High quality abstracts were selected and discussed as oral or poster presentations. The aim of this review is to distribute the scientific highlights of this workshop outside the group as analyzed and represented by experts in retrovirology, immunology and clinical research.The December 2011 5 Maarten from December 6-9 and featured presentations from 210 scientists representing approximately 10 countries. Since its inception, the goal of the workshop has been to provide a forum for research aimed at understanding the mechanism by which HIV-1 persists in the face of antiretroviral therapy (ART) and to develop strategies with which to curtail viral persistence and accelerate the objective of viral eradication. While ART has fundamentally impacted the health of individuals living with HIV infection and effects durable suppression of plasma viral RNA to undetectable levels, current treatment regimens are unable to eradicate the virus [The 5+ T cells [Planelles et al. [in vitro differentiated central memory cells. The system uses peripheral blood mononuclear cells from healthy human donors. Following isolation of naïve CD4+ cells by negative selection, cells are activated with CD3 and CD28 antibodies in medium containing TGF-beta, anti interleukin (IL)-12 and anti IL4 antibodies followed by culture in IL-2. Following HIV-1 infection, cells return to a quiescent state where the majority of infected cells are latent. Planelles et al. have previously demonstrated that these latently infected cells can be re-activated by incubation with CD3/CD28 antibodies. The investigators went on to use this system to screen for anti-latency agents. The investigators demonstrated that incubation with IL-2 + IL-7 can induce homeostatic proliferation and reactivation in about 10% of the latently infected cells. However, full reactivation of all latently infected cells in the cultures required antigenic stimulation. While this study describes a system that can be very useful for the screening of anti-latency agents, it highlights the concern that agents inducing homeostatic proliferation may have the potential to expand integrated proviruses through gene duplication at mitosis. Nonetheless, this won't impact the majority of latent genomes that fail to become reactivated during homeostatic proliferation and therefore, not achieve the goal of eradicating latent provirus. An important point to consider is whether homeostatic proliferation favors the duplication of archival and defective provirus rather than latent provirus. It is unclear whether latent provirus duplication by mitosis during homeostatic proliferation leads to expression of the provirus and subsequent clearance by cytopathic effects or by immune clearance. Defective provirus would be less likely to be cleared by these mechanisms. Therefore it would be important to determine to what extent homeostatic proliferation allows duplication of latent provirus without subsequent clearance.The development of strategies to eliminate HIV-1 reservoirs that persist in the face of ART will require a complete understanding of the nature of viral persistence and latency and how these processes are regulated. While latency is considered the single biggest obstacle to viral eradication, how latency is established and regulated is still not well understood. Most of the studies conducted to date on viral latency have focused on models that employ established cell lines. They have demonstrated various forms of latency regulation and the role of host cell cycle, epigenetic effects and other host cell factors that can regulate viral latency. However, it is unclear as to the extent to which these cell line models of viral latency reflect the true nature of latency as it exists in memory CD4 T cells . For this et al.  describeGarcia et al. [ex vivo to produce virus. The frequency of latently infected, resting cells was determined to be in the reaching of 9.9 per million CD4+ T cells, which is in the range observed for patients on suppressive therapy. The availability of an in vivo model of viral latency extends the tools available for the analysis of anti-latency drugs.Continuing on with the theme of primary models of viral latency, a et al.  describeWhile the majority of research has focused on the role of lymphoid reservoirs in viral persistence and latency, several presentations focused on the role of myeloid cells in viral persistence. A large body of research has demonstrated the important association of animal lentiviruses with myeloid-lineage cells and, in particular, macrophages. However, very little attention has focused on the role of myeloid-lineage cells in the biology of primate lentiviruses. Early studies demonstrated infection of macrophage in the tissues such as the lung and central nervous system (CNS) but, beyond that, there is no clear understanding of whether macrophage support viral persistence in patients on antiretroviral therapy or whether latency is also manifest in macrophage or other myeloid-lineage cells. Arguably, the strongest piece of experimental evidence supporting an essential role for macrophage in primate lentivirus biology is the demonstration that myeloid-lineage cells harbor an antiviral restriction that is not exhibited by T cells and further, that the virus has evolved a strategy to circumvent this restriction.Clements et al. [Russell et al. [s et al.  discussel et al.  who examLewin et al. [+ T-cells incubated with CCL19 and CCL21 receptor chemokines, CXCL 10 and CCR 6 rendered them permissive to HIV-1 infection and provirus establishment. Incubation with CCL 19 prior to infection activated RhoA signaling and alteration of the actin network that was sufficient for establishment of the integrated provirus. Furthermore, integration of HIV-1 in resting cells was also dependent upon PI3K pathway activation and inhibitors of the PI3K pathway did not affect nuclear localization of viral cDNA but prevented cDNA integration. Lewin et al. went on further to describe how dendritic cells may play a role in the infection of resting memory CD4+ cells. Monocyte-derived dendritic cells (myeloid DCs) promoted latent infection of resting memory CD4+ cells when in co-culture but less efficiently when cells were incubated separately. This suggests the presentence of soluble factors that may play a role in the promotion of CD4 latency by myeloid DCs.In addition to the viral mechanisms of HIV-1 persistence, it is well established that the immune system plays a critical role in the establishment and persistence of a viral reservoir during therapy . Interesn et al.  presenteLieberman et al. [+ T cells, monocytes and macrophages, she demonstrated that the knockdown of CCR5, HIV Vif and gag or Trex1 inhibit HIV transmission in tissue explants and humanized mice.n et al.  outlinedLaguette et al. [e et al.  discussee et al. ,12. SAMHManel et al. [l et al.  gave furO'Doherty et al. [in vitro assay. Her data suggested that latently infected cells continuously express low levels of viral proteins and constitute targets for HIV-specific T cell responses. In an in vitro killing assay, EC displayed more effective removal of latently infected CD4+ T cells than chronic progressors.y et al.  quantifiy et al. ,16. She Lichterfeld et al. [+ T cells from EC to HIV-specific CD8+ T cell mediated killing. In a cytotoxicity assay, CD4+ T cells from EC showed an increased susceptibility to CD8 mediated killing. Interestingly, a reduced susceptibility of naïve CD4+ T cells to CD8+ T cells mediated killing was associated with a lower viral reservoir in EC.d et al.  also preDeeks et al. [Deeks et al. summarized the recent results from two Raltegravir intensification trials indicating that this intervention did not modify the levels of T cells activation in blood and rectum but may reduce them in the terminal ileum [s et al.  focused s et al. . Inflammal ileum ,21.Chomont et al. [+ T cell nadir significantly predicted levels of HIV proviral DNA. The evolution in the TCR repertoire of virally suppressed subjects was correlated with the genetic evolution of the viral reservoir, supporting a model in which the dynamic of the memory CD4+ T cell compartment drives the dynamic of the latent reservoir. In the second part of his talk, Chomont presented a novel assay aimed at monitoring HIV persistence during ART. The assay, which uses authentic CD4+ T cells from virally suppressed subjects, could also be used to identify novel compounds aimed at reactivating HIV production in latently infected cells.t et al.  listed bVandergeeten et al. [+ T cells and hypothesized that they may also contribute to the persistence of latently infected cells. Both cytokines induced proliferation, activation and survival of CD4+ T cells in vitro. Similarly, both IL-7 and IL-15 enhanced viral production in productively infected CD4+ T cells isolated from HIV infected subjects. Strikingly, the two cytokines differed in their ability to induce HIV production in latently infected cells, with IL-15 being much more efficient than IL-7. These results suggested that IL-15 should be considered as a possible candidate to force viral expression of the latent reservoir to achieve HIV eradication.n et al.  outlinedChirullo et al. [o et al.  started o et al.  showing o et al. .Kuritzkes et al. [s et al.  mentioneIt will undeniably be concerted decisions to make between patients scientists, ethic committees and funding agencies.The immunological and virological benefits of ART initiation during the early steps of HIV infection have been reported for more than 10 years ,29. StarMarkowitz et al. [z et al.  presenteAt 48 weeks, from 26 patients randomized in the 5-drug arm, 23 patients remained on study; and from 