Reliable, Semi-Automated Wound Healing Rate Determination in Muscle

In the field of Regenerative and Sport Medicine, there is great interest in the development and validation of compounds and devices with the potential to accelerate wound healing and muscle regeneration. In vitro, this effect can be evaluated in a scratch test model, in which a pipette removes a line of cells from a confluent monolayer of cells with high regenerative capacity and the time to close this injury measured. PURPOSE: To develop a reliable, dynamic, and quantitative process with a shorter duty cycle and semi-automatic operation for the determination of wound healing rate, as compared to fully manual operation. METHODS: C2C12 murine myoblasts were cultured to confluence under standard conditions. A 200 µL pipette tip was used to make a scratch across each well, and 0 and 0.5mM of pro-myogenic Si-ions were added to the media. A Keyence BZX-710 microscope was used to capture images every 183 seconds over 36 hours at 10x magnification with 0.7 pixels/μm and 4 µm pitch. An enclosed cell culture stage contained a cell incubator system keeping cells at 37°C with a 5% CO2 humidified air. For the manual operation, one image was randomly selected from the automated images every 12hr. ImageJ Macro WH_NJ was used to quantify the percent area of the field (scratched) of interest without cells and was normalized as needed per experimental conditions. RESULTS: The manual and automatic slopes for the 0 and 0.5mM Si-ion treatments were -4.87E-06, -4.84E-06, -6.01E-06, and -5.98E-06, respectively, for the full 0-36hr. There was a high degree of correlation between the manual and semi-automatic rates for both the 0 and 0.5mM Si-ions, at r=.84 and .98, respectively. There were no statistically significant differences between healing rates (i.e., closure times) for the automatic or manual 0 or 0.5mM Si-ions, or within either method, following a two-tailed student’s t-test with alpha level of p\u3c.05. Within 12hr periods, the semi-automatic method provided greater detail for the healing rate, such as the faster initial rate seen in 0.5mM Si-ion, not discernible in 12-hour increments for the manual method. CONCLUSION: These data support the functionality of our new methodology described here. The descriptive and inferential statistics shown here demonstrate agreement between the two analyses, while the semi-automated method presented additional dynamics and kinetics information beyond the manual method in early-test behavior that could not be measured manually. Further development in this area will focus on continuing to shorten duty cycles for higher fidelity and the quantitative analysis of dynamic behaviors. Potential clinical-translational applications of our new method are to screen libraries of compounds with putative muscle regeneration capacity using human muscle cells. We also plan to test basal differences in muscle cells from biopsies of sedentary and active individuals, as well as healthy individuals vs. those with various metabolic and musculoskeletal and cardiovascular disorders, and aging sarcopenia. Our new methodology coupled with these translational studies will help advance new compounds and devices with early promise for the field of Regenerative and Sport Medicine into the pre-clinical animal phases of validation

A pedagogic appraisal of the Priority Heuristic

We have explored how science and mathematics teachers made decisions when confronted with a dilemma in which a fictitious young woman, Deborah, may choose to have an operation that might address a painful spinal condition. We sought to explore the extent to which psychological heuristic models, in particular the Priority Heuristic, might successfully describe the decision-making process of these teachers and how an analysis of the role of personal and emotional factors in shaping the decision-making process might inform pedagogical design. A novel aspect of this study is that the setting in which the decision-making process is examined contrasts sharply with those used in psychological experiments. We found that to some extent, even in this contrasting setting, the Priority Heuristic could describe these teachers' decision-making. Further analysis of the transcripts yielded some insights into limitations on scope as well the richness and complexity in how personal factors were brought to bear. We see these limitations as design opportunities for educational intervention

Modeling of Polymer Clay Nanocomposite for a Multiscale Approach

The mechanical property enhancement of polymer reinforced with nano-thin clay platelets (of high aspect ratio) is associated with a high polymer-filler interfacial area per unit volume. The ideal case of fully separated (exfoliated) platelets is generally difficult to achieve in practice: a typical nanocomposite also contains multilayer stacks of intercalated platelets. Here we use numerical modelling to investigate how the platelet properties affect the overall mechanical properties. The configuration of platelets is modelled using a statistical interpretation of the Representative Volume Element (RVE) approach, in which an ensemble of "sample" heterogeneous material is generated (with periodic boundary conditions). A simple Monte Carlo algorithm is used to place non-intersecting platelets in the RVE according to a specified set of statistical distributions. The effective stiffness of the platelet-matrix system is determined by measuring the stress (using standard Finite Element analysis) produced as a result of applying a small deformation to the boundaries, and averaging over the entire statistical ensemble. In this work we determine the way in which the platelet properties (curvature, filling fraction, stiffness, aspect ratio) and the number of layers in the stack affect the overall stiffness enhancement of the nanocomposite. Thus, we bridge the gap between behaviour on the macroscopic scale with that on the scale of the nano-reinforcement, forming part of a multi-scale modelling framework.Comment: 39 pages, 19 figure

