (ABM) is an in silico technique that is being used in a variety of research areas such as in social sciences, economics and increasingly in biomedicine as an interdisciplinary tool to study the dynamics of complex systems. Here, we describe its applicability to integrative tumor biology research by introducing a multi-scale tumor modeling platform that understands brain cancer as a complex dynamic biosystem. We summarize significant findings of this work, and discuss both challenges and future directions for ABM in the field of cancer research.     

From in silico to in vitro: modelling and production of Trichoderma reesei endoglucanase 1 and its mutant in Pichia pastoris

In this study, a major cellulase, namely endoglucanase 1 (EGI) from Trichoderma reesei was mutated by the introduction of four different lysine and glycine rich loops to create a hotspot for directed crosslinking of EGI away from the active site. The impact of the inserted loops on the stability of the enzyme was analyzed using molecular dynamics (MD) and the effect on the active site was studied using molecular mechanics (MM) simulations. The best loop mutation predicted in silico (EGI_L5) was introduced to EGI via site directed mutagenesis. The loop mutant EGI_L5 and EGI were both expressed in Pichia pastoris. Enzymes were characterized and their activities against soluble substrates such as CMC and 4-MUC were determined. Both enzymes exhibited similar pH and temperature activity and thermal stability profiles. Moreover, specific activity of EGI_L5 against 4-MUC was found to be the same as the native enzyme.     

A Study of Early Afterdepolarizations in a Model for Human Ventricular Tissue

Sudden cardiac death is often caused by cardiac arrhythmias. Recently, special attention has been given to a certain arrhythmogenic condition, the long-QT syndrome, which occurs as a result of genetic mutations or drug toxicity. The underlying mechanisms of arrhythmias, caused by the long-QT syndrome, are not fully understood. However, arrhythmias are often connected to special excitations of cardiac cells, called early afterdepolarizations (EADs), which are depolarizations during the repolarizing phase of the action potential. So far, EADs have been studied mainly in isolated cardiac cells. However, the question on how EADs at the single-cell level can result in fibrillation at the tissue level, especially in human cell models, has not been widely studied yet. In this paper, we study wave patterns that result from single-cell EAD dynamics in a mathematical model for human ventricular cardiac tissue. We induce EADs by modeling experimental conditions which have been shown to evoke EADs at a single-cell level: by an increase of L-type Ca currents and a decrease of the delayed rectifier potassium currents. We show that, at the tissue level and depending on these parameters, three types of abnormal wave patterns emerge. We classify them into two types of spiral fibrillation and one type of oscillatory dynamics. Moreover, we find that the emergent wave patterns can be driven by calcium or sodium currents and we find phase waves in the oscillatory excitation regime. From our simulations we predict that arrhythmias caused by EADs can occur during normal wave propagation and do not require tissue heterogeneities. Experimental verification of our results is possible for experiments at the cell-culture level, where EADs can be induced by an increase of the L-type calcium conductance and by the application of I blockers, and the properties of the emergent patterns can be studied by optical mapping of the voltage and calcium.     

The Heat Is On! Using Particle Models to Change Students' Conceptions of Heat and Temperature

Elementary, middle-level, and high school science teachers commonly find their students have misconceptions about heat and temperature. Unfortunately, student misconceptions are difficult to modify or change and can prevent students from learning the accurate scientific explanation. In order to improve our students' understanding of heat and temperature, we created a three-part instructional activity designed to engage them in the development of kinetic particle models. First, the students use household materials—food coloring and water—to investigate and create particle models depicting motion at the molecular level. Second, the students complete a similar activity designed to help them visualize the connections between heat and particle motion. Third, the students build a thermometer, observe it in action, and create particle models explaining how the thermometer works. By the end of the series of activities, the students are able develop their own definitions for heat and temperature. Through this hands-on and minds-on series of activities, students learn to define and differentiate the concepts of heat and temperature.     

Accelerated molecular dynamics simulations of the octopamine receptor using GPUs: discovery of an alternate agonist‐binding position

Octopamine receptors (OARs) perform key biological functions in invertebrates, making this class of G‐protein coupled receptors (GPCRs) worth considering for insecticide development. However, no crystal structures and very little research exists for OARs. Furthermore, GPCRs are large proteins, are suspended in a lipid bilayer, and are activated on the millisecond timescale, all of which make conventional molecular dynamics (MD) simulations infeasible, even if run on large supercomputers. However, accelerated Molecular Dynamics (aMD) simulations can reduce this timescale to even hundreds of nanoseconds, while running the simulations on graphics processing units (GPUs) would enable even small clusters of GPUs to have processing power equivalent to hundreds of CPUs. Our results show that aMD simulations run on GPUs can successfully obtain the active and inactive state conformations of a GPCR on this reduced timescale. Furthermore, we discovered a potential alternate active‐state agonist‐binding position in the octopamine receptor which has yet to be observed and may be a novel GPCR agonist‐binding position. These results demonstrate that a complex biological system with an activation process on the millisecond timescale can be successfully simulated on the nanosecond timescale using a simple computing system consisting of a small number of GPUs. Proteins 2016; 84:1480–1489. © 2016 Wiley Periodicals, Inc.     

Planning a lab lesson on the law of thermal conduction with learning by preparing to teach design

Lab activities have been innovated and progressed to fulfill the purpose that students need to experience physics concepts through hands-on and minds-on interactions. However, student-centered and conceptual activities in physics labs are challenged with time and material constraints that degrades the merits of the learning environment. This study adopts a pedagogical design, “learning by preparing to teach”, to address the challenges with conducting a satisfactory number of activities in physics labs and therefore promoting student hands-on and minds-on experiences with the activities. Eight activities covering the conceptual variables of thermal conduction in a two-hour lab session were studied by freshmen students at a university. To implement the pedagogical design, the students were arranged into the learning and teaching groups that paved way to a lab design that student was first learners and then teachers. Lab activities were configured that each group studied a certain set of activities. Upon completing the activities, members of the learning groups were designated to the teaching groups in which students cooperated to achieve conceptual objectives of the lab. The results showed that a usual lab duration becomes effective to cover numerous activities.     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.