The Role Of RNA-Binding Protein Pumilio In Regulation Of Nociceptive Sensation

Nociception is the sensation of potentially tissue damaging stimuli, and is necessary for the survival of all animals. Without it, organisms would not be able to navigate their environment safely and efficiently, both avoiding potentially dangerous situations and not wasting energy responding to every stimulus like a possible threat. Many of the underlying nociceptive processes are conserved throughout metazoan systems, and the overlap between Drosophila melanogaster and humans is extensive. Characterizing the regulatory processes behind nociceptive sensation is important to provide avenues for treatment of chronic pain in the human population, and one potential point of regulation is the Pumilio (Pum)  protein. Flies with decreased and increased expression of pumilio were tested for nociceptive defects to mechanical and thermal stimuli, and the effects of changed Pumilio expression on dendrite morphology were also quantified. Reduction of pumilio expression using RNA interference (RNAi) led to hypersensitivity to mechanical and thermal stimuli, and a reduced dendrite phenotype. Increase of pumilio expression by overexpression of pumilio cDNA in nociceptor neurons led to an insensitive phenotype to mechanical stimuli. To study the possible downstream effectors of Pumilio, a fluorescent live-imaging tool will be created that will allow visualization of paralytic mRNA in the nociceptor neurons. These experiments begin to elucidate the role Pumilio plays in the regulation of nociception and the molecular mechanisms by which it regulates nociception.

Distinct responses to rare codons in select Drosophila tissues

Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index (taCAI), to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in an evolutionarily young testis-specific gene, RpL10Aa. Optimizing RpL10Aa codons disrupts female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology

Electroencephalography during general anaesthesia differs between term-born and premature-born children

OBJECTIVES: Premature birth is associated with a wide range of complications in later life, including structural and functional neurological abnormalities and altered pain sensitivity. We investigated whether during anaesthesia premature-born children display different patterns of background EEG activity and exhibit increased responses to nociceptive stimuli. METHODS: We examined background EEG and time-locked responses to clinical cannulation in 45 children (mean age (±SD) at study: 4.9(±3.0)years) under sevoflurane monoanaesthesia maintained at a steady-state end-tidal concentration of 2.5%. 15 were born prematurely (mean gestational age at birth: 29.2 ± 3.9 weeks) and 30 were age-matched term-born children. RESULTS: Background levels of alpha and beta power were significantly lower in the premature-born children compared to term-born controls (p=0.048). Clinical cannulation evoked a significant increase in delta activity (p=0.032), which was not significantly different between the two groups (p=0.44). CONCLUSIONS: The results indicate that whilst under anaesthesia premature-born children display different patterns of background brain activity compared to term-born children. SIGNIFICANCE: As electrophysiological techniques are increasingly used by anaesthetists to gauge anaesthetic depth, differences in background levels of electrophysiological brain activity between premature and term-born children may be relevant when considering titration of anaesthetic dose

Noxious stimulation evokes ipsilateral and contralateral flexion activity in preterm and term infants.

<p>A: Evoked activity on the two sides are not significantly different in A, term infants (n = 13) and B: preterm infants (n = 11). Inset: Experiment set-up, surface EMG leads were placed over the ipsilateral (filled lines) and contralateral (dashed lines) biceps femoris to record flexion reflex EMG activity evoked by a heel lance. Example trace from term and preterm infant below, arrow indicates time of noxious stimulus.</p

Summary of the experimental groups, stimulation type and EMG recording set-up used in the study.

<p>Experimental groups are grouped by stimulation type (A, noxious stimulation – clinically required heel lance; B, tactile stimulation; C, punctate stimulation) and EMG recording set-up (ipsilateral or paired ipsi-contralateral). Schematic of the EMG recording set-up shown on left side of figure panel: ipsilateral (top), or paired ipsi¬contralateral (bottom); grey circles represent surface EMG electrode location over the infant limb.</p

Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo.

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator

Single and repeated punctate stimulation evokes flexion activity that is gestational age dependent.

<p>A, Cutaneous sensory threshold to punctate stimulation significantly increases with gestational age; B, threshold force required to evoke leg withdrawal and example flexion reflex EMG traces from term and preterm infants are shown. C, Threshold flexion reflex EMG responses in preterm infants are significantly greater than in term (two-way ANOVA, ***p<0.0001 age group, **p = <0.0001 time; Fisher's unpaired t-test, 250 ms, *p = 0.02; 500 ms, ***p<0.0001, 750 ms, ***p<0.0001). Note- two EMG recordings excluded from the analysis due to technical artefact. D, Brief, low frequency repeated punctate stimulation sensitised infant flexion reflex EMG activity as shown by a significantly larger flexion response to a threshold hair after 10 seconds of repeated stimulation, compared to the responses to the same threshold hair when tested 10 s before the train (two-way ANOVA, *p = 0.04 treatment,***p = 0.002, time; Fisher's paired t-test, 250 ms, *p = 0,002, n = 11, pooled term and preterm.</p

Demographic characterisation of the neonatal population.

<p>SDs are in parentheses. GA, Gestational Age.</p>*<p>Information unavailable for 2 infants. ⁺Information unavailable for 4 infants.</p

Flexion withdrawal reflex biceps femoris EMG activity evoked by noxious and tactile stimulation.

<p>Only data from those infants that displayed a paired tactile and lance response is shown here. A. term (n = 7) and B. preterm infants (n = 5). Data is expressed as fold increase in EMG activity measured in 250 ms time epochs for 4 secs post stimulus. While in term infants (A) the flexor reflex response to noxious lance stimulation is significantly greater than to tactile stimulation, in preterm infants, the two responses are not significantly different. [Term infants two-way ANOVA, **p = 0.006 time, ****p<0.0001 stimulus. Student's t-test, at 250 ms, *p = 0.014, at 500 ms, ***p = 0.0005, and at 750 ms, **p = 0.006].</p