Fly with the flock: immersive solutions for animal movement visualization and analytics

Understanding the movement of animals is important for a wide range of scientific interests including migration, disease spread, collective movement behaviour and analysing motion in relation to dynamic changes of the environment such as wind and thermal lifts. Particularly, the three-dimensional (3D) spatial–temporal nature of bird movement data, which is widely available with high temporal and spatial resolution at large volumes, presents a natural option to explore the potential of immersive analytics (IA). We investigate the requirements and benefits of a wide range of immersive environments for explorative visualization and analytics of 3D movement data, in particular regarding design considerations for such 3D immersive environments, and present prototypes for IA solutions. Tailored to biologists studying bird movement data, the immersive solutions enable geo-locational time-series data to be investigated interactively, thus enabling experts to visually explore interesting angles of a flock and its behaviour in the context of the environment. The 3D virtual world presents the audience with engaging and interactive content, allowing users to ‘fly with the flock’, with the potential to ascertain an intuitive overview of often complex datasets, and to provide the opportunity thereby to formulate and at least qualitatively assess hypotheses. This work also contributes to ongoing research efforts to promote better understanding of bird migration and the associated environmental factors at the global scale, thereby providing a visual vehicle for driving public awareness of environmental issues and bird migration patterns

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

The p38/MK2/Hsp25 Pathway Is Required for BMP-2-Induced Cell Migration

Background: Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. Methodology/Principal Findings: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2¿s physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38¿ or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. Conclusions: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration

A Reform Strategy for Germany

In this chapter, we outline a reform strategy to promote a more entrepreneurial society in Germany. Germany has developed a successful model of capitalism in which high productivity growth is driven by on-the-job learning and firm-specific skill accumulation. The economy is rooted in a strong and regionally embedded Mittelstand, which supports an export-oriented industry mainly based on incremental innovations, but which is less conducive to more radical innovation. We, therefore, suggest a reform agenda for Germany that encourages more entrepreneurial experimentation with the aim of facilitating radical innovation, both in incumbent and new firms. Germany’s entrepreneurial talent should be encouraged to take on more risk, the education system could promote initiative, creativity and a willingness to experiment, and a more equal playing field between dependent employment and self-employment/employer could be created

Temporal changes in the epidemiology, management, and outcome from acute respiratory distress syndrome in European intensive care units: a comparison of two large cohorts

Background: Mortality rates for patients with ARDS remain high. We assessed temporal changes in the epidemiology and management of ARDS patients requiring invasive mechanical ventilation in European ICUs. We also investigated the association between ventilatory settings and outcome in these patients. Methods: This was a post hoc analysis of two cohorts of adult ICU patients admitted between May 1–15, 2002 (SOAP study, n = 3147), and May 8–18, 2012 (ICON audit, n = 4601 admitted to ICUs in the same 24 countries as the SOAP study). ARDS was defined retrospectively using the Berlin definitions. Values of tidal volume, PEEP, plateau pressure, and FiO2 corresponding to the most abnormal value of arterial PO2 were recorded prospectively every 24 h. In both studies, patients were followed for outcome until death, hospital discharge or for 60 days. Results: The frequency of ARDS requiring mechanical ventilation during the ICU stay was similar in SOAP and ICON (327[10.4%] vs. 494[10.7%], p = 0.793). The diagnosis of ARDS was established at a median of 3 (IQ: 1–7) days after admission in SOAP and 2 (1–6) days in ICON. Within 24 h of diagnosis, ARDS was mild in 244 (29.7%), moderate in 388 (47.3%), and severe in 189 (23.0%) patients. In patients with ARDS, tidal volumes were lower in the later (ICON) than in the earlier (SOAP) cohort. Plateau and driving pressures were also lower in ICON than in SOAP. ICU (134[41.1%] vs 179[36.9%]) and hospital (151[46.2%] vs 212[44.4%]) mortality rates in patients with ARDS were similar in SOAP and ICON. High plateau pressure (> 29 cmH2O) and driving pressure (> 14 cmH2O) on the first day of mechanical ventilation but not tidal volume (> 8 ml/kg predicted body weight [PBW]) were independently associated with a higher risk of in-hospital death. Conclusion: The frequency of and outcome from ARDS remained relatively stable between 2002 and 2012. Plateau pressure > 29 cmH2O and driving pressure > 14 cmH2O on the first day of mechanical ventilation but not tidal volume > 8 ml/kg PBW were independently associated with a higher risk of death. These data highlight the continued burden of ARDS and provide hypothesis-generating data for the design of future studies