Fluorescent Protein Labeling Research Articles

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia(1,2) . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)(3). This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell. In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

BackgroundMalaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP.MethodsDetection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry.ResultsA new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites.DiscussionFilter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events.

Metabolic labeling with noncanonical amino acids and visualization by chemoselective fluorescent tagging.

Fluorescent labeling of proteins by genetically encoded fluorescent protein tags has enabled an enhanced understanding of cell biological processes but is restricted to the analysis of a limited number of identified proteins. This approach does not permit, e.g., the unbiased visualization of a full proteome in situ. We describe here a fluorescence-based method to follow proteome-wide patterns of newly synthesized proteins in cultured cells, tissue slices, and a whole organism. This technique is compatible with immunohistochemistry and in situ hybridization. Key to this method is the introduction of a small bio-orthogonal reactive group by metabolic labeling. This is accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) in a step very similar to classical radioisotope labeling. Subsequently, an alkyne-bearing fluorophore is covalently attached to the group by "click chemistry"--a copper(I)-catalyzed [3+2]azide-alkyne cycloaddition. By similar means, metabolic labeling can also be performed with the alkyne-bearing homopropargylglycine (HPG) and clicked to an azide-functionalized fluorophore.

Comparison of Spheroids Formed by Rat Glioma Stem Cells and Neural Stem Cells Reveals Differences in Glucose Metabolism and Promising Therapeutic Applications

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

The Effect of the Microenvironment Created by a Titanium Mesh Cage on Subcutaneous Experimental Bone Formation and Inhibition of Absorption

We attempted to form ectopic bone under the skin of rats without adding any extrinsic bone-inducing growth factors or cytokines using bone marrow stromal cells (BMSCs), a collagen scaffold and a titanium mesh cage. We set up a space made up of a cage inserted into the subcutaneous region of rats’ backs, where we could eliminate the possible influence of residual bone tissue on bone induction. We filled this space with a collagen matrix containing BMSCs. At week 8 and month 6 after implantation, the specimens were removed and observed histologically, histochemically and enzyme histochemically. As a result, bone tissue was identified in each case within the titanium cages, even though we had not used bone-inducing chemical substances. Bone generation was not found in test cases without a cage. Enhanced green fluorescence protein (EGFP) labeling of the implanted BMSCs clearly showed that these cells differentiated into osteoblasts and subsequently into osteocytes in the formed bone tissue. Host cells without EGFP labeling were also confirmed to be involved in bone formation. Six months after transplantation, the implanted cells were still present in the generated bone, and no significant resorption of the generated bone was observed. These results indicate that the physically stable spatial microenvironment created by the cage in vivo plays an important role in bone formation and inhibition of its resorption, which we refer to as the ‘cage effect’.

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes. In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts. In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Selective N-terminal fluorescent labeling of proteins using 4-chloro-7-nitrobenzofurazan: A method to distinguish protein N-terminal acetylation

A fluorogenic derivatization method was developed to distinguish the protein N-terminal acetylation status. The unacetylated protein selectively reacted with 4-chloro-7-nitrobenzofurazan (NBD–Cl) at neutral pH to provide high fluorescence. In contrast, the protein with N-terminal acetylation was essentially nonfluorescent under the same conditions despite the presence of many internal lysine residues. Fluorescence of the NBD-labeled protein was very stable, and only micromolar concentrations of proteins were required for reliable detection. This method also provides a general and practical way to quantify proteins when their N-terminal amino group is available.

Context Surrounding Processing Sites Is Crucial in Determining Cleavage Rate of a Subset of Processing Sites in HIV-1 Gag and Gag-Pro-Pol Polyprotein Precursors by Viral Protease

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.

Internal Jugular Vein Compression Mitigates Traumatic Axonal Injury in a Rat Model by Reducing the Intracranial Slosh Effect

Traumatic brain injury (TBI) remains a devastating condition for which extracranial protection traditionally has been in the form of helmets, which largely fail to protect against intracranial injury. To determine whether the pathological outcome after traumatic brain injury can be improved via slosh mitigation by internal jugular vein (IJV) compression. Two groups of 10 adult male Sprague-Dawley rats were subjected to impact-acceleration traumatic brain injury. One group underwent IJV compression via application of a collar before injury; the second group did not. Intracranial pressure and intraocular pressure were measured before and after IJV compression to assess collar performance. All rats were killed after a 7-day recovery period, and brainstem white matter tracts underwent fluorescent immunohistochemical processing and labeling of β-amyloid precursor protein, a marker of axonal injury. Digital imaging and statistical analyses were used to determine whether IJV compression resulted in a diminished number of injured axons. Compression of the IJV resulted in an immediate 30% increase in intraocular and intracranial pressures. Most notably, IJV compression resulted in > 80% reduction in the number of amyloid precursor protein-positive axons as indicated by immunohistochemical analysis. Using a standard acceleration-deceleration laboratory model of mild traumatic brain injury, we have shown successful prevention of axonal injury after IJV compression as indicated by immunohistochemical staining of amyloid precursor protein. We argue that IJV compression reduces slosh-mediated brain injury by increasing intracranial blood volume, which can be indirectly measured by intracranial and intraocular pressures.

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42±0.92mm(3) after 3days, 1.24±0.76mm(3) after 7days, 0.33±0.03mm(3) after 14days, and 0.09±0.05mm(3) after 28days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.

Top-Down FTICR MS for the Identification of Fluorescent Labeling Efficiency and Specificity of the Cu-Protein Azurin

Fluorescent protein labeling has been an indispensable tool in many applications of biochemical, biophysical, and cell biological research. Although detailed information about the labeling stoichiometry and exact location of the label is often not necessary, for other purposes, this information is crucial. We have studied the potential of top-down electrospray ionization (ESI)-15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to study the degree and positioning of fluorescent labeling. For this purpose, we have labeled the Cu-protein azurin with the fluorescent label ATTO 655-N-hydroxysuccinimide(NHS)-ester and fractionated the sample using anion exchange chromatography. Subsequently, individual fractions were analyzed by ESI-15T FTICR to determine the labeling stoichiometry, followed by top-down MS fragmentation, to locate the position of the label. Results showed that, upon labeling with ATTO 655-NHS, multiple different species of either singly or doubly labeled azurin were formed. Top-down fragmentation of different species, either with or without the copper, resulted in a sequence coverage of approximately 50%. Different primary amine groups were found to be (potential) labeling sites, and Lys-122 was identified as the major labeling attachment site. In conclusion, we have demonstrated that anion exchange chromatography in combination with ultrahigh resolution 15T ESI-FTICR top-down mass spectrometry is a valuable tool for measuring fluorescent labeling efficiency and specificity.

Heterogeneity in Pseudomonas aeruginosa Biofilms Includes Expression of Ribosome Hibernation Factors in the Antibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response in the Metabolically Active Population

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.

Determinants of the Drosophila Odorant Receptor Pattern

In most olfactory systems studied to date, neurons that express the same odorant receptor (Or) gene are scattered across sensory epithelia, intermingled with neurons that express different Or genes. In Drosophila, olfactory sensilla that express the same Or gene are dispersed on the antenna and the maxillary palp. Here we show that Or identity is specified in a spatially stereotyped pattern by the cell-autonomous activity of the transcriptional regulators Engrailed and Dachshund. Olfactory sensilla then become highly motile and disperse beneath the epidermis. Thus, positional information and cell motility underlie the dispersed patterns of Drosophila Or gene expression.

Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells.

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.

A cysteine probe with high selectivity and sensitivity promoted by response-assisted electrostatic attraction

A new turn-on fluorescent probe for fast detection of cysteine in physiological conditions, based on a new response-assisted electrostatic attraction strategy, is reported. The practical utility of the probe in fluorescent protein labeling and subcellular imaging is also demonstrated.

Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging

Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging

The Development of Methods for the Site-Specific Fluorescent Labeling of Spliceosomal Proteins for use in Single-Molecule Studies

Spliceosomes are multi-megadalton ribonucleoprotein (RNP) complexes responsible for catalyzing the removal of noncoding introns from eukaryotic precursor messenger RNA (pre-mRNA) transcripts and ligating the flanking coding exon sequences to produce a mature messenger RNA (mRNA). Spliceosomal assembly and catalysis require a highly dynamic coordination of proteins and RNAs with the purpose of producing mature mRNA that can be properly transcribed by the ribosome. Due to the complexity of the process and a lack of suitable tools, there is still much unknown about the assembly of the spliceosome. Single molecule fluorescence microscopy tools have recently been developed for studies of spliceosome assembly and dynamics of the pre-mRNA substrate throughout splicing. In an effort to advance the study of splicing and delineate the exact timing and dynamics required to achieve splicing, we are currently investigating methods for the site-specific labeling of proteins, one of these methods being protein trans-splicing (PTS) using a split intein. Protein splicing is a naturally occurring process in which a protein editor, called an intein, excises itself out of a host protein in which it is embedded creating a new peptide bond between its two flanking regions, the exteins. PTS uses an artificially or naturally split intein to create a new peptide bond between flanking exteins similar to protein splicing. Upon incubation of the two components, protein splicing will take place, resulting in the ligation of the two extein regions. By fluorescently labeling one of the extein components, we hope to use PTS to site-specifically label several spliceosomal proteins with the ultimate goal of correlating the association and dissociation of spliceosomal components with pre-mRNA dynamics in order to gain a better understanding of the mechanism of pre-mRNA splicing.

Anabolic Steroids and Head Injury

The suggestion has been made that neurological changes seen in the syndrome of chronic traumatic encephalopathy may be due to exogenous anabolic steroid use rather than traumatic brain injury. To determine whether administration of anabolic steroids alters the pathophysiology of traumatic brain injury. Sixty adult male Sprague-Dawley rats and a linear acceleration model of traumatic brain injury were used. Experimental groups were (1) preinjury anabolic steroids, (2) preinjury placebo carrier, (3) anabolic steroids without injury, (4) no steroids and no injury, (5) postinjury placebo carrier, and (6) postinjury anabolic steroids. Following a 30-day recovery, rats were euthanized, and brainstem white matter tracts underwent fluorescent immunohistochemical processing and labeling of β-amyloid precursor protein (APP), a marker of axonal injury. Digital imaging and statistical analyses were used to determine whether anabolic steroid administration resulted in a significant change in the number of injured axons. There was no statistically significant difference in number of APP-positive axons by immunohistochemical analysis between respective anabolic steroid and placebo groups. Using a standard acceleration-deceleration model of mild traumatic brain injury, we have shown successful visualization of traumatically injured axons with antibody staining of APP. Our results indicate no statistically significant effect of anabolic steroids on the number of APP-positive axons. With the use of this model, and within its limitations, we see no adverse effect or causative role of anabolic steroid administration on the brain following mild traumatic brain injury using APP counts as a marker for anatomic injury.

Fluorogenic Protein Labeling through Photoinduced Electron Transfer-Based BL-Tag Technology

Keeping quenchers on a short leash: Shortening of the linker chain length combined with modification of the quencher moiety was found to improve fluorogenic probes useful for protein labeling by the mutant β-lactamase-tag (BL-tag) technology. The most effective probe, CC3 DNB, displays fluorescence quenching by photoinduced electron transfer (PET) and results in fluorescent protein labeling with fast kinetics.