14 patients randomized to the 3-drug arm, 11 patients remained on study.At 96 weeks, 18 patients remained on study in the 5-drug arm, and 10 patients in the 3-drug arm. As expected the 5-drug regimen achieved < 50 copies/ml of plasma viremia faster, but at week 48 there were 3 virological failures in the 5 drug arm, and none in the 3-drug arm. No differences were found in terms of proviral DNA levels or infectious virus titers between the 2 arms. Overall, these results are quite disappointing but concern a population of patients treated after a mean duration of symptoms of acute infection of about 50 days.Ananworanich et al. [On the contrary, h et al.  treated +CCR5+ T cells in sigmoid colonincreased from Fiebig I to III stages (p = 0.0014). After treatment initiation, a rapid and equivalent plasma HIV RNA decline was observed with both regimens.Before ART initiation, total HIV DNA in PBMC was significantly lower in Fiebig I versus Fiebig III patients (p = 0.007) and the loss of CD4+CCR5+ T cells in sigmoid colon significantly increased after antiretroviral treatment in Fiebig III and IV subjects.The percentage of CD4Total and integrated HIV DNA in PBMCs declined significantly after Mega-ART and ART. Total HIV DNA by week 24 was undetectable in 40% of patients. Integrated HIV DNA by week 24 was undetectable in 80% of patients.Total and integrated HIV DNA in sigmoid colon declined significantly after Mega-ART. Total HIV DNA by week 24 was undetectable in 35% of patients. Integrated HIV DNA by week 24 was undetectable in 78% of patients.The frequency of PBMCs harboring HIV DNA during acute HIV predicted HIV reservoir size after 24 weeks of treatment (p < 0.0001).Consequently, these studies tend to show that a smaller reservoir size is obtained when ART is initiated very early at acute infection, but no clear benefit is found by adding more than 3 drugs in the combination.Zack et al. [in vitro on reactivation of latent HIV.k et al.  proposedk et al. , they prMargolis et al. [+ cells showed increased viral RNA expression in vitro following the presence of Vorinostat. From oncologic studies, it is known that the peak of Vorinostat in plasma occurs between 4 and 8 hours after a single administration. A mean increase in vivo of 4.4 fold of resting CD4+ T cell associated full-length RNA was observed after the administration of 400 mg of Vorinostat and sampling patients at around 6 hours post administration.s et al.  presenteHernandez et al. [Provocative data were presented by z et al.  suggestiChen et al. [in vitro in ACH-1 and U1 cells. In these models, Gnidimacrin was at least 2,000-fold more potent than Prostratin.n et al. ,38 showein vitro demonstration that ART is quite ineffective at preventing direct HIV cell-to-cell transfer [Whether or not ongoing HIV replication or propagation persists during effective ART is not a new topic, but it has recently been fuelled by the transfer .Schacker et al. [6 cells. Although plasma viremia became undetectable within 2 months in all patients, the authors demonstrated that HIV continued to infect new cells in lymphoid tissues and that the drugs rarely reached effective concentrations in these tissues [Bringing the problem at the clinical level, r et al.  analyzed tissues .It will therefore be important to develop new ways of drug delivery in lymphoid tissues, in particular for strategies purging HIV reservoirs, in order to protect uninfected cells in every compartment.Deeks et al. [+ cells expressing PD-1 contain more proviral DNA than those who do not. Immune therapies based on the blockade of PD-1 interaction with its ligands are supposed to increase HIV production and enhance anti-HIV specific immune responses. The first clinical trial using anti PD-1 antibodies (ACTG 5301) is under approval. It is a single-arm pilot study to evaluate the safety, pharmacokinetic profile, and effects of a single dose of anti-PD-1 antibody in 40 chronically HIV-infected patients receiving effective ART for more than 36 months.s et al.  argued tVandergeeten et al. [in vitro for reactivating the reservoir while inducing much less cell proliferation. Consequently, IL-15 is a possible candidate to achieve HIV eradication, although IL-7 increases the number of CD4+ T cells harboring HIV integrated DNA.As mentioned previously, n et al.  presenteJune et al. [3 and undetectable viremia received a single infusion of 1 × 1010 modified cells. Four weeks after the infusion, a structured treatment interruption (STI) was planned for a maximum of 12 weeks. Although plasma viremia rebounded in all cases within 4 weeks following the STI, it began to decrease in 5 cases before ART was resumed. In particular, "patient 205" who was heterozygote for the CCR5 delta 32 mutation before ZFN therapy, reached undetectable viral load by day 112. Consequently, ZFN modified cells distributed and