Further analysis of the effects of baffles on combustion instability

A computerized analytical model, developed to predict the effects of baffles on combustion instability, was modified in an effort to improve the ability to properly predict stability effects. The model was modified: (1) to replace a single spatially-averaged response factor by separate values for each baffle compartment; (2) to calculate the axial component of the acoustic energy flux, and (3) to permit analysis of traveling waves in a thin annular chamber. Allowance for separate average response factors in each baffle compartment was found to significantly affect the predicted results. With this modification, an optimum baffle length was predicted which gave maximum stability

Analysis of combustion instability in liquid propellant engines with or without acoustic cavities

Analytical studies have been made of the relative combustion stability of various propellant combinations when used with hardware configurations representative of current design practices and with or without acoustic cavities. Two combustion instability models, a Priem-type model and a modification of the Northern Research and Engineering (NREC) instability model, were used to predict the variation in engine stability with changes in operating conditions, hardware characteristics or propellant combination, exclusive of acoustic cavity effects. The NREC model was developed for turbojet engines but is applicable to liquid propellant engines. A steady-state combustion model was used to predict the needed input for the instability models. In addition, preliminary development was completed on a new model to predict the influence of an acoustic cavity with specific allowance for the effects the nozzle, steady flow and combustion

Analysis of the effects of baffles on combustion instability

An analytical model has been developed for predicting the effects of baffles on combustion instability. This model has been developed by coupling an acoustic analysis of the wave motion within baffled chambers with a model for the oscillatory combustion response of a propellant droplet developed by Heidmann. A computer program was developed for numerical solution of the resultant coupled equations. Diagnostic calculations were made to determine the reasons for the improper prediction. These calculations showed that the chosen method of representing the combustion response was a very poor approximation. At the end of the program, attempts were made to minimize this effect but the model still improperly predicts the stability trends. Therefore, it is recommended that additional analysis be done with an improved approximation

Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-PAINT

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments

Teaching old NCATs new tricks: using non-canonical amino acid tagging to study neuronal plasticity

The non-canonical amino acid labeling techniques BONCAT (bioorthogonal non-canonical amino acid tagging) and FUNCAT (fluorescent non-canonical amino acid tagging) enable the specific identification and visualization of newly synthesized proteins. Recently, these techniques have been applied to neuronal systems to elucidate protein synthesis dynamics during plasticity, identify stimulation-induced proteomes and subproteomes and to investigate local protein synthesis in specific subcellular compartments. The next generation of tools and applications, reviewed here, includes the development of new tags, the quantitative identification of newly synthesized proteins, the application of NCAT to whole animals, and the ability to genetically restrict NCAT labeling. These techniques will enable not only improved detection but also allow new scientific questions to be tackled

A genetically encodable cell-type-specific protein synthesis inhibitor

Chemical inhibitors have revealed requirements for protein synthesis that drive cellular plasticity. We developed a genetically encodable protein synthesis inhibitor (gePSI) to achieve cell-type-specific temporal control of protein synthesis. Controlled expression of the gePSI in neurons or glia resulted in rapid, potent and reversible cell-autonomous inhibition of protein synthesis. Moreover, gePSI expression in a single neuron blocked the structural plasticity induced by single-synapse stimulation

Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition

The azide−alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide−alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na_2S_2O_4, 2% HOCH_2CH_2SH, 10% HCO_2H, 95% CF_3CO_2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO_2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies

Fluorescence Visualization of Newly Synthesized Proteins in Mammalian Cells

Modern proteomic methods enable efficient identification of the hundreds or thousands of proteins present in whole cells or in isolated organelles. However, a thorough understanding of the proteome requires insight into protein localization as well as protein identity. Recently, visualization of newly synthesized proteins in bacterial cells was demonstrated through co-translational introduction of an alkynyl amino acid followed by selective CuI-catalyzed ligation of the alkynyl side chain to the fluorogenic dye 3-azido-7-hydroxycoumarin. Here we report that selective fluorescence labeling and imaging of newly synthesized proteins can be accomplished in a diverse set of mammalian cells

Axonal Translation of -Catenin Regulates Synaptic Vesicle Dynamics

Many presynaptic transcripts have been observed in axons, yet their role in synapse development remains unknown. Using visually and pharmacologically isolated presynaptic terminals from dissociated rat hippocampal neurons, we found that ribosomes and β-catenin mRNA preferentially localize to recently formed boutons. Locally translated β-catenin accumulates at presynaptic terminals where it regulates synaptic vesicle release dynamics. Thus, local translation of β-catenin is a newly described mechanism for axons to independently functionalize nerve terminals at great distances from cellular somata

Neuronal ribosomes exhibit dynamic and context-dependent exchange of ribosomal proteins

Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation was independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis