The skin conductance response indicating pain relief is independent of self or social influence on pain

Pain relief is defined as the ease of pain and is thus highly relevant for clinical applications and everyday life. Given that pain relief is based on the cessation of an aversive pain experience, it is reasonable to assume that pain relief learning would also be shaped by factors that alter subjective and physiological pain responses, such as social presence or a feeling of control. To date, it remains unclear whether and how factors that shape autonomic pain responses might affect pain relief learning. Here, we investigated how pain relief learning is shaped by two important factors known to modulate pain responses, i.e. social influence and controllability of pain. Skin conductance responses (SCRs) were recorded while participants learned to associate a formerly neutral stimulus with pain relief under three different pain conditions. In the social-influence condition (N = 34), the pain stimulation could be influenced by another person’s decisions. In the self-influence condition (N = 31), the participants themselves could influence the pain stimulation. Finally, in the no-influence condition (N = 32), pain stimulation was simply delivered without any influence. According to our results, the SCRs elicited by the stimulus that was associated with pain relief were significantly smaller compared to the SCRs elicited by a neutral control stimulus, indicating pain relief learning. However, there was no significant difference in the pain relief learning effect across the groups. These results suggest that physiological pain relief learning in humans is not significantly influenced by social influence and pain controllability

Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in <i>Plasmodium falciparum</i> Protein Export

<div><p>Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite <i>Plasmodium falciparum</i>. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.</p></div

Genomic position based screen for new PNEPs.

<p>(A) Venn diagram of selection of PNEPs based on a subtelomeric gene location (<200 kb from chromosome end). (B, C) Fluorescence pattern of non-exported (B) or exported (C) GFP fusion proteins. Protein structure of each candidate is indicated to the left of each panel as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1</a>. Nuclei were stained with DAPI. Size bars: 5 µm. (D) The fluorescence in the host cell of PF08_0005-GFP represents full length soluble protein as determined by Western blot as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1D</a>, except that GAPDH was used as the parasite internal control.</p

Soluble PNEPs need to be unfolded to reach the host cell.

<p>(A) Double transgenic parasites expressing MSRP6-GFP as well as MSRP6 fused to mDHFR and mCherry (constructs indicated on top) grown either with (+wr) or without (control) WR99210. The DIC image with DAPI stained nuclei (DIC/DAPI) and the merged GFP and mCherry signal (merge) are also presented. (B) As in (A) but with parasites expressing MSRP7-GFP and MSRP7-mDHFR-mCherry. The fluorescence internal to the parasite represents re-internalised protein in the food vacuole. Size bars: 5 µm. (C) Western blots of Percoll enriched double transgenic parasites shown in (A) and grown in the absence (control, exported) or presence (+wr, fluorescence remaining in parasite periphery) of WR99210 that were treated with saponin and separated into supernatant (SN) and pellet (P). SERP is found soluble in the PV and was used to demonstrate the action of saponin. GAPDH is the parasite internal control. The intensity of the mCherry, GFP and SERP signals in the extracts of parasites treated with wr is less than that of parasites not treated with wr, which may reflect either a somewhat slower growth cycle or slight differences in the stage composition of the parasite population after sample preparation. MSRP7-mDHFR-mCherry in the PV may also be more efficiently re-internalised and degraded in the food vacuole, reducing its amount in the blocked compared to the unblocked state.</p

Further PNEPs encoded by genes at the <i>msp7</i> gene locus.

<p>(A) Fluorescence pattern of PF13_0192-GFP. (B) Co-localisation IFA of PF13_0192-GFP with SBP1. (C) Fluorescence pattern of PF13_0191-GFP. Two panels are shown to demonstrate cells with (yellow arrows) and without additional foci of fluorescence in the host cell (ratio indicated in %, at least 50 cells were analysed on 3 occasions, standard deviation in brackets). (D) Bodipy-TR-C₅-ceramide (Bodipy) stained parasites expressing PF13_0191-GFP. Protein structure in A and C indicated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1</a>. Nuclei were stained with DAPI. Size bars: 5 µm.</p

MSRP6 is a Maurer's clefts protein expressed in trophozoites and schizonts.

<p>(A) Western blot with parental 3D7 (3D7), MSRP6-GFP expressing parasites and MSRP6 knock out parasites (Δ<i>msrp6</i>) demonstrates specificity of the antibodies. Molecular weight standards are indicated in kDa. Parasite GAPDH was used as a loading control. (B) Western blot with specific MSRP6 serum and extracts from stage specific parasites (hours post invasion indicated above each lane). GAPDH was used as a loading control. (C) Co-localisation IFA using anti-REX1 and anti-MSRP6 antibodies with MSRP6 knock out parasites (top) and parental 3D7 parasites (bottom). Size bar: 5 µm. (D) Pre-embedding immuno-EM of infected RBC, where the host cell cytosol has been released with tetanolysin, reacted with anti-MSRP6 antisera. Small panels show overviews with boxes highlighting individual Maurer's clefts for which enlargements are shown. Gold label is indicated with arrowheads. Size bars: 1 µm.</p