trafficked normally compared to endogenous CD4+ cells and showed antiviral activity during STI.Trying to mimic the "Berlin patient", e et al.  updated Elimination of CCR5 expression is not expected to directly impact the size of the latent reservoir. If the stability of the latent reservoir is extended by reservoir replenishment, limiting target cell availability would, in turn, reduce potential sources of the virus that drives the replenishment and, in this scenario, an accelerated decay of the latent reservoir would be expected. However if the intrinsic stability of the latent reservoir is indeed measured in decades and there is no replenishment, then introduction of CCR5-negative cells would not alter the decay of the latent reservoir. This should still produce a functional cure since any virus released from reactivated latent cells would be unable to establish new infections. Either scenario would be a significant step forward since patients transduced with CCR5-negative stem cell would be predicted to control the virus in the absence of ART.Jerome et al. [e et al.  have beeth International Workshop on HIV Persistence led to the presentation and discussion of exciting new data from research groups working towards an HIV cure. Over the years, the control of HIV reservoirs is progressively moving from bench to bedside. The next edition of the workshop will be held in Miami, Fl, 3-6 December, 2013.As anticipated, the 5The authors declare that they have no competing interests. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of their institutions.All the authors contributed equally to the manuscre therapeutic session. All authors read and approved the final manuscript.


In the special issue “Signaling Molecules and Signal Transduction in Cells” authors were invited to submit papers regarding important and novel aspects of extra- and intracellular signaling which have implications on physiological and pathophysiological processes. These aspects included compounds which are involved in these processes, elucidation of signaling pathways, as well as novel techniques for the analysis of signaling pathways. In response, various novel and important topics are elucidated in this special issue. Several of the manuscripts presented discuss compounds which might be involved in cellular apoptosis and thereby influence cancer or embryonic development.Ginsenoside Rh2 (G-Rh2), derived from the plant Ginseng, acts anti-proliferative and pro-apoptotic. Its intracellular effects through apoptotic pathways were analyzed by Guo et al. Rapamycin is an inhibitor of mTOR  and thereby acts antiproliferative on some tumors [et al. studied the anti-tumor effect of rapamycin inducing apoptosis and autophagy on pancreatic cancer cells [et al. proffer that JRS-15, a derivative of xylocydine which was a novel cyclin-dependent kinase inhibitor, induced mitochondrial apoptosis in several cancer cell lines [et al. reviewed the effect of various bioactive compounds from marine organisms including sponges, actinomycetes and soft corals on the diverse apoptotic pathways of cancer cells [The compound e tumors . Dai et ll lines . Kalimuter cells .ochratoxin A (OTA) is nephrotoxic, hepatotoxic and immunotoxic. The cytotoxic effects of OTA on mouse embryonic development inducing reactive oxygen species and mitochondrial apoptosis were studied by Hsuuw et al. [et al. reviewed the role of reggie/flotillin proteins for Wnt secretion and gradient formation and its effect on development [et al. reported the osteogenic potential of diverse signaling pathways including Wnt, BMP, FGF and TGFβ [et al. analyzed the toxic effect of cesium in plants. High concentrations of cesium inhibited plant growth inducing the jasmonate pathway and thereby probably modified potassium uptake machineries [unfolded protein response (UPR) and the steroid response element (SREBP) was studied by Bedoya-Perez et al. in the mosquito Aedes aegypti using the Cry11Aa toxin [UPR signaling pathways in mammalian and their implications were reviewed by Carrara et al. [The common mycotoxin w et al. . Wnt morelopment . Furtherand TGFβ . Adams ehineries . The difAa toxin . Furthera et al. .et al. described prostaglandin E2 GPCR signaling in dendritic cells in respect to the cellular life cycle [Resolvin (resolution-phase interaction products) is a member of a novel family of aspirin-triggered short-lived autacoids synthesized during inflammation. Keinan et al. presented resolvin signaling pathways which could be used in oral health treatment [G-Protein coupled receptors (GPCR) represent the most abundant class of mammalian membrane-bound receptors and are valuable pharmacological targets. The review by De Kejzer fe cycle . Resolvireatment .EGFR to the intracellular dynein IC2 was described by Pullikuth et al. [spatial regulation of EGFR signaling including endocytosis were elucidated by Ceresa et al. [et al. [EGFR Y845 phosphorylation probably interacted with Mucin-1 and cleaved Galectin-3 which could serve as a diagnostic tool for differentiation of benign and malign tumors. Regulation of endocytosis and cell signaling is an emerging role of intersectins which were summarized by Hunter et al. [Growth factors are important mediators of developmental processes. Mutations in the tyrosine kinase growth factor receptors are known to induce severe diseases, including the susceptibility to cancer. Therefore, the regulation of growth factor receptor signaling is essential for the understanding of physiology and pathophysiology of these proteins. In this regard, the link of h et al. . Furthera et al. . The mec [et al. . They for et al. . There wet al. explained the role of fibroblast growth factor (FGF) and the FGF receptors Heartless(Htl) and Breathless (Btl) for development and differentiation in Drosophila [et al. described that interference of peptide apatamers with growth factors e.g., TGFβ or EGFR could be suitable for the analysis of their signaling pathways in high throughput screening studies [Formyl peptide receptor 2 agonists, their distinct signaling pathways and their involvement in immunological responses and cancer were reviewed by Cattaneo et al. [Muha osophila . Conidi  studies . Formyl o et al. .et al. discussed the effect of EPO, its derivatives and the serine/threonine kinase receptor EPO-R in endothelial cells, regarding desensitization/resensitization/expression using an in vitro model [et al. discussed the role of cytokines in healthy and inflammatory skin diseases [Erythropoietin (EPO) induces erythropoiesis and is used as a pharmacological drug, e.g., as biosimilars for long-term treatment of anemia. However, EPO also acts on other types of cells, e.g., endothelial mediating proliferative and angiogenic effects and might be important for the therapeutic outcome. Trincavelli ro model . Hänel ediseases .retinoid nuclear orphan receptor RORα were reviewed by Du et al. implicating its role as tumor suppressor [Functions of the ppressor .the secretory pathway calcium (Ca2+)-ATPase pump (SPCA1). Micaroni et al. hypothesized that the gene ASTE1 influences ATP2C1 gene expression. ASTE1 dysregulation might induce cell death and tumor transformation [Mutations in the gene ATP2C cause the Hailey-Hailey skin disease in humans. ATP2C1 encodes ormation .Nitric oxide is an important signaling molecule which exerts pleiotropic functions. Its regulatory function in skeletal muscle during exercise was summarized by Suhr et al. [in vivo is an emerging field which was presented in comparison to cAMP by Sprenger et al. in a featured review paper [r et al. . Solubleew paper .MAP kinase scaffold was reviewed by Meister et al. [et al. presented a study regarding the gene En-MAPK1 which was activated during pheromone signaling of the polar ciliate Euplotes nobili [et al. described the regulation of T-Cell activation and function by diacylglycerol kinases [Kinase cascades are essential for the intracellular signal transduction. In this regard, the r et al.  coordinas nobili . Further kinases .et al. set its focus on the atypical phosphatases eyes absent (EYA) which acted as dual Thr/Tyr-phosphatase and members of the phosphoglycerate mutase (PGAM) family  which exerted His-based Tyr-phosphatase activity [Phosphorylation is controlled by protein phosphatases. Recently, atypical protein phosphatases were discovered which were structurally different from the known families of Ser/Thr- and Tyr-phosphatases. Sadatomi activity .ubiquitination of Notch and its signaling intracellular function was reviewed by Moretti et al. Small GTP-binding proteins are important regulators of intracellular signaling [RhoA in the intestinal epithelial barrier was summarized by Tong et al. [SUMOylation, e.g., on ATF3. Its role inhibiting prostate cancer cells was presented by Wang et al. [The ubiquitination of proteins is a proteasomal degradation motif. However, ubiquitination is also used as an intracellular receptor signaling motif. In this regard, the ignaling . As an eg et al. . A furthg et al. .exosomes for (patho)physiological processes is a topic which was reviewed in detail by Corrado et al. [et al. [The controlled release of compounds from cells is important for intercellular signaling and communication. Beyond various exocytosis mechanisms, the analysis and implication of o et al. . The com [et al. .In summary, several important and novel aspects of intracellular and intercellular signaling in health and disease were highlighted in this special issue. However, signal transduction in and from cells is a huge field which can only partly be touched on in one special issue. Therefore, a further special issue covering more aspects of this engrossing field will follow in 2014.


