DALMATIAN: An Algorithm for Automatic Cell Detection and Counting in 3D

Current 3D imaging methods, including optical projection tomography, light-sheet microscopy, block-face imaging, and serial two photon tomography enable visualization of large samples of biological tissue. Large volumes of data obtained at high resolution require development of automatic image processing techniques, such as algorithms for automatic cell detection or, more generally, point-like object detection. Current approaches to automated cell detection suffer from difficulties originating from detection of particular cell types, cell populations of different brightness, non-uniformly stained, and overlapping cells. In this study, we present a set of algorithms for robust automatic cell detection in 3D. Our algorithms are suitable for, but not limited to, whole brain regions and individual brain sections. We used watershed procedure to split regional maxima representing overlapping cells. We developed a bootstrap Gaussian fit procedure to evaluate the statistical significance of detected cells. We compared cell detection quality of our algorithm and other software using 42 samples, representing 6 staining and imaging techniques. The results provided by our algorithm matched manual expert quantification with signal-to-noise dependent confidence, including samples with cells of different brightness, non-uniformly stained, and overlapping cells for whole brain regions and individual tissue sections. Our algorithm provided the best cell detection quality among tested free and commercial software

On the superfluidity of classical liquid in nanotubes

In 2001, the author proposed the ultra second quantization method. The ultra second quantization of the Schr\"odinger equation, as well as its ordinary second quantization, is a representation of the N-particle Schr\"odinger equation, and this means that basically the ultra second quantization of the equation is the same as the original N-particle equation: they coincide in 3N-dimensional space. We consider a short action pairwise potential V(x_i -x_j). This means that as the number of particles tends to infinity, $N\to\infty$, interaction is possible for only a finite number of particles. Therefore, the potential depends on N in the following way: $V_N=V((x_i-x_j)N^{1/3})$. If V(y) is finite with support $\Omega_V$, then as $N\to\infty$ the support engulfs a finite number of particles, and this number does not depend on N. As a result, it turns out that the superfluidity occurs for velocities less than $\min(\lambda_{\text{crit}}, \frac{h}{2mR})$, where $\lambda_{\text{crit}}$ is the critical Landau velocity and R is the radius of the nanotube.Comment: Latex, 20p. The text is presented for the International Workshop "Idempotent and tropical mathematics and problems of mathematical physics", Independent University of Moscow, Moscow, August 25--30, 2007 and to be published in the Russian Journal of Mathematical Physics, 2007, vol. 15, #

Radiation Induces Distinct Changes in Defined Subpopulations of Neural Stem and Progenitor Cells in the Adult Hippocampus

While irradiation can effectively treat brain tumors, this therapy also causes cognitive impairments, some of which may stem from the disruption of hippocampal neurogenesis. To study how radiation affects neurogenesis, we combine phenotyping of subpopulations of hippocampal neural stem and progenitor cells with double- and triple S-phase labeling paradigms. Using this approach, we reveal new features of division, survival, and differentiation of neural stem and progenitor cells after exposure to gamma radiation. We show that dividing neural stem cells, while susceptible to damage induced by gamma rays, are less vulnerable than their rapidly amplifying progeny. We also show that dividing stem and progenitor cells that survive irradiation are suppressed in their ability to replicate 0.5–1 day after the radiation exposure. Suppression of division is also observed for cells that entered the cell cycle after irradiation or were not in the S phase at the time of exposure. Determining the longer term effects of irradiation, we found that 2 months after exposure, radiation-induced suppression of division is partially relieved for both stem and progenitor cells, without evidence for compensatory symmetric divisions as a means to restore the normal level of neurogenesis. By that time, most mature young neurons, born 2–4 weeks after the irradiation, still bear the consequences of radiation exposure, unlike younger neurons undergoing early stages of differentiation without overt signs of deficient maturation. Later, 6 months after an exposure to 5 Gy, cell proliferation and neurogenesis are further impaired, though neural stem cells are still available in the niche, and their pool is preserved. Our results indicate that various subpopulations of stem and progenitor cells in the adult hippocampus have different susceptibility to gamma radiation, and that neurogenesis, even after a temporary restoration, is impaired in the long term after exposure to gamma rays. Our study provides a framework for investigating critical issues of neural stem cell maintenance, aging, interaction with their microenvironment, and post-irradiation therapy

