Surface sticking and lateral diffusion of lipids in supported bilayers

The diffusion of fluorescently labeled lipids in supported bilayers is studied using two different methods: Z-scan fluorescence correlation spectroscopy (z-scan FCS) and two-focus fluorescence correlation spectroscopy (2f-FCS). It is found that the data can be fitted consistently only when taking into account partial sticking of the labeled lipids to the supporting glass surface. A kinetic reaction-diffusion model is developed and applied to the data. We find a very slow sticking rate which, however, when neglected, leads to strongly varying estimates of the free diffusion coefficient. The study reveals a strong sensitivity of FCS on even slight binding/unbinding kinetics of the labeled molecules, which has significance for related diffusion measurements in cellular lipid membranes

Super-resolution provided by the arbitrarily strong superlinearity of the blackbody radiation

Blackbody radiation is a fundamental phenomenon in nature, and its explanation by Planck marks a cornerstone in the history of Physics. In this theoretical work, we show that the spectral radiance given by Planck's law is strongly superlinear with temperature, with an arbitrarily large local exponent for decreasing wavelengths. From that scaling analysis, we propose a new concept of super-resolved detection and imaging: if a focused beam of energy is scanned over an object that absorbs and linearly converts that energy into heat, a highly nonlinear thermal radiation response is generated, and its point spread function can be made arbitrarily smaller than the excitation beam focus. Based on a few practical scenarios, we propose to extend the notion of super-resolution beyond its current niche in microscopy to various kinds of excitation beams, a wide range of spatial scales, and a broader diversity of target objects

Specific Heat Study of the Magnetic Superconductor HoNi2B2C

The complex magnetic transitions and superconductivity of HoNi2B2C were studied via the dependence of the heat capacity on temperature and in-plane field angle. We provide an extended, comprehensive magnetic phase diagram for B // [100] and B // [110] based on the thermodynamic measurements. Three magnetic transitions and the superconducting transition were clearly observed. The 5.2 K transition (T_{N}) shows a hysteresis with temperature, indicating the first order nature of the transition at B=0 T. The 6 K transition (T_{M}), namely the onset of the long-range ordering, displays a dramatic in-plane anisotropy: T_{M} increases with increasing magnetic field for B // [100] while it decreases with increasing field for B // [110]. The anomalous anisotropy in T_{M} indicates that the transition is related to the a-axis spiral structure. The 5.5 K transition (T^{*}) shows similar behavior to the 5.2 K transition, i.e., a small in-plane anisotropy and scaling with Ising model. This last transition is ascribed to the change from a^{*} dominant phase to c^{*} dominant phase.Comment: 9 pages, 11 figure

Building on the past, shaping the future: The environmental mutagenesis and genomics society

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97167/1/em21765.pd

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background: We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner

Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup.

The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig-a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described

Intercellular communication in spheroids

This chapter has shown that the response of spheroid cells to gap junctional communication may lead to certain metabolic and cell physiological changes. It has also become apparent that the functions of the gap junctions are very complex. They may, for example, be related to the fundamental effects of cAMP and/or Ca 2+. These lines of evidence should be pursued further. However, further insight into these functions may also be gained from a study of the structure and function of the gap-junctional proteins, as well as from a genetic approach (e.g., Willecke et al. 1982, 1983). In this context, the spheroids are of particular importance as test systems, since they perfectly simulate the three dimensional arrangement of cells encountered in a tissue. Indeed, the results presented in the sections "Biophysical and Biochemical Effects Associated with Intercellular Communications" and "Intercellular Communication and Radiosensitivity" have revealed clear cut differences between cells growing as spheroids or as monolayers in response to communication dependent processes, which indicate that the response of the monolayers could be somewhat trivial. The advantage of multicellular spheroid systems with three-dimensional growth over monolayer cultures is unquestionable. Cells growing in three-dimensional multicell spheroids may re-establish their regulatory activities and, therefore, match the in vivo conditions more closely. Multicell spheroids allow in vitro investigations on differentiating systems and on interactions between normal and malignant cells, thus substituting costly in vivo experiments

Assessment of Systemic Genetic Damage in Pediatric Inflammatory Bowel Disease.

The etiology of distal site cancers in inflammatory bowel disease (IBD) is not well understood and requires further study. We investigated whether pediatric IBD patients' blood cells exhibit elevated levels of genomic damage by measuring the frequency of mutant phenotype (CD59-/CD55-) reticulocytes (MUT RET) as a reporter of PIG-A mutation, and the frequency of micronucleated reticulocytes (MN-RET) as an indicator of chromosomal damage. IBD patients (n = 18 new onset disease, 46 established disease) were compared to age-matched controls (constipation or irritable bowel syndrome patients from the same clinic, n = 30) and young healthy adults age 19 - 24 (n = 25). IBD patients showed no indication of elevated MUT RET relative to controls (mean ± std. dev. = 3.1 ± 2.3 x 10-6 versus 3.6 ± 5.6 x 10-6 , respectively). In contrast, of 59 IBD patients where %MN-RET measurements were obtained, 10 exceeded the upper bound 90% tolerance interval derived from control subjects (i.e., 0.42%). Furthermore, each of the 10 IBD patients with elevated MN-RET had established disease (10/42), none were new onset (0/17) (p = 0.049). Interestingly, each of the subjects with increased chromosomal damage was receiving anti-TNF based monotherapy at the time blood was collected (10/10, 100%), whereas this therapy was less common (20/32, 63%) among patients that exhibited ≤ 0.42% MN-RET (p = 0.040). The results clearly indicate the need for further work to understand whether the results presented herein are reproducible, and if so, to elucidate the causative factor(s) responsible for elevated MN-RET frequencies in some IBD patients

Live Imaging of Xwnt5A-ROR2 Complexes

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging