A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

BACKGROUND: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. RESULTS: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. CONCLUSIONS: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. AVAILABILITY: RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).

Exploring unfrequent events with accelerated molecular dynamics simulations: from photochemistry to drug design

La meva incorporació al grup de recerca del Prof. McCammon (University of California San Diego) en qualitat d’investigador post doctoral amb una beca Beatriu de Pinós, va tenir lloc el passat 1 de desembre de 2010; on vaig dur a terme les meves tasques de recerca fins al darrer 1 d’abril de 2012. El Prof. McCammon és un referent mundial en l’aplicació de simulacions de dinàmica molecular (MD) en sistemes biològics d’interès humà. La contribució més important del Prof. McCammon en la simulació de sistemes biològics és el desenvolupament del mètode de dinàmiques moleculars accelerades (AMD). Les simulacions MD convencionals, les quals estan limitades a l’escala de temps del nanosegon (~10-9s), no son adients per l’estudi de sistemes biològics rellevants a escales de temps mes llargues (μs, ms...). AMD permet explorar fenòmens moleculars poc freqüents però que son clau per l’enteniment de molts sistemes biològics; fenòmens que no podrien ser observats d’un altre manera. Durant la meva estada a la “University of California San Diego”, vaig treballar en diferent aplicacions de les simulacions AMD, incloent fotoquímica i disseny de fàrmacs per ordinador. Concretament, primer vaig desenvolupar amb èxit una combinació dels mètodes AMD i simulacions Car-Parrinello per millorar l’exploració de camins de desactivació (interseccions còniques) en reaccions químiques fotoactivades. En segon lloc, vaig aplicar tècniques estadístiques (Replica Exchange) amb AMD en la descripció d’interaccions proteïna-lligand. Finalment, vaig dur a terme un estudi de disseny de fàrmacs per ordinador en la proteïna-G Rho (involucrada en el desenvolupament de càncer humà) combinant anàlisis estructurals i simulacions AMD. Els projectes en els quals he participat han estat publicats (o estan encara en procés de revisió) en diferents revistes científiques, i han estat presentats en diferents congressos internacionals. La memòria inclosa a continuació conté més detalls de cada projecte esmentat.

Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

Colonic movements in healthy subjects as monitored by a Magnet Tracking System.

The Magnet Tracking System (MTS) is a minimally-invasive technique of continuous evaluation of gastrointestinal motility. In this study, MTS was used to analyse colonic propulsive dynamics and compare the transit of a magnetic pill with that of standard radio-opaque markers. MTS monitors the progress in real time of a magnetic pill through the gut. Ten men and 10 women with regular daily bowel movements swallowed this pill and 10 radio-opaque markers at 8 pm. Five hours of recordings were conducted during 2 following mornings. Origin, direction, amplitude and velocity of movements were analysed relative to space-time plots of the pill trajectory. Abdominal radiographs were taken to compare the progress of both pill and markers. The magnetic pill lay idle for 90% of its sojourn in the colon; its total retrograde displacement accounted for only 20% of its overall movement. Analysis of these movements showed a bimodal distribution of velocities: around 1.5 and 50 cm min(-1), the latter being responsible for 2/3 of distance traversed. There were more movements overall and more mass movements in males. Net hourly forward progress was greater in the left than right colon, and greater in males. The position of the magnetic pill correlated well with the advancement of markers. MTS showed patterns and propulsion dynamics of colonic segments with as yet unmet precision. Detailed analysis of slow and fast patterns of colonic progress makes it possible to specify the motility of colonic segments, and any variability in gender. Such analysis opens up promising avenues in studies of motility disorders.

A wearable system for multi-segment foot kinetics measurement.

This study aims to design a wearable system for kinetics measurement of multi-segment foot joints in long-distance walking and to investigate its suitability for clinical evaluations. The wearable system consisted of inertial sensors (3D gyroscopes and 3D accelerometers) on toes, forefoot, hindfoot, and shank, and a plantar pressure insole. After calibration in a laboratory, 10 healthy elderly subjects and 12 patients with ankle osteoarthritis walked 50m twice wearing this system. Using inverse dynamics, 3D forces, moments, and power were calculated in the joint sections among toes, forefoot, hindfoot, and shank. Compared to those we previously estimated for a one-segment foot model, the sagittal and transverse moments and power in the ankle joint, as measured via multi-segment foot model, showed a normalized RMS difference of less than 11%, 14%, and 13%, respectively, for healthy subjects, and 13%, 15%, and 14%, for patients. Similar to our previous study, the coronal moments were not analyzed. Maxima-minima values of anterior-posterior and vertical force, sagittal moment, and power in shank-hindfoot and hindfoot-forefoot joints were significantly different between patients and healthy subjects. Except for power, the inter-subject repeatability of these parameters was CMC>0.90 for healthy subjects and CMC>0.70 for patients. Repeatability of these parameters was lower for the forefoot-toes joint. The proposed measurement system estimated multi-segment foot joints kinetics with acceptable repeatability but showed difference, compared to those previously estimated for the one-segment foot model. These parameters also could distinguish patients from healthy subjects. Thus, this system is suggested for outcome evaluations of foot treatments.

