Formation and removal of alkylthiolate self-assembled monolayers on gold in aqueous solutions

We report the development of novel reagents and approaches for generating recyclable biosensors. The use of aqueous media for the formation of protein binding alkylthiolate monolayers on Au surfaces results in accelerated alkylthiolate monolayer formation and improvement in monolayer integrity as visualized by fluorescence microscopy and CV techniques. We have also developed an electrocleaning protocol that is compatible with microfluidics devices, and this technique serves as an on-chip method for cleaning Au substrates both before and after monolayer formation. The techniques for the formation and dissociation of biotinylated SAMs from aqueous solvents reported here may be applied towards the development of Au-based sensor devices and microfluidics chips in the future. A potential use of these devices includes the specific capture and triggered release of target cells, proteins, or small molecules from liquid samples

Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Four-dimensional fluorescent imaging of embryonic quail development

Traditionally, our understanding of developmental biology has been based on the fixation and study of embryonic samples. Detailed microscopic scrutiny of static specimens at varying ages allowed for anatomical assessment of tissue development. The advent of confocal and two-photon excitation (PE) microscopy enables researchers to acquire volumetric images in three dimensions (x, y, and z) plus time (t). Here, we present techniques for acquisition and analysis of three-dimensional time-lapse data. Both confocal microscopy and 2PE microscopy techniques are used. In addition, data processing for tiled image stitching and time-lapse analysis is discussed. Additionally, the development of a new transgenic Japanese quail has provided an embryonic model system that is more easily accessible than mammalian models and more efficient to breed than the classic avian model, the chicken. Here we also outline the preparation in vitro of these transgenic quail embryos for imaging

Distinct Pools of Non-Glycolytic Substrates Differentiate Brain Regions and Prime Region-Specific Responses of Mitochondria

<div><p>Many hereditary diseases are characterized by region-specific toxicity, despite the fact that disease-linked proteins are generally ubiquitously expressed. The underlying basis of the region-specific vulnerability remains enigmatic. Here, we evaluate the fundamental features of mitochondrial and glucose metabolism in synaptosomes from four brain regions in basal and stressed states. Although the brain has an absolute need for glucose <i>in vivo,</i> we find that synaptosomes prefer to respire on non-glycolytic substrates, even when glucose is present. Moreover, glucose is metabolized differently in each brain region, resulting in region-specific “signature” pools of non-glycolytic substrates. The use of non-glycolytic resources increases and dominates during energy crisis, and triggers a marked region-specific metabolic response. We envision that disease-linked proteins confer stress on all relevant brain cells, but region-specific susceptibility stems from metabolism of non-glycolytic substrates, which limits how and to what extent neurons respond to the stress.</p></div

Distinct brain regions harbor discrete “signature” pools of non-glycolytic substrates for energy production.

<p>(<b>A</b>) Schematic representation of chemical classes grouping of metabolites from MetaMapp analysis. (<b>B</b>) Metabolic networks of biochemical reaction pairs (dark blue edges) and chemical similarity (light blue edges) show the regulation of all identified metabolites in four brain regions. Blue = down regulated metabolites, red = up regulated metabolites with a median false discovery rate <0.5% from SAM (n = 5 or 6). Ball sizes reflect magnitude of differential metabolite expression. Metabolites that were not significantly different were left unnamed in order to keep visual clarity.</p

Partial least squared (PLS) statistics and clustering analysis of region-specific brain indicate differences in metabolomes.

<p>(<b>A</b>) PC 1 (t1) and PC 2 (t2) shows the separation of the metabolome among four different brain regions. PC 1 (30.5% total explained variance) discriminated metabolite profiles of STR and HIP from CRT and CBL. PC 2 (6.0% total explained variance) primarily separated clusters between STR and HIP (n = 5 or 6). Red circle indicates 3×S.E. (<b>B</b>) Hierarchical clustering analysis showing chemically/biochemically classifies metabolites clustered according to different brain regions (n = 5 or 6). The color code; CBL: teal, HIP: blue, STR: red, CTX: light green.</p

Energy deficits induce region-specific OCR that depends on use of non-glycolytic substrates in synaptosomes.

<p>(<b>A</b>) Schematic representation of experimental design for bioenergetics analysis of mitochondrial respiration on exogenous glucose and endogenous non-glycolytic substrates on various conditions. (<b>B</b>) Representative profiles of brain regional OCR under basal conditions and upon injections of 2-DG and FCCP. Each profile represents one independent biological experiment analyzed in triplicate. Data are means ± SEM (n = 3). The arrows indicate the injection of the inhibitors. Three independent experiments were performed to obtain quantification of OCR presented in (<b>C</b>). (<b>C</b>) Synaptosomal OCR under basal condition and upon inhibitions with 2-DG and FCCP. Upon inhibition with 2-DG, glycolysis is inhibited and energy arises only from endogenous pyruvate and non-glycolytic substrates. OCR is highest in the hippocampus, moderate in the striatum and cortex, and the lowest in the cerebellum. Upon FCCP injection after 2-DG, the same OCR pattern is observed. The OCR increase was short-lived and decreased at approximately 16 minutes upon FCCP treatment. Three independent experiments were performed. Data are means ± SEM (n = 3). *<i>P</i><0.05, **<i>P</i><0.01 with one-way ANOVA and Fisher’s LSD.</p

Magnified visual table of the observed six chemical classes in each region of the brain.

<p>The metabolic network is sub-categorized according to the resultant network topology. The clustered sub-network provides unique expression pattern of each brain region. Columns indicate six sub-clusters of the metabolic network and rows represent the four brain regions analyzed. Blue = down regulated metabolites, red = up regulated metabolites at a median false discovery rate <0.5% from SAM (n = 5 or 6). Ball sizes reflect magnitude of differential metabolite expression. Metabolites that were not significantly different were left unnamed in order to keep visual clarity.</p