Adult onset pan-neuronal human tau tubulin kinase 1 expression causes severe cerebellar neurodegeneration in mice.

The kinase TTBK1 is predominantly expressed in the central nervous system and has been implicated in neurodegenerative diseases including Alzheimer's disease, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis through its ability to phosphorylate the proteins tau and TDP-43. Mutations in the closely related gene TTBK2 cause spinocerebellar ataxia, type 11. However, it remains unknown whether altered TTBK1 activity alone can drive neurodegeneration.

Altered splicing of ATP6AP2 causes X-linked parkinsonism with spasticity (XPDS).

We report a novel gene for a parkinsonian disorder. X-linked parkinsonism with spasticity (XPDS) presents either as typical adult onset Parkinson's disease or earlier onset spasticity followed by parkinsonism. We previously mapped the XPDS gene to a 28 Mb region on Xp11.

2,1,3-Benzothiadiazole (BTD)-moiety-containing red emitter conjugated amphiphilic poly(ethylene glycol)-block-poly(epsilon-caprolactone) copolymers for bioimaging.

2,1,3-Benzothiadiazole (BTD)-containing red emitter was chemically conjugated onto amphiphilic poly(ethylene glycol)-block-poly(epsilon-caprolactone) (PEG-b-PCL) copolymers to form two new fluorophore-conjugated block copolymers (P5 and P7). P5 is a cationic amino group-containing polymer, whereas, P7 is a neutral polymer. The polymers formed micelles in aqueous solution with average diameters of 45 nm (P7) and 78 nm (P5), which were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM).

A microwell array device capable of measuring single-cell oxygen consumption rates.

Due to interest in cell population heterogeneity, the development of new technology and methodologies for studying single cells has dramatically increased in recent years. The ideal single cell measurement system would be high throughput for statistical relevance, would measure the most important cellular parameters, and minimize disruption of normal cell function. We have developed a microwell array device capable of measuring single cell oxygen consumption rates (OCR).

Utilization of micelles formed from poly(ethylene glycol)-block-poly(epsilon-caprolactone) block copolymers as nanocarriers to enable hydrophobic red two-photon absorbing emitters for cells imaging.

A hydrophobic two-photon absorbing (2PA) red emitter (R) was successfully incorporated into micelles formed from two block copolymers, poly(epsilon-caprolactone)-block-poly(ethylene glycol)s, for imaging and toxicity studies. In micelles, the chromophore R exhibits a 2PA cross-section of 400 GM (1 GM = 1 x 10(-50) cm(4) s photon(-1) molecule(-1)) at 820 nm, which is among the highest values reported for red 2PA emitters. The micelles with a cationic amino moiety-containing poly(ethylene glycol) corona showed an enhancement of cell internalization and delivered the dye into the cytoplasmic regions of the mouse macrophage RAW 264.

Population heterogeneity in Methylobacterium extorquens AM1.

Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter (P(mxaF)) and gfp(uv) was used with flow cytometry to analyse the distribution of gene expression in populations grown on either succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing.

A cellular isolation system for real-time single-cell oxygen consumption monitoring.

The development of a cellular isolation system (CIS) that enables the monitoring of single-cell oxygen consumption rates in real time is presented. The CIS was developed through a multidisciplinary effort within the Microscale Life Sciences Center (MLSC) at the University of Washington. The system comprises arrays of microwells containing Pt-porphyrin-embedded polystyrene microspheres as the reporter chemistry, a lid actuator system and a gated intensified imaging camera, all mounted on a temperature-stabilized confocal microscope platform.

Cell-to-cell heterogeneity in growth rate and gene expression in Methylobacterium extorquens AM1.

Cell-to-cell heterogeneity in gene expression and growth parameters was assessed in the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methylotrophy promoter (P(mxaF)) and gfp(uv) (encoding a variant of green fluorescent protein [GFPuv]) was used to assess single-cell gene expression. Using a flowthrough culture system and laser scanning microscopy, data on fluorescence and cell size were obtained over time through several growth cycles for cells grown on succinate or methanol.

Measurement of respiration rates of Methylobacterium extorquens AM1 cultures by use of a phosphorescence-based sensor.

Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotroph Methylobacterium extorquens AM1 to demonstrate subtle mutant phenotypes.

Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry.

The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquens AM1 with methanol as the sole organic carbon and energy source and growth-limiting substrate. Label tracing experiments were carried out using 70% (13)C-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm.