Single fluorescent protein-based Ca2+ sensors with increased dynamic range

<p>Abstract</p> <p>Background</p> <p>Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).</p> <p>Results</p> <p>Here we report significant progress on the development of the latter type of Ca²⁺sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺response to a prolonged glutamate treatment in cortical neurons.</p> <p>Conclusion</p> <p>We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.</p

Towards Machine Wald

The past century has seen a steady increase in the need of estimating and predicting complex systems and making (possibly critical) decisions with limited information. Although computers have made possible the numerical evaluation of sophisticated statistical models, these models are still designed \emph{by humans} because there is currently no known recipe or algorithm for dividing the design of a statistical model into a sequence of arithmetic operations. Indeed enabling computers to \emph{think} as \emph{humans} have the ability to do when faced with uncertainty is challenging in several major ways: (1) Finding optimal statistical models remains to be formulated as a well posed problem when information on the system of interest is incomplete and comes in the form of a complex combination of sample data, partial knowledge of constitutive relations and a limited description of the distribution of input random variables. (2) The space of admissible scenarios along with the space of relevant information, assumptions, and/or beliefs, tend to be infinite dimensional, whereas calculus on a computer is necessarily discrete and finite. With this purpose, this paper explores the foundations of a rigorous framework for the scientific computation of optimal statistical estimators/models and reviews their connections with Decision Theory, Machine Learning, Bayesian Inference, Stochastic Optimization, Robust Optimization, Optimal Uncertainty Quantification and Information Based Complexity.Comment: 37 page

Scaling properties of protein family phylogenies

One of the classical questions in evolutionary biology is how evolutionary processes are coupled at the gene and species level. With this motivation, we compare the topological properties (mainly the depth scaling, as a characterization of balance) of a large set of protein phylogenies with a set of species phylogenies. The comparative analysis shows that both sets of phylogenies share remarkably similar scaling behavior, suggesting the universality of branching rules and of the evolutionary processes that drive biological diversification from gene to species level. In order to explain such generality, we propose a simple model which allows us to estimate the proportion of evolvability/robustness needed to approximate the scaling behavior observed in the phylogenies, highlighting the relevance of the robustness of a biological system (species or protein) in the scaling properties of the phylogenetic trees. Thus, the rules that govern the incapability of a biological system to diversify are equally relevant both at the gene and at the species level.Comment: Replaced with final published versio

Содержание фактора активации тромбоцитов в плазме крови больных бронхиальной астмой. Влияние тромбоцитафереза

Levels of serum PA F were investigated in patients with various bronchial asthma (BA) forms and in various forms of the clinical manifestation of the disease before and after plateletapheresis (PtA). 15 patients with BA (9 atopic and 6 aspirin-sensitive ones) and 4 healthy donors were examined. The examination demonstrated that only tracks of PA F in the healthy donors are registered but in BA patients the PAF levels were significantly increased. It was found, that in patients with atopic BA the PAF levels were almost by two times higher than in aspirinic BA ones. Moreover, the PAF level in a great extent depends on the disease degree. The serum PAF level was decreased in 42% in average after trobmocytapheresis.The study demonstrates the features of thrombocytapheresis action and develops the imagination about its action mechanisms.Исследовался уровень фактора активации тромбоцитов (ФАТ) в крови больных различными формами бронхиальной астмы разной степени тяжести и влияние тромбоцитафереза на этот показатель. Обследовано 15 больных с БА (9 с атопической и 6 с аспириновой) и 4 здоровых донора. Исследования показали, что в крови здоровых доноров регистрируются лишь следы ФАТ, тогда как у больных БА содержание ФАТ значительно повышено. Выявлено, что у больных аспириновой астмой уровень ФАТ почти в 2 раза ниже, чем в группе больных атопической астмой. Кроме того, чем тяж елее протекает заболевание, тем вы ш е уровень ФАТ. После тромбоцитафереза уровень ФАТ в крови больных, получавших данную терапию , снизился в среднем на 42%.Исследования раскрывают особенности действия тромбоцитафереза, углубляют представления о механизме действия этой процедуры

Glutamate-induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones

The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and mitochondrial potential (ΔΨm) in individual hippocampal neurones in culture coloaded with fura-2 AM or fura-2FF AM and rhodamine 123 (Rh 123).In most cells (96 %) at 6-7 days in vitro (DIV) and in a small proportion of cells (29 %) at 11-17 DIV the [Ca2+]i increase induced by exposure to 100 μm glutamate for 10 min was associated with a small mitochondrial depolarisation, followed by mitochondrial repolarisation, and a degree of recovery of [Ca2+]i following glutamate washout. In the majority of neurones at 11-17 DIV (71 %), exposure to glutamate for 10 min induced a profound mono- or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca2+]i plateau despite the removal of glutamate.Addition of glutamate receptor antagonists (15 μm MK-801 plus 75 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)) to the washout solution did not affect the post-glutamate [Ca2+]i plateau in neurones exhibiting a profound mitochondrial depolarisation but greatly improved [Ca2+]i recovery in those neurones undergoing only a small mitochondrial depolarisation, suggesting that the release of endogenous glutamate delays [Ca2+]i recovery in the postglutamate period.Cyclosporin A (500 nM) or N-methyl Val-4-cyclosporin A (200 nM) delayed or even prevented the development of the second phase of mitochondrial depolarisation in cells at 11-17 DIV and increased the proportion of neurones exhibiting a small monophasic mitochondrial depolarisation and [Ca2+]i recovery upon glutamate removal.We have thus described a striking correlation between mitochondrial depolarisation and the failure of cells to restore [Ca2+]i following a toxic glutamate challenge. These data suggest that mitochondrial dysfunction plays a major role in the deregulation of [Ca2+]i associated with glutamate toxicity