Identification of biomolecule mass transport and binding rate parameters in living cells by inverse modeling

BACKGROUND: Quantification of in-vivo biomolecule mass transport and reaction rate parameters from experimental data obtained by Fluorescence Recovery after Photobleaching (FRAP) is becoming more important. METHODS AND RESULTS: The Osborne-Moré extended version of the Levenberg-Marquardt optimization algorithm was coupled with the experimental data obtained by the Fluorescence Recovery after Photobleaching (FRAP) protocol, and the numerical solution of a set of two partial differential equations governing macromolecule mass transport and reaction in living cells, to inversely estimate optimized values of the molecular diffusion coefficient and binding rate parameters of GFP-tagged glucocorticoid receptor. The results indicate that the FRAP protocol provides enough information to estimate one parameter uniquely using a nonlinear optimization technique. Coupling FRAP experimental data with the inverse modeling strategy, one can also uniquely estimate the individual values of the binding rate coefficients if the molecular diffusion coefficient is known. One can also simultaneously estimate the dissociation rate parameter and molecular diffusion coefficient given the pseudo-association rate parameter is known. However, the protocol provides insufficient information for unique simultaneous estimation of three parameters (diffusion coefficient and binding rate parameters) owing to the high intercorrelation between the molecular diffusion coefficient and pseudo-association rate parameter. Attempts to estimate macromolecule mass transport and binding rate parameters simultaneously from FRAP data result in misleading conclusions regarding concentrations of free macromolecule and bound complex inside the cell, average binding time per vacant site, average time for diffusion of macromolecules from one site to the next, and slow or rapid mobility of biomolecules in cells. CONCLUSION: To obtain unique values for molecular diffusion coefficient and binding rate parameters from FRAP data, we propose conducting two FRAP experiments on the same class of macromolecule and cell. One experiment should be used to measure the molecular diffusion coefficient independently of binding in an effective diffusion regime and the other should be conducted in a reaction dominant or reaction-diffusion regime to quantify binding rate parameters. The method described in this paper is likely to be widely used to estimate in-vivo biomolecule mass transport and binding rate parameters

Dopamine Inhibits Mitochondrial Motility in Hippocampal Neurons

The trafficking of mitochondria within neurons is a highly regulated process. In an earlier study, we found that serotonin (5-HT), acting through the 5-HT1A receptor subtype, promotes axonal transport of mitochondria in cultured hippocampal neurons by increasing Akt activity, and consequently decreasing glycogen synthase kinase (GSK3beta) activity. This finding suggests a critical role for neuromodulators in the regulation of mitochondrial trafficking in neurons. In the present study, we investigate the effects of a second important neuromodulator, dopamine, on mitochondrial transport in hippocampal neurons.Here, we show that dopamine, like 5-HT, regulates mitochondrial motility in cultured hippocampal neurons through the Akt-GSK3beta signaling cascade. But, in contrast to the stimulatory effect of 5-HT, administration of exogenous dopamine or bromocriptine, a dopamine 2 receptor (D2R) agonist, caused an inhibition of mitochondrial movement. Moreover, pretreatment with bromocriptine blocked the stimulatory effect of 5-HT on mitochondrial movement. Conversely, in cells pretreated with 5-HT, no further increases in movement were observed after administration of haloperidol, a D2R antagonist. In contrast to the effect of the D2R agonist, addition of SKF38393, a dopamine 1 receptor (D1R) agonist, promoted mitochondrial transport, indicating that the inhibitory effect of dopamine was actually the net summation of opposing influences of the two receptor subtypes. The most pronounced effect of dopamine signals was on mitochondria that were already moving directionally. Western blot analysis revealed that treatment with either a D2R agonist or a D1R antagonist decreased Akt activity, and conversely, treatment with either a D2R antagonist or a D1R agonist increased Akt activity.Our observations strongly suggest a role for both dopamine and 5-HT in regulating mitochondrial movement, and indicate that the integrated effects of these two neuromodulators may be important in determining the distribution of energy sources in neurons

Inhibition of Electrical Activity by Retroviral Infection with Kir2.1 Transgenes Disrupts Electrical Differentiation of Motoneurons

Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca2+-dependent K+ channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K+ currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na+ channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons

Effects of Chronic Calorie Restriction or Dietary Resveratrol Supplementation on Insulin Sensitivity Markers in a Primate, Microcebus murinus

The prevalence of diabetes and hyperinsulinemia increases with age, inducing metabolic failure and limiting lifespan. Calorie restriction (CR) without malnutrition delays the aging process, but its long-term application to humans seems difficult. Resveratrol (RSV), a dietary polyphenol, appears to be a promising CR mimetic that can be easily administered in humans. In this work, we hypothesized that both CR and RSV impact insulin sensitivity in a non-human primate compared to standard-fed control (CTL) animals. Four- to five-year-old male grey mouse lemurs (Microcebus murinus) were assigned to three dietary groups: a CTL group, a CR group receiving 30% fewer calories than the CTL and a RSV group receiving the CTL diet supplemented with RSV (200 mg·day−1·kg−1). Insulin sensitivity and glycemia were assessed using an oral glucose tolerance test (OGTT) and the homeostasis model assessment of insulin resistance (HOMA-IR index) evaluation after 21 or 33 months of chronic treatment. Resting metabolic rate was also measured to assess the potential relationships between this energy expenditure parameter and insulin sensitivity markers. No differences were found after a 21-month period of treatment, except for lower glucose levels 30 min after glucose loading in CR animals. After 33 months, CR and RSV decreased glycemia after the oral glucose loading without decreasing fasting blood insulin. A general effect of treatment was observed on the HOMA-IR index, with an 81% reduction in CR animals and 53% in RSV animals after 33 months of treatment compared to CTL. Chronic CR and dietary supplementation with RSV affected insulin sensitivity by improving the glucose tolerance of animals without disturbing their baseline insulin secretion. These results suggest that both CR and RSV have beneficial effects on metabolic alterations, although these effects are different in amplitude between the two anti-aging treatments and potentially rely on different metabolic changes