A behavioral ecology of shermen: hidden stories from trajectory data in the Northern Humboldt Current System

This work proposes an original contribution to the understanding of shermen spatial behavior, based on the behavioral ecology and movement ecology paradigms. Through the analysis of Vessel Monitoring System (VMS) data, we characterized the spatial behavior of Peruvian anchovy shermen at di erent scales: (1) the behavioral modes within shing trips (i.e., searching, shing and cruising); (2) the behavioral patterns among shing trips; (3) the behavioral patterns by shing season conditioned by ecosystem scenarios; and (4) the computation of maps of anchovy presence proxy from the spatial patterns of behavioral mode positions. At the rst scale considered, we compared several Markovian (hidden Markov and semi-Markov models) and discriminative models (random forests, support vector machines and arti cial neural networks) for inferring the behavioral modes associated with VMS tracks. The models were trained under a supervised setting and validated using tracks for which behavioral modes were known (from on-board observers records). Hidden semi-Markov models performed better, and were retained for inferring the behavioral modes on the entire VMS dataset. At the second scale considered, each shing trip was characterized by several features, including the time spent within each behavioral mode. Using a clustering analysis, shing trip patterns were classi ed into groups associated to management zones, eet segments and skippers' personalities. At the third scale considered, we analyzed how ecological conditions shaped shermen behavior. By means of co-inertia analyses, we found signi cant associations between shermen, anchovy and environmental spatial dynamics, and shermen behavioral responses were characterized according to contrasted environmental scenarios. At the fourth scale considered, we investigated whether the spatial behavior of shermen re ected to some extent the spatial distribution of anchovy. Finally, this work provides a wider view of shermen behavior: shermen are not only economic agents, but they are also foragers, constrained by ecosystem variability. To conclude, we discuss how these ndings may be of importance for sheries management, collective behavior analyses and end-to-end models.

Fertilizer 15N balance in a coffee cropping system: a case study in Brazil

Knowledge about the fate of fertilizer nitrogen in agricultural systems is essential for the improvement of management practices in order to maximize nitrogen (N) recovery by the crop and reduce N losses from the system to a minimum. This study involves fertilizer management practices using the 15N isotope label applied in a single rate to determine the fertilizer-N balance in a particular soil-coffee-atmosphere system and to deepen the understanding of N plant dynamics. Five replicates consisting of plots of about 120 plants each were randomly defined within a 0.2 ha coffee plantation planted in 2001, in Piracicaba, SP, Brazil. Nine plants of each plot were separated in sub-plots for the 15N balance studies and treated with N rates of 280 and 350 kg ha-1 during 2003/2004 and 2004/2005, respectively, both of them as ammonium sulfate enriched to a 15N abundance of 2.072 atom %. Plant shoots were considered as separate parts: the orthotropic central branch, productive branches, leaves of productive branches, vegetative branches, leaves of vegetative branches and fruit. Litter, consisting of dead leaves accumulated below the plant canopy, was measured by the difference between leaves at harvest and at the beginning of the following flowering. Roots and soil were sampled down to a depth of 1.0 at intervals of 0.2 m. Samples from the isotopic sub-plots were used to evaluate total N and 15N, and plants outside sub-plots were used to evaluate dry matter. Volatilization losses of NH3 were estimated using special collectors. Leaching of fertilizer-N was estimated from deep drainage water fluxes and 15N concentrations of the soil solution at 1 m soil depth. At the end of the 2-year evaluation, the recovery of 15N applied as ammonium sulfate was 19.1 % in aerial plant parts, 9.4 % in the roots, 23.8 % in the litter, 26.3 % in the fruit and 12.6 % remaining in the 0_1.0 m soil profile. Annual leaching and volatilization losses were very small (2.0 % and 0.9 %, respectively). After two years, only 6.2 % N were missing in the balance (100 %) which can be attributed to other non-estimated compartments and experimental errors. Results show that an enrichment of only 2 % atom 15N allows the study of the partition of fertilizer-N in a perennial crop such as coffee during a period of two years.