Combined 1H-Detected solid-state NMR spectroscopy and electron cryotomography to study membrane proteins across resolutions in native environments

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted system

Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of \u3b1-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels

YidC in Nanodiscs

Biological cells form basis of every living organism consists on Earth. The number of cells can range from just a single one (bacteria, amoeba) up to 150 trillion (Elephant). These cells contain a number of different components. Some of the most important components are proteins. Being linear polymers of amino-acids, they are folded into specific compact structures and facilitate important functions such as energy production, signal transduction, protein trafficking, molecular transport, and also host-pathogen interactions.1 An important class of proteins are membrane proteins. These proteins are embedded into anisotropic lipid bilayers at the cell boundaries, where they can be used for the interaction of the cell with the "outer" world. Be it the transport of nutrients across the membrane or signal transduction, they can handle everything. Membrane proteins have a large hydrophobic area. This area is normally located within the hydrophobic core of the membrane formed by lipid acyl chains. Hydrophobic interactions allow the protein to integrate into the membrane and to acquire the correct structure. Due to the complex environment at the membrane interface, most membrane proteins need some help with folding and positioning inside the membrane. For this insertases are used. In E.coli there are two complexes used for this: YidC and SecYEG. This is also because it is in itself a membrane protein so it cannot be analyzed outside of a membrane. In a membrane vesicle the exact number and composition of proteins is unknown. The size and shape of the vesicles can differ greatly and it is possible for interactions to occur that can normally not be seen. Also it is hard to analyze both sides of a protein at once since most synthetic bilayers form micelles2. The SecYEG complex has seen extensive research but YidC is still relatively unknown. is why the structure and mechanism of YidC is still largely unknown. This is true for most membrane proteins. Normally detergents are used to extract membrane proteins. However, this unnatural environment may destabilize and/or dissociate protein complexes and render the protein non-functional1. This makes analytical methods relying on detergent micells very unreliable. To combat this difficulty a solution has been found. A nanodisc is a small disc of a mixture of lipids surrounded by a scaffold protein to keep the layer intact. This size can be controlled and is homogeneous is shape. A nanodisc (Reference) can imitate a normal membrane while still allowing single molecule analysis. In the following sections more will be explained about YidC, Nanodiscs and the research preformed to incorporate a YidC protein in a nanodisc.

Research Report 1 : Role of YidC C-terminus and loop domains in interaction with the ribosome

De begeleider en/of auteur heeft geen toestemming gegeven tot het openbaar maken van de scriptie. The supervisor and/or the author did not authorize public publication of the thesis.

Differentiating Ligand and Inhibitor Interactions of a Single Antiporter

Regulatory mechanisms of ion and solute transporters are in focus of biomedical and biochemical studies and build a key for disease therapies. Inhibition of sodium/proton exchangers efficiently prevents ischemic heart disease and reperfusion development in humans, but molecular mechanisms behind are not clear. Using single-molecule force spectroscopy we observe the binding of the inhibitor 2-aminoperimidine (AP) to sodium/proton antiporters NhaA from Escherichia coli. Deactivating interactions were significantly suppressed at enhanced sodium concentrations of 200 mM as well as in the pH-locked inactive conformation of NhaA. New molecular interactions were quantified and localized within the protein occurring upon a competitive inhibitor binding. The inhibitor, which was targeted and bound to the ligand-binding pocket, altered interactions established at alpha-helix IX. These molecular mechanisms deactivating the antiporter were different to those established upon ligand binding and activation of NhaA