Multiphoton and Fluorescence Lifetime Imaging Microscopy in Studying Nanoparticle Pharmacokinetics in Skin and Liver

The use of nanoparticles has increased in consumer products in recent decades; however, concerns regarding their safety remain. Zinc oxide is used in sunblocking and may generate free radicals in response to UV illumination, leading to DNA damage and an immunological response. With high‐resolution, high‐contrast imaging in biological tissue, multiphoton microscopy is able to separate nanoparticles signals from endogenous fluorophores. It has been proven to be very useful in imaging penetration of zinc oxide nanoparticles in skin and in combination with fluorescence lifetime imaging microscopy study cellular function as well. This chapter aims to review the use of these imaging techniques in studying the uptake and distribution of nanoparticles in skin and liver. Due to the questionable clinical use and possible toxicity of nanoparticles, it is important to study their pharmacokinetics. Some nanomaterials have been identified as relatively toxic to humans and a few metal nanoparticles have been reported to penetrate and be detected in blood. Multiphoton microscopy has high resolution and is able to visualize nanoparticles, due to their optical properties, in vivo. The addition of fluorescence lifetime imaging makes it possible to measure the physiochemical environment, with outputs that can be statistically analyzed, posing an advantage over fluorescence intensity imaging only

Concentration and composition of natural urine of some michigan small mammals

Analysis of bladder urine of small mammals collected during the summer in Michigan shows that: 1. 1. Snap-trapping rodents and insectivores can be an effective technique for collecting natural urine.2. 2. Individuals not immediately killed by the trap produce abnormally concentrated urine.3. 3. Natural urine is very dilute compared to maximum concentrating abilities of the species tested.4. 4. Brief periods of captivity (Eptesicus fuscus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23382/1/0000327.pd

A Revised Design for Microarray Experiments to Account for Experimental Noise and Uncertainty of Probe Response

Background Although microarrays are analysis tools in biomedical research, they are known to yield noisy output that usually requires experimental confirmation. To tackle this problem, many studies have developed rules for optimizing probe design and devised complex statistical tools to analyze the output. However, less emphasis has been placed on systematically identifying the noise component as part of the experimental procedure. One source of noise is the variance in probe binding, which can be assessed by replicating array probes. The second source is poor probe performance, which can be assessed by calibrating the array based on a dilution series of target molecules. Using model experiments for copy number variation and gene expression measurements, we investigate here a revised design for microarray experiments that addresses both of these sources of variance. Results Two custom arrays were used to evaluate the revised design: one based on 25 mer probes from an Affymetrix design and the other based on 60 mer probes from an Agilent design. To assess experimental variance in probe binding, all probes were replicated ten times. To assess probe performance, the probes were calibrated using a dilution series of target molecules and the signal response was fitted to an adsorption model. We found that significant variance of the signal could be controlled by averaging across probes and removing probes that are nonresponsive or poorly responsive in the calibration experiment. Taking this into account, one can obtain a more reliable signal with the added option of obtaining absolute rather than relative measurements. Conclusion The assessment of technical variance within the experiments, combined with the calibration of probes allows to remove poorly responding probes and yields more reliable signals for the remaining ones. Once an array is properly calibrated, absolute quantification of signals becomes straight forward, alleviating the need for normalization and reference hybridizations

Aspects of nitrogen and mineral nutrition in Icelandic reindeer, Rangifer tarandus

Nitrogen and mineral (Fe, Mg, Na, K, and Ca) compositions of foodstuffs consumed by and dung produced by male, pregnant and lactating female adult Icelandic reindeer and calves were determined during May of 1992. Iron levels in foodstuffs are consistently above the reported toxicity level for similar-sized sheep (0.5 ppt) and may lead to iron toxicity when consumed by reindeer during periods of high lean body mass catabolism. Male and female reindeer meet nutrient requirements for all measured elements and nitrogen with the possible exception of calcium for males during antler growth.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31343/1/0000253.pd

The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase

RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria

DNA sequencing by multiple capillaries that form a waveguide

A 12-capillary prototype electrophoresis system for DNA sequencing has been constructed. Laser illumination is introduced into an optical waveguide that is formed by an array of individual capillaries that serve both as the optical elements of the periodic array and as the channels containing sieving media for electrophoresis. A theoretical framework and experimental data will be presented to illustrate the viability of this approach

Effect of temperature on intrinsic intestinal motility in a hibernator

In vitro lowering and raising of the temperature of the initial segment of the small intestine of a homeotherm, the laboratory mouse; Mus musculus, and a hibernator, the big brown bat; Eptesicus fuscus indicate: 1. 1.|A direct relationship exists between frequency of contractions and gut temperature which is essentiallty linear in mice, with contractions ceasing at about 16[deg]C, and is curvilinear in gut segments from hibernationg Eptesicus, with contractions ceasing at about 7[deg]C2. 2.|Amplitude of contractions in both species are generally independent of temperature flux; however, in bat gut contraction amplitude drops to very low levels at low temperatures3. 3.|Relationships to temperature are similar during both cooling and warming of the intestine.4. 4.|Based on motility, the intestine of a hibernator continues to function at a much lower temperature than the gut of a homeotherm.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21797/1/0000196.pd

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other α/β-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

Dynamic assembly of primers on nucleic acid templates

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of ∼1 μM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy