Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies

Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94 % of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was ～2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types.

Electrophoretic behavior of individual nuclear species as determined by capillary electrophoresis with laser-induced fluorescence detection

We determined the feasibility of using capillary electrophoresis with postcolumn laser-induced fluorescence (CE-LIF) detection to characterize electrophoretic properties of isolated cell nuclei and impurities present in nuclear fractions. These fractions were isolated from NS-1 mouse hybridoma cells, stained with hexidium iodide, a DNA intercalating dye, and analyzed by CE-LIF detection. The corresponding electropherograms display two features: (i) broad peaks (6-90 s wide) caused by the cell culturing media and by free-DNA intercalated with hexidium iodide, and (ii) a large number of narrow peaks (180 ms wide), resulting from DNA associated with individual intact or disrupted nuclei. We confirmed that the narrow peaks were not caused by contaminating mitochondria. The overall electrophoretic mobility range of disrupted nuclei is 0 to -5 x 10(-4)cm(2)/Vs, while intact nuclei seem to have mobilities in the -1.5 to -3.5 x 10(-4)cm(2)/Vs range. Furthermore, the highly sensitive CE-LIF method reveals a high abundance of disrupted nuclei that cannot be directly observed by confocal microscopy.     

Activation energy of single-stranded DNA moving through cross-linked polyacrylamide gels at 300 V/cm effect of temperature on sequencing rate in high-electric-field capillary gel electrophoresis

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration.     

Pt dendrimer nanocomposites for oxygen reduction reaction in direct methanol fuel cells

Dendrimer-encapsulated Pt nanoparticles (G4OHPt) were prepared by chemical reduction at room temperature. The G4OHPt, with average diameters of ca. 2.7 nm, were characterized by X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis. Electrocatalytic behavior for oxygen reduction reaction was investigated using a rotating disk electrode configuration in an acidic medium, with and without the presence of methanol (0.01, 0.1, and 1 M). Kinetic studies showed that electrodes based on Pt nanoparticles encapsulated inside the dendrimer display a higher selectivity for ORR in the presence of methanol than electrodes based on commercial Pt black catalysts. Also, the dendritic polymer confers a protective effect on the Pt in the presence of methanol, which allows its use as a cathode in a direct methanol fuel cell operating at different temperatures. Good performance was obtained at 90 °C and 2 bar of pressure with a low platinum loading on the electrode surface.     

Detection of heteroplasmy in individual mitochondrial particles

Mitochondrial DNA (mtDNA) mutations have been associated with disease and aging. Since each cell has thousands of mtDNA copies, clustered into nucleoids of five to ten mtDNA molecules each, determining the effects of a given mtDNA mutation and their connection with disease phenotype is not straightforward. It has been postulated that heteroplasmy (coexistence of mutated and wild-type DNA) follows simple probability rules dictated by the random distribution of mtDNA molecules at the nucleoid level. This model has been used to explain how mutation levels correlate with the onset of disease phenotype and loss of cellular function. Nonetheless, experimental evidence of heteroplasmy at the nucleoid level is scarce. Here, we report a new method to determine heteroplasmy of individual mitochondrial particles containing one or more nucleoids. The method uses capillary cytometry with laser-induced fluorescence detection to detect individual mitochondrial particles stained with PicoGreen, which makes it possible to quantify the mtDNA copy number of each particle. After detection, one or more particles are collected into polymerase chain reaction (PCR) wells and then subjected to real-time multiplexed PCR amplification. This PCR strategy is suitable to obtain the relative abundance of mutated and wild-type mtDNA. The results obtained here indicate that individual mitochondrial particles and nucleoids contained within these particles are not heteroplasmic. The results presented here suggest that current models of mtDNA segregation and distribution (i.e., heteroplasmic nucleoids) need further consideration.     

On-column labeling for capillary electrophoretic analysis of individual mitochondria directly sampled from tissue cross sections

This technical note reports on a new procedure to on-column-label organelles sampled from a tissue cross section into a fused silica capillary. These organelles are then analyzed by capillary electrophoresis with postcolumn laser-induced fluorescence detection. In this procedure, the fluorescent label does not come in contact with the tissue, which facilitates visualization of the sampled tissue cross section. In addition, on-column labeling allows for better control of the reaction time and fluorescent label concentrations. As a proof-of-principle, we show results of mitochondria from rat gastrocnemius muscle cross sections that were on-column-labeled with 10-N-nonyl acridine orange (NAO), a mitochondrion-specific probe, and compare them with results for NAO in-tissue labeling of the same tissue. The new organelle labeling procedure reported here may easily be extended to the analysis of individual organelles in other biological samples and may become a valuable tool in studies investigating the role of mitochondria in muscle aging and exercise physiology.      

Psychoacoustics of in-car switch buttons: From feelings to engineering parameters

The car interior is becoming quieter and other sounds are now exposed to user perception, such as the sound produced by interface buttons when actuated. So, the functional role of the button sound on interface operation and its aesthetic and emotional role on the user experience are now more important than before. However, little research and design effort has been paid to understand how to design buttons that produce a pleasant sound. Moreover, the button’s sound requirements received by interface manufacturers are ill-defined, insufficient or even inexistent, and consequently their conversion into specifications for manufacturing is problematic and leads to long and costly development processes. The purpose of this paper is to contribute to identify relevant acoustic parameters that explain the users sound preferences. Data on preference subjective judgments were collected and buttons acoustic signals were measured allowing the development of preference models based on partial least squares regression and neural networks methods. The former was successful in selecting the relevant parameters to describe the preference ratings of the buttons sound. The later, dealing with the non-linear nature of acoustic perception, was able to predict preferences based on the relevant parameters.     

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

The number of particles in a sample heavily influences the shape of a distribution corresponding to the individual particle measurements. Selecting an adequate number of particles that prevents biases due to sample size is particularly difficult for complex biological systems in which statistical distributions are not normal. Quantile analysis is a powerful statistical technique that can rapidly compare differences between multiple distributions of individual particles. This report utilizes quantile analysis to show that the number of events detected affects the mobility distributions for rat liver and mouse liver mitochondria, sample individual particles, when analyzed via capillary electrophoresis with laser-induced fluorescence. When the mitochondrial sample is small (e.g. <78), there are not enough events to obtain statistically relevant mobility data. Adsorption to the capillary surface also significantly affects the mobility distribution at a small number of events in uncoated and dynamically coated capillaries. These adsorption effects can be overcome when the mitochondrial load on the capillary is sufficiently large (i.e. >609 and >1426 events for mouse liver on uncoated capillaries and rat liver on dynamically coated capillaries, respectively). It is anticipated that quantile analysis can be used to study other distributions of individual particles, such as nanoparticles, organelles, and biomolecules, and that distributions of these particles will also be dependent on sample size.