Deficit, human capital and economic growth dynamics

Long-run economic growth arouses a great interest since it can shed light on the income-path of an economy and try to explain the large differences in income we observe across countries and over time. The neoclassical model has been followed by several endogenous growth models which, contrarily to the former, seem to predict that economies with similar preferences and technological level, do not necessarily tend to converge to similar per capita income levels. This paper attempts to show a possible mechanismthrough which macroeconomic disequilibria and inefficiencies, represented by budget deficits, may hinder human capital accumulation and therefore economic growth. Using a mixed education system, deficit is characterized as a bug agent which may end up sharply reducing the resources devoted to education and training. The paper goes a step further from the literature on deficit by introducing a rich dynamic analysis of the effects of a deficit reduction on different economic aspects.Following a simple growth model and allowing for slight changes in the law of human capital accumulation, we reach a point where deficit might sharply reduce human capital accumulation. On the other hand, a deficit reduction carried on for a long time, taking that reduction as a more efficient management of the economy, may prove useful in inducing endogenous growth. Empirical evidence for a sample of countries seems to support the theoretical assumptions in the model: (1) evidence on an inverse relationship betweendeficit and human capital accumulation, (2) presence of a strongly negative associationbetween the quantity of deficit in the economy and the rate of growth. They may prove a certain role for budget deficit in economic growth

Size-dependent recombination dynamics in ZnO nanowires.

A deep understanding of the recombination dynamics of ZnO nanowires NWs is a natural step for a precise design of on-demand nanostructures based on this material system. In this work we investigate the influence of finite-size on the recombination dynamics of the neutral bound exciton around 3.365 eV for ZnO NWs with different diameters. We demonstrate that the lifetime of this excitonic transition decreases with increasing the surface-to-volume ratio due to a surface induced recombination process. Furthermore, we have observed two broad transitions around 3.341 and 3.314 eV, which were identified as surface states by studying the dependence of their life time and intensitiy with the NWs dimensions.

Deficit, human capital and economic growth dynamics

Long-run economic growth arouses a great interest since it can shed light on the income-path of an economy and try to explain the large differences in income we observe across countries and over time. The neoclassical model has been followed by several endogenous growth models which, contrarily to the former, seem to predict that economies with similar preferences and technological level, do not necessarily tend to converge to similar per capita income levels. This paper attempts to show a possible mechanismthrough which macroeconomic disequilibria and inefficiencies, represented by budget deficits, may hinder human capital accumulation and therefore economic growth. Using a mixed education system, deficit is characterized as a bug agent which may end up sharply reducing the resources devoted to education and training. The paper goes a step further from the literature on deficit by introducing a rich dynamic analysis of the effects of a deficit reduction on different economic aspects.Following a simple growth model and allowing for slight changes in the law of human capital accumulation, we reach a point where deficit might sharply reduce human capital accumulation. On the other hand, a deficit reduction carried on for a long time, taking that reduction as a more efficient management of the economy, may prove useful in inducing endogenous growth. Empirical evidence for a sample of countries seems to support the theoretical assumptions in the model: (1) evidence on an inverse relationship betweendeficit and human capital accumulation, (2) presence of a strongly negative associationbetween the quantity of deficit in the economy and the rate of growth. They may prove a certain role for budget deficit in economic growth

Comparison between molecular dynamics abd Monte Carlo simulations of an ordering process in a binary alloy

Ordering in a binary alloy is studied by means of a molecular-dynamics (MD) algorithm which allows to reach the domain growth regime. Results are compared with Monte Carlo simulations using a realistic vacancy-atom (MC-VA) mechanism. At low temperatures fast growth with a dynamical exponent x>1/2 is found for MD and MC-VA. The study of a nonequilibrium ordering process with the two methods shows the importance of the nonhomogeneity of the excitations in the system for determining its macroscopic kinetics.

