Dataset Information

Electroosmotic Perfusion, External Microdialysis: Simulation and Experiment.

ABSTRACT: Information about the rates of hydrolysis of neuropeptides by extracellular peptidases can lead to a quantitative understanding of how the steady-state and transient concentrations of neuropeptides are controlled. We have created a small microfluidic device that electroosmotically infuses peptides into, through, and out of the tissue to a microdialysis probe outside the head. The device is created by two-photon polymerization (Nanoscribe). Inferring quantitative estimates of a rate process from the change in concentration of a substrate that has passed through tissue is challenging for two reasons. One is that diffusion is significant, so there is a distribution of peptide substrate residence times in the tissue. This affects the product yield. The other is that there are multiple paths taken by the substrate as it passes through tissue, so there is a distribution of residence times and thus reaction times. Simulation of the process is essential. The simulations presented here imply that a range of first order rate constants of more than 3 orders of magnitude is measurable and that 5-10 min is required to reach a steady state value of product concentration following initiation of substrate infusion. Experiments using a peptidase-resistant d-amino acid pentapeptide, yaGfl, agree with simulations.

SUBMITTER: Rerick MT

PROVIDER: S-EPMC10360060 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Electroosmotic Perfusion, External Microdialysis: Simulation and Experiment.

Rerick Michael T MT Chen Jun J Weber Stephen G SG

ACS chemical neuroscience 20230628 14

Information about the rates of hydrolysis of neuropeptides by extracellular peptidases can lead to a quantitative understanding of how the steady-state and transient concentrations of neuropeptides are controlled. We have created a small microfluidic device that electroosmotically infuses peptides into, through, and out of the tissue to a microdialysis probe outside the head. The device is created by two-photon polymerization (Nanoscribe). Inferring quantitative estimates of a rate process from ...[more]

PMID: 37379416

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Project description:The most widely-used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms and analysis algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. All commercial tiling array platforms performed well, although each platform and analysis algorithm had distinct performance and cost characteristics. Simple sequence repeats and genome redundancy tend to result in false positives on oligonucleotide platforms. The spike-in DNA samples and the resulting array data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: chip-ChIP simulation For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf For each replicate array, we labeled 1Âµg of spike-in sample and 1Âµg of control sample. The Bioprime Plus Array CGH labeling Module (Invitrogen Catolog number 18095-014) was used. The spike-in was labeled with the red channel dye (Alexa Fluor-647) and the control was labeled with the green channel dye (Alexa Fluor-555) in two of the replicates. A dye swap was performed for the third replicate. These samples were then competitively hybridized to three replicate 244k arrays from Agilent, (AMADID 25150451). We hybridized the samples to the arrays using the Agilent Array CGH protocol, with some modifications. Briefly, labeled DNA was combined with Cot-1 DNA, blocking reagent, and hybridization buffer. The hybridizations were carried out at 60Â°C. The arrays were scanned using an Agilent dual laser scanner, and the images were processed using Agilent Feature Extraction Software.

Project description:We demonstrate here a method that perfuses a small region of an organotypic hippocampal culture with a solution containing an enzyme substrate, a neuropeptide. Perfusate containing hydrolysis products is continually collected and subsequently analyzed for the products of the enzymatic degradation of the peptide substrate. The driving force for perfusion is an electric field. The fused silica capillaries used as "push" and "pull" or "source" and "collection" capillaries have a ζ-potential that is negative and greater in magnitude than the tissue's ζ-potential. Thus, depending on the magnitudes of particular dimensions, the electroosmotic flow in the capillaries augments the fluid velocity in the tissue. The flow rate is not directly measured; however, we determine it using a finite-element approach. We have determined the collection efficiency of the system using an all d-amino acid internal standard. The flow rates are low, in the nL/min range, and adjustable by controlling the current or voltage in the system. The collection efficiency of the d-amino acid peptide internal standard is variable, increasing with increased current and thus electroosmotic flow rate. The collection efficiency can be rationalized in the context of a Peclet number. Electroosmotic push-pull perfusion of the neuropeptide galanin (gal1-29) through the extracellular space of an organotypic hippocampal culture results in its hydrolysis by ectopeptidase reactions occurring in the extracellular space. The products of hydrolysis were identified by MALDI-MS. Experiments at two levels of current (8-12 μA and 19-40 μA) show that the probability of seeing hydrolysis products (apparently from aminopeptidases) is greater in the Cornu Ammonis area 3 (CA3) than in the Cornu Ammonis area 1 (CA1) in the higher current experiments. In the lower current experiments, shorter peptide products of aminopeptidases (gal13-29 to gal20-19) are seen with greater frequency in CA3 than in CA1 but there is no statistically significant difference for longer peptides (gal3-29 to gal12-29).

Dataset Information

Electroosmotic Perfusion, External Microdialysis: Simulation and Experiment.

Publications

Electroosmotic Perfusion, External Microdialysis: Simulation and Experiment.

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