A novel method of dynamic culture surface expansion improves mesenchymal stem cell proliferation and phenotype.

Repeated passaging in conventional cell culture reduces pluripotency and proliferation capacity of human mesenchymal stem cells (MSC). We introduce an innovative cell culture method whereby the culture surface is dynamically enlarged during cell proliferation. This approach maintains constantly high cell density while preventing contact inhibition of growth. A highly elastic culture surface was enlarged in steps of 5% over the course of a 20-day culture period to 800% of the initial surface area. Nine weeks of dynamic expansion culture produced 10-fold more MSC compared with conventional culture, with one-third the number of trypsin passages. After 9 weeks, MSC continued to proliferate under dynamic expansion but ceased to grow in conventional culture. Dynamic expansion culture fully retained the multipotent character of MSC, which could be induced to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. Development of an undesired fibrogenic myofibroblast phenotype was suppressed. Hence, our novel method can rapidly provide the high number of autologous, multipotent, and nonfibrogenic MSC needed for successful regenerative medicine.

Dynamic modulation of CCR7 expression and function on naive T lymphocytes in vivo.

The chemokine receptor CCR7 is critical for the recirculation of naive T cells. It is required for T cell entry into secondary lymphoid organs (SLO) and for T cell motility and retention within these organs. How CCR7 activity is regulated during these processes in vivo is poorly understood. Here we show strong modulation of CCR7 surface expression and occupancy by the two CCR7 ligands, both in vitro and in vivo. In contrast to blood, T cells in SLO had most surface CCR7 occupied with CCL19, presumably leading to continuous signaling and cell motility. Both ligands triggered CCR7 internalization in vivo as shown in Ccl19(-/-) and plt/plt mice. Importantly, CCR7 occupancy and down-regulation led to strongly impaired chemotactic responses, an effect reversible by CCR7 resensitization. Therefore, during their recirculation, T cells cycle between states of free CCR7 with high ligand sensitivity in blood and occupied CCR7 associated with continual signaling and reduced ligand sensitivity within SLO. We propose that these two states of CCR7 are important to allow the various functions CCR7 plays in T cell recirculation.

Simulation of interface dynamics across pore discontinuities

We study the dynamics of a water-oil meniscus moving from a smaller to a larger pore. The process is characterised by an abrupt change in the configuration, yielding a sudden energy release. A theoretic study for static conditions provides analytical solutions of the surface energy content of the system. Although the configuration after the sudden energy release is energetically more convenient, an energy barrier must be overcome before the process can happen spontaneously. The energy barrier depends on the system geometry and on the flow parameters. The analytical results are compared to numerical simulations that solve the full Navier-Stokes equation in the pore space and employ the Volume Of Fluid (VOF) method to track the evolution of the interface. First, the numerical simulations of a quasi-static process are validated by comparison with the analytical solutions for a static meniscus, then numerical simulations with varying injection velocity are used to investigate dynamic effects on the configuration change. During the sudden energy jump the system exhibits an oscillatory behaviour. Extension to more complex geometries might elucidate the mechanisms leading to a dynamic capillary pressure and to bifurcations in final distributions of fluid phases in porous

Improving plant functional groups for dynamic models of biodiversity: at the crossroads between functional and community ecology

The pace of on-going climate change calls for reliable plant biodiversity scenarios. Traditional dynamic vegetation models use plant functional types that are summarized to such an extent that they become meaningless for biodiversity scenarios. Hybrid dynamic vegetation models of intermediate complexity (hybrid-DVMs) have recently been developed to address this issue. These models, at the crossroads between phenomenological and process-based models, are able to involve an intermediate number of well-chosen plant functional groups (PFGs). The challenge is to build meaningful PFGs that are representative of plant biodiversity, and consistent with the parameters and processes of hybrid-DVMs. Here, we propose and test a framework based on few selected traits to define a limited number of PFGs, which are both representative of the diversity (functional and taxonomic) of the flora in the Ecrins National Park, and adapted to hybrid-DVMs. This new classification scheme, together with recent advances in vegetation modeling, constitutes a step forward for mechanistic biodiversity modeling.

Mynodbcsv: Lightweight Zero-Config Database Solution for Handling Very Large CSV Files.

Volumes of data used in science and industry are growing rapidly. When researchers face the challenge of analyzing them, their format is often the first obstacle. Lack of standardized ways of exploring different data layouts requires an effort each time to solve the problem from scratch. Possibility to access data in a rich, uniform manner, e.g. using Structured Query Language (SQL) would offer expressiveness and user-friendliness. Comma-separated values (CSV) are one of the most common data storage formats. Despite its simplicity, with growing file size handling it becomes non-trivial. Importing CSVs into existing databases is time-consuming and troublesome, or even impossible if its horizontal dimension reaches thousands of columns. Most databases are optimized for handling large number of rows rather than columns, therefore, performance for datasets with non-typical layouts is often unacceptable. Other challenges include schema creation, updates and repeated data imports. To address the above-mentioned problems, I present a system for accessing very large CSV-based datasets by means of SQL. It's characterized by: "no copy" approach - data stay mostly in the CSV files; "zero configuration" - no need to specify database schema; written in C++, with boost [1], SQLite [2] and Qt [3], doesn't require installation and has very small size; query rewriting, dynamic creation of indices for appropriate columns and static data retrieval directly from CSV files ensure efficient plan execution; effortless support for millions of columns; due to per-value typing, using mixed text/numbers data is easy; very simple network protocol provides efficient interface for MATLAB and reduces implementation time for other languages. The software is available as freeware along with educational videos on its website [4]. It doesn't need any prerequisites to run, as all of the libraries are included in the distribution package. I test it against existing database solutions using a battery of benchmarks and discuss the results.

Objective assessment of image quality in conventional and digital mammography taking into account dynamic range.

The goal of this work is to develop a method to objectively compare the performance of a digital and a screen-film mammography system in terms of image quality. The method takes into account the dynamic range of the image detector, the detection of high and low contrast structures, the visualisation of the images and the observer response. A test object, designed to represent a compressed breast, was constructed from various tissue equivalent materials ranging from purely adipose to purely glandular composition. Different areas within the test object permitted the evaluation of low and high contrast detection, spatial resolution and image noise. All the images (digital and conventional) were captured using a CCD camera to include the visualisation process in the image quality assessment. A mathematical model observer (non-prewhitening matched filter), that calculates the detectability of high and low contrast structures using spatial resolution, noise and contrast, was used to compare the two technologies. Our results show that for a given patient dose, the detection of high and low contrast structures is significantly better for the digital system than for the conventional screen-film system studied. The method of using a test object with a large tissue composition range combined with a camera to compare conventional and digital imaging modalities can be applied to other radiological imaging techniques. In particular it could be used to optimise the process of radiographic reading of soft copy images.

Reticulocyte dynamic and hemoglobin variability in hemodialysis patients treated with Darbepoetin alfa and C.E.R.A.: a randomized controlled trial.

BACKGROUND: In a simulation based on a pharmacokinetic model we demonstrated that increasing the erythropoiesis stimulating agents (ESAs) half-life or shortening their administration interval decreases hemoglobin variability. The benefit of reducing the administration interval was however lessened by the variability induced by more frequent dosage adjustments. The purpose of this study was to analyze the reticulocyte and hemoglobin kinetics and variability under different ESAs and administration intervals in a collective of chronic hemodialysis patients. METHODS: The study was designed as an open-label, randomized, four-period cross-over investigation, including 30 patients under chronic hemodialysis at the regional hospital of Locarno (Switzerland) in February 2010 and lasting 2 years. Four subcutaneous treatment strategies (C.E.R.A. every 4 weeks Q4W and every 2 weeks Q2W, Darbepoetin alfa Q4W and Q2W) were compared with each other. The mean square successive difference of hemoglobin, reticulocyte count and ESAs dose was used to quantify variability. We distinguished a short- and a long-term variability based respectively on the weekly and monthly successive difference. RESULTS: No difference was found in the mean values of biological parameters (hemoglobin, reticulocytes, and ferritin) between the 4 strategies. ESAs type did not affect hemoglobin and reticulocyte variability, but C.E.R.A induced a more sustained reticulocytes response over time and increased the risk of hemoglobin overshooting (OR 2.7, p = 0.01). Shortening the administration interval lessened the amplitude of reticulocyte count fluctuations but resulted in more frequent ESAs dose adjustments and in amplified reticulocyte and hemoglobin variability. Q2W administration interval was however more favorable in terms of ESAs dose, allowing a 38% C.E.R.A. dose reduction, and no increase of Darbepoetin alfa. CONCLUSIONS: The reticulocyte dynamic was a more sensitive marker of time instability of the hemoglobin response under ESAs therapy. The ESAs administration interval had a greater impact on hemoglobin variability than the ESAs type. The more protracted reticulocyte response induced by C.E.R.A. could explain both, the observed higher risk of overshoot and the significant increase in efficacy when shortening its administration interval.Trial registrationClinicalTrials.gov NCT01666301.

Dynamic models of viral replication and latency.

PURPOSE OF REVIEW: HIV targets primary CD4(+) T cells. The virus depends on the physiological state of its target cells for efficient replication, and, in turn, viral infection perturbs the cellular state significantly. Identifying the virus-host interactions that drive these dynamic changes is important for a better understanding of viral pathogenesis and persistence. The present review focuses on experimental and computational approaches to study the dynamics of viral replication and latency. RECENT FINDINGS: It was recently shown that only a fraction of the inducible latently infected reservoirs are successfully induced upon stimulation in ex-vivo models while additional rounds of stimulation make allowance for reactivation of more latently infected cells. This highlights the potential role of treatment duration and timing as important factors for successful reactivation of latently infected cells. The dynamics of HIV productive infection and latency have been investigated using transcriptome and proteome data. The cellular activation state has shown to be a major determinant of viral reactivation success. Mathematical models of latency have been used to explore the dynamics of the latent viral reservoir decay. SUMMARY: Timing is an important component of biological interactions. Temporal analyses covering aspects of viral life cycle are essential for gathering a comprehensive picture of HIV interaction with the host cell and untangling the complexity of latency. Understanding the dynamic changes tipping the balance between success and failure of HIV particle production might be key to eradicate the viral reservoir.

Dynamic impacts of the inhibition of the molecular chaperone hsp90 on the T-cell proteome have implications for anti-cancer therapy.

The molecular chaperone Hsp90-dependent proteome represents a complex protein network of critical biological and medical relevance. Known to associate with proteins with a broad variety of functions termed clients, Hsp90 maintains key essential and oncogenic signalling pathways. Consequently, Hsp90 inhibitors are being tested as anti-cancer drugs. Using an integrated systematic approach to analyse the effects of Hsp90 inhibition in T-cells, we quantified differential changes in the Hsp90-dependent proteome, Hsp90 interactome, and a selection of the transcriptome. Kinetic behaviours in the Hsp90-dependent proteome were assessed using a novel pulse-chase strategy (Fierro-Monti et al., accompanying article), detecting effects on both protein stability and synthesis. Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects. As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients. Most likely, consequences of the role of Hsp90 in gene expression determined a global reduction in net de novo protein synthesis. This decrease appeared to be greater in magnitude than a concomitantly observed global increase in protein decay rates. Several novel putative Hsp90 clients were validated, and interestingly, protein families with critical functions, particularly the Hsp90 family and cofactors themselves as well as protein kinases, displayed strongly increased decay rates due to Hsp90 inhibitor treatment. Remarkably, an upsurge in survival pathways, involving molecular chaperones and several oncoproteins, and decreased levels of some tumour suppressors, have implications for anti-cancer therapy with Hsp90 inhibitors. The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions. Data are available via ProteomeXchange with identifier PXD000537.

Use of a dynamic foot pressure index to monitor the effects of treatment for equinus gait in children with cerebral palsy.

The purpose of this study is to introduce and describe a newly developed index using foot pressure analysis to quantify the degree of equinus gait in children with cerebral palsy before and after injection with botulinum toxin. Data were captured preinjection and 12 weeks postinjection. Ten children aged 2(1/2) to 6(1/2) years took part (5 boys and 5 girls). Three of them had a diagnosis of spastic diplegia and 7 of congenital hemiplegia. In total, 13 limbs were analyzed. After orientation and segmentation of raw pedobarographic data, we determined a dynamic foot pressure index graded 0 to 100 that quantified the relative degree of heel and forefoot contact during stance. These data were correlated (Pearson correlation) with clinical measurements of dorsiflexion at the ankle (on a slow and fast stretch) and video observation (using the Observational Gait Scale). Pedobarograph data were strongly correlated with both the Observational Gait Scale scores (R = 0.79, P < 0.005) and clinical measurements of dorsiflexion on a fast stretch, which is reflective of spasticity (R = 0.70, P < 0.005). We demonstrated the index's sensitivity in detecting changes in spasticity and good correlation with video observations seems to indicate this technique's potential validity. When manipulated and segmented appropriately, and with the development of a simple ordinal index, we found that foot pressure data provided a useful tool in tracking changes in patients with spastic equinus.

A dynamic model of keratinocyte stem cell renewal and differentiation: role of the p21WAF1/Cip1 and Notch1 signaling pathways.

We present here a dynamic model of functional equilibrium between keratinocyte stem cells, transit amplifying populations and cells that are reversibly versus irreversibly committed to differentiation. According to this model, the size of keratinocyte stem cell populations can be controlled at multiple levels, including relative late steps in the sequence of events leading to terminal differentiation and by the influences of a heterogeneous extra-cellular environment. We discuss how work in our laboratory, on the interconnection between the cyclin/CDK inhibitor p21WAF1/Cip1 and the Notch1 signaling pathways, provides strong support to this dynamic model of stem cell versus committed and/or differentiated keratinocyte populations.

Proton NMR of (15)N-choline metabolites enhanced by dynamic nuclear polarization.

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from (15)N to methyl- and methylene protons, using the small (2,3)J((1)H,(15)N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T(1)((15)N) approximately 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.