Paul A. Fleming

The Caenorhabditis elegans Choline Transporter CHO-1 Sustains Acetylcholine Synthesis and Motor Function in an Activity-Dependent Manner

Cholinergic neurotransmission supports motor, autonomic, and cognitive function and is compromised in myasthenias, cardiovascular diseases, and neurodegenerative disorders. Presynaptic uptake of choline via the sodium-dependent, hemicholinium-3-sensitive choline transporter (CHT) is believed to sustain acetylcholine (ACh) synthesis and release. Analysis of this hypothesis in vivo is limited in mammals because of the toxicity of CHT antagonists and the early postnatal lethality of CHT−/− mice (Ferguson et al., 2004). In Caenorhabditis elegans, in which cholinergic signaling supports motor activity and mutant alleles impacting ACh secretion and response can be propagated, we investigated the contribution of CHT (CHO-1) to facets of cholinergic neurobiology. Using the cho-1 promoter to drive expression of a translational, green fluorescent protein-CHO-1 fusion (CHO-1:GFP) in wild-type and kinesin (unc-104) mutant backgrounds, we establish in the living nematode that the transporter localizes to cholinergic synapses, and likely traffics on synaptic vesicles. Using embryonic primary cultures, we demonstrate that CHO-1 mediates hemicholinium-3-sensitive, high-affinity choline uptake that can be enhanced with depolarization in a Ca2+-dependent manner supporting ACh synthesis. Although homozygous cho-1 null mutants are viable, they possess 40% less ACh than wild-type animals and display stress-dependent defects in motor activity. In a choline-free liquid environment, cho-1 mutants demonstrate premature paralysis relative to wild-type animals. Our findings establish a requirement for presynaptic choline transport activity in vivo in a model amenable to a genetic dissection of CHO-1 regulation.

In‐process gap detection in friction stir welding

Purpose – This paper aims to investigate methods of implementing in‐process fault avoidance in robotic friction stir welding (FSW).Design/methodology/approach – Investigations into the possibilities for automatically detecting gap‐faults in a friction stir lap weld were conducted. Force signals were collected from a number of lap welds containing differing degrees of gap faults. Statistical analysis was carried out to determine whether these signals could be used to develop an automatic fault detector/classifier.Findings – The results demonstrate that the frequency spectra of collected force signals can be mapped to a lower dimension through discovered discriminant functions where the faulty welds and control welds are linearly separable. This implies that a robust and precise classifier is very plausible, given force signals.Research limitations/implications – Future research should focus on a complete controller using the information reported in this paper. This should allow for a robotic friction stir ...

Misalignment detection and enabling of seam tracking for friction stir welding

Abstract This paper describes a technique for determining the position of a friction stir welding (FSW) tool with respect to the weld seam during welding. Forces are used as a feedback signal, and a general regression neural network is trained to predict offset position given weld forces. Experimental results demonstrate the accuracy of the developed position predictor. This technique is proposed for online misalignment detection or as a position estimator for in-process tracking of the weld seam for FSW and robotic FSW.

An open-source analytical platform for analysis of C. elegans swimming-induced paralysis.

BACKGROUND The nematode Caenhorhabditis elegans offers great power for the identification and characterization of genes that regulate behavior. In support of this effort, analytical methods are required that provide dimensional analyses of subcomponents of behavior. Previously, we demonstrated that loss of the presynaptic dopamine (DA) transporter, dat-1, evokes DA-dependent Swimming-Induced Paralysis (Swip) (Mcdonald et al., 2007), a behavior compatible with forward genetic screens (Hardaway et al., 2012). NEW METHOD Here, we detail the development and implementation of SwimR, a set of tools that provide for an automated, kinetic analysis of C. elegans Swip. SwimR relies on open source programs that can be freely implemented and modified. RESULTS We show that SwimR can display time-dependent alterations of swimming behavior induced by drug-treatment, illustrating this capacity with the dat-1 blocker and tricyclic antidepressant imipramine (IMI). We demonstrate the capacity of SwimR to extract multiple kinetic parameters that are impractical to obtain in manual assays. COMPARISON WITH EXISTING METHODS Standard measurements of C. elegans swimming utilizes manual assessments of the number of animals exhibiting swimming versus paralysis. Our approach deconstructs the time course and rates of movement in an automated fashion, offering a significant increase in the information that can be obtained from swimming behavior. CONCLUSIONS The SwimR platform is a powerful tool for the deconstruction of worm thrashing behavior in the context of both genetic and pharmacological manipulations that can be used to segregate pathways that underlie nematode swimming mechanics.