Computerized cognitive bias modification for social anxiety disorder has in several well conducted trials shown great promise with as many as 72% no longer fulfilling diagnostic criteria after a 4 week training program. To test if the same program can be transferred from a clinical setting to an internet delivered home based treatment the authors conducted a randomized, double-blind placebo-controlled trial.After a diagnostic interview 79 participants were randomized to one of two attention training programs using a probe detection task. In the active condition the participant was trained to direct attention away from threat, whereas in the placebo condition the probe appeared with equal frequency in the position of the threatening and neutral faces.Results were analyzed on an intention-to-treat basis, including all randomized participants. Immediate and 4-month follow-up results revealed a significant time effect on all measured dimensions . However, there were no time x group interactions. The lack of differences in the two groups was also mirrored by the infinitesimal between group effect size both at post test and at 4-month follow-up.We conclude that computerized attention bias modification may need to be altered before dissemination for the Internet.ISRCTN01715124 A recent development in the treatment of anxiety disorders is attention bias modification , which dMoreover, there is evidence that successful treatment for social phobia may lead to a normalization of attention bias for threat . This fiIn a meta-analysis on the effects of attention bias modification by Hakamata and coworkers , 10 rande.g., [via the internet that could open the door for millions of people in need. Another study that also found positive outcomes was carried out by Schmidt and coworkers [Attention bias modification is a potentially effective computerized treatment that most often has been delivered in a laboratory setting with subclinical samples e.g., . The trae.g., . The tree.g., . Up untie.g., . Both cooworkers . At termoworkers  have invvia computer we decided to investigate if this novel treatment could be delivered via the Internet with no physical contact with the study participants. This has been done once in a recent trial by Boettcher and co-workers [via the Internet was superior to a placebo condition (random attention training) in a group of participants with diagnosed social anxiety disorder recruited from the community. We hypothesized that the treatment would be better than the control condition and that it would be possible to present attention bias modification via the Internet.Another recent development in the treatment of social anxiety disorder is the possibility to deliver CBT over the Internet . A large-workers . In facte.g. psychosis, substance misuse) that could be expected to influence the outcome of the study; j) having a primary diagnosis of social anxiety disorder according to the Structured Clinical Interview for DSM–IV Axis I Disorders . The laAs evident from the CONSORT flowchart Figure , of the via the Internet [The following social anxiety scales constituted the outcome measures in the study: the Liebowitz Social Anxiety Scale self-report version , theInternet ,41).et al.[via the Internet.Participants were either assigned to the real attention modification program or to a placebo version. Everything was identical in both conditions except for the location of the probe. Hence, in both conditions a trial began with a fixation cross (“+”) presented in the center of the screen for 500 ms. Immediately following termination of the fixation cue, the web based flash program in full screen mode presented two faces of the same person, one face on the top and one on the bottom, with each pair displaying one of two combinations of emotions. Either neutral-disgust, or neutral-neutral. After presentation of the faces for 500 ms, a probe appeared in the location of one of the two faces. Participants were instructed to indicate whether the probe was the letter E or F by pressing the corresponding arrow on the keyboard using their dominant hand. The probe remained on the screen until a response was given, after which the next trial began. During each session, 160 trials with various combinations of probe type (E/F), probe position (top/bottom), face type  and person . There are a