Multiple edges of a quantum Hall system in a strong electric

In this paper we show that if the electrons in a quantum Hall sample are subjected to a constant electric field in the plane of the material, comparable in magnitude to the background magnetic field on the system of electrons, a multiplicity of edge states localized at different regions of space is produced in the sample. The actions governing the dynamics of these edge states are obtained starting from the well-known Schrödinger field theory for a system of nonrelativistic electrons, where on top of the constant background electric and magnetic fields, the electrons are further subject to slowly varying weak electromagnetic fields. In the regions between the edges, dubbed as the "bulk," the fermions can be integrated out entirely and the dynamics expressed in terms of a local effective action involving the slowly varying electromagnetic potentials. It is further shown how the bulk action is gauge noninvariant in a particular way, and how the edge states conspire to restore the U(1) electromagnetic gauge invariance of the system. In the edge action we obtain a heretofore unnoticed gauge-invariant term that depends on the particular edge. We argue that this term may be detected experimentally as different edges respond differently to a monochromatic probe due to this term

Hysteresis and avalanches in the T=0 random field ising model with 2-spin flip dynamics

We study the nonequilibrium behavior of the three-dimensional Gaussian random-field Ising model at T=0 in the presence of a uniform external field using a two-spin-flip dynamics. The deterministic, history-dependent evolution of the system is compared with the one obtained with the standard one-spin-flip dynamics used in previous studies of the model. The change in the dynamics yields a significant suppression of coercivity, but the distribution of avalanches (in number and size) stays remarkably similar, except for the largest ones that are responsible for the jump in the saturation magnetization curve at low disorder in the thermodynamic limit. By performing a finite-size scaling study, we find strong evidence that the change in the dynamics does not modify the universality class of the disorder-induced phase transition.

Microbial biomass and soil fauna during the decomposition of cover crops in no-tillage system

The decomposition of plant residues is a biological process mediated by soil fauna, but few studies have been done evaluating its dynamics in time during the process of disappearance of straw. This study was carried out in Chapecó, in southern Brazil, with the objective of monitoring modifications in soil fauna populations and the C content in the soil microbial biomass (C SMB) during the decomposition of winter cover crop residues in a no-till system. The following treatments were tested: 1) Black oat straw (Avena strigosa Schreb.); 2) Rye straw (Secale cereale L.); 3) Common vetch straw (Vicia sativa L.). The cover crops were grown until full flowering and then cut mechanically with a rolling stalk chopper. The soil fauna and C content in soil microbial biomass (C SMB) were assessed during the period of straw decomposition, from October 2006 to February 2007. To evaluate C SMB by the irradiation-extraction method, soil samples from the 0-10 cm layer were used, collected on eight dates, from before until 100 days after residue chopping. The soil fauna was collected with pitfall traps on seven dates up to 85 days after residue chopping. The phytomass decomposition of common vetch was faster than of black oat and rye residues. The C SMB decreased during the process of straw decomposition, fastest in the treatment with common vetch. In the common vetch treatment, the diversity of the soil fauna was reduced at the end of the decomposition process.

Nitrogen dynamics in soil management systems. I - flux of inorganic nitrogen (NH4+ and NO3-)

In agricultural systems the N-NH4+ and N-NO3- contents is significantly affected by soil management. This study investigated the dynamics of inorganic nitrogen (N; NH4+ and NO3-) in an experimental evaluation of soil management systems (SMSs) adopted in 1988 at the experimental station of the ABC Foundation in Ponta Grossa, in the Central South region of the State of Paraná. The objective of this study was to evaluate the changes in N-NH4+ and N-NO3- flux in the surface layer of a Red Latosol arising from SMSs over a 12-month period. The experiment was arranged in a completely randomized block design in split plots, in three replications. The plots consisted of the following SMSs: 1) conventional tillage (CT); 2) minimum tillage (MT); 3) no-tillage with chisel plow every three years (NT CH); and 4) continuous no-tillage (CNT). To evaluate the dynamics of inorganic N, the subplots represented samplings (11 sampling times, T1 - T11). The ammonium N (N-NH4+) and nitric N (N-NO3-) contents were higher in systems with reduced tillage (MT and NT CH) and without tillage (CNT) than in the CT system. In the period from October 2003 to February 2004, the N-NH4+ was higher than the N-NO3- soil content. Conversely, in the period from May 2004 to July 2004, the N-NO3- was higher than the N-NH4+ content. The greatest fluctuation in the N-NH4+ and N-NO3- contents occurred in the 0-2.5 cm layer, and the highest peak in the N-NH4+ and N-NO3- concentrations occurred after the surface application of N. Both N-NH4+ and N-NO3- were strongly correlated with the soil organic C content, which indicated that these properties vary together in the system.