Disruption of Mitochondrial DNA Replication in Drosophila Increases Mitochondrial Fast Axonal Transport In Vivo

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases

Advancing a Conceptual Model of Evidence-Based Practice Implementation in Public Service Sectors

Implementation science is a quickly growing discipline. Lessons learned from business and medical settings are being applied but it is unclear how well they translate to settings with different historical origins and customs (e.g., public mental health, social service, alcohol/drug sectors). The purpose of this paper is to propose a multi-level, four phase model of the implementation process (i.e., Exploration, Adoption/Preparation, Implementation, Sustainment), derived from extant literature, and apply it to public sector services. We highlight features of the model likely to be particularly important in each phase, while considering the outer and inner contexts (i.e., levels) of public sector service systems

The role of the mitochondria and the endoplasmic reticulum contact sites in the development of the immune responses

Abstract Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are dynamic modules enriched in subset of lipids and specialized proteins that determine their structure and functions. The MERCs regulate lipid transfer, autophagosome formation, mitochondrial fission, Ca2+ homeostasis and apoptosis. Since these functions are essential for cell biology, it is therefore not surprising that MERCs also play a critical role in organ physiology among which the immune system stands by its critical host defense function. This defense system must discriminate and tolerate host cells and beneficial commensal microorganisms while eliminating pathogenic ones in order to preserve normal homeostasis. To meet this goal, the immune system has two lines of defense. First, the fast acting but unspecific innate immune system relies on anatomical physical barriers and subsets of hematopoietically derived cells expressing germline-encoded receptors called pattern recognition receptors (PRR) recognizing conserved motifs on the pathogens. Second, the slower but very specific adaptive immune response is added to complement innate immunity. Adaptive immunity relies on another set of specialized cells, the lymphocytes, harboring receptors requiring somatic recombination to be expressed. Both innate and adaptive immune cells must be activated to phagocytose and process pathogens, migrate, proliferate, release soluble factors and destroy infected cells. Some of these functions are strongly dependent on lipid transfer, autophagosome formation, mitochondrial fission, and Ca2+ flux; this indicates that MERCs could regulate immunity

Incorporating non-equilibrium dynamics into demographic history inferences of a migratory marine species

ELC was supported while writing this paper by a EU Horizon 2020 Marie Slodowska Curie Fellowship, project BEHAVIOUR-CONNECT, by a Newton Fellowship from the Royal Society of London and Bayesian statistical training was supported by National Science Foundation (award DEB- 1145200). Laboratory analyses conducted by ELC were funded by a small grant from the British Ecological Society 5076 / 6118 and Bayesian analysis was supported by training from the National Science Foundation under Grant No. DEB-1145200. OEG was supported by the Marine Alliance for Science and Technology for Scotland (MASTS) funded by the Scottish Founding Council (grant reference HR09011). Genetic data from the South African right whale samples were generated by MB and PJP with the support of UC Berkeley, University of Stockholm and University of Groningen. Computational Biology analyses were supported by the University of St Andrews Bioinformatics Unit which is funded by a Wellcome Trust ISSF award.Understanding how dispersal and gene flow link geographically separated populations over evolutionary history is challenging, particularly in migratory marine species. In southern right whales (SRWs, Eubalaena australis), patterns of genetic diversity are likely influenced by the glacial climate cycle and recent history of whaling. Here we use a dataset of mitochondrial DNA (mtDNA) sequences (n=1,327) and nuclear markers (17 microsatellite loci, n=222) from major wintering grounds to investigate circumpolar population structure, historical demography, and effective population size. Analyses of nuclear genetic variation identify two population clusters that correspond to the South Atlantic and Indo-Pacific ocean basins that have similar effective breeder estimates. In contrast, all wintering grounds show significant differentiation for mtDNA, but no sex-biased dispersal was detected using the microsatellite genotypes. An approximate Bayesian computation (ABC) approach with microsatellite markers compared scenarios with gene flow through time, or isolation and secondary contact between ocean basins, while modeling declines in abundance linked to whaling. Secondary-contact scenarios yield the highest posterior probabilities, implying that populations in different ocean basins were largely isolated and came into secondary contact within the last 25,000 years, but the role of whaling in changes in genetic diversity and gene flow over recent generations could not be resolved. We hypothesis that these findings are driven by factors that promote isolation, such as female philopatry, and factors that could promote dispersal, such oceanographic changes. These findings highlight the application of ABC approaches to infer connectivity in mobile species with complex population histories and currently low levels of differentiation.PostprintPeer reviewe