total of 8 persons showing 2 different facial expressions; 4 male and 4 female showing disgust or neutral. In the real condition the probe was always presented  at the location of the neutral face if there also was a disgust face present . In contrast, in the placebo condition the location of the probe could not be predicted since the probe appeared with equal frequency in the position of the disgust face and the neutral face. The remaining 32 trials were neutral-neutral with the probe randomly presented at the top/bottom. For a more detailed description of the two conditions see Amir et al.), since Participants were encouraged to do the training on Tuesdays and Thursdays. They received an email and a SMS reminding them to do the training on the training days. If a session was missed a reminder was sent the following day. The participants could only do the training between 5 AM and 11 PM, and there should always be least one day between the sessions.The participants were divided into two groups; treatment or control by an online true random-number service independent of the investigators. The study protocol was approved by the regional ethics committee, and written informed consent was obtained from all participants by surface mail.All self-report scales were administered before the start of the treatment. During the treatment LSAS-SR once a week (Sundays). Following the four weeks of training LSAS-SR, SPS, SIAS, SPSQ, MADRS-S and QOLI was readministered. Immediately following the training phase, a clinical global impression of improvement (CGI-I) was mapped on a 7-point scale CGI; ) after aIn accordance with the intention-to-treat principle, all participants were asked to complete post-treatment and follow-up assessments, regardless of how many training sessions they had completed. Independent t-tests and chi-2 were used to check if randomization had resulted in a balanced distribution across both conditions. As evident from Figure d = 0.15; placebo d = 0.19).Most participants (74 of 79) completed all 8 training session for a mean of 7.8. Tables vs. placebo group: very much improved (5.0% vs. 2.6%), much improved (2.5% vs. 20.5%), small improvement (32.5% vs. 35.9%), unchanged (57.5% vs. 38.5%) and small deterioration (2.5% vs. 2.6%)  = 7.5; p = 0.112). In addition to the self-report measures and the blind CGI-I rating a SCID interview was conducted with the results that three (7.5%) in the treatment group and two (5.1%) in the placebo group no longer met DSM-IV criteria for social phobia.The CGI-I rating at post-treatment (n = 79) showed the following non-significant results for the treatment group Of the 79 participants who commenced treatment 74 (93.7%) completed all 8 training sessions as scheduled. However, five (6.3%) dropped out after only finishing M = 4.8 (SD = 1.0) sessions.An analysis of the proportion of correct trials revealed no difference between the groups. The overall average was M = 98.7% correct indications of the letters presented. In addition, there was no difference in the average response time per trial M = 733 ms (SD = 290 ms).To get a measure of attention bias we followed the suggested procedure from the previous Amir study . That inTo assess whether participants remained blind to their respective experimental condition, we asked the participants at post treatment whether they thought they had received the active or the placebo intervention. Of the participants who provided responses (n = 76) 18.9% (7/37) in the treatment group and 28.2% (11/39) in the placebo condition thought they had been randomized to the treatment program  = 0.9, p = .42).et al.[et al.[et al.[et al.[i.e., internet based flash program) vs. personal computer delivery was a difference between current study and previous research.This was a double blind randomized controlled trial with the aim to transfer the promising results of the Amir et al. and Schml.[et al. studies l.[et al.. Specifil.[et al., while Bl.[et al.. The prol.[et al.,18). Whel.[et al. is explal.[et al. simply eAn explanation of the null findings could be that this study also included participants with non-generalized social phobia. However, the mean scores on LSAS-SR were almost identical . In addition, when running the analysis with the NGSP and GSP groups separated, no differences in the treatment effect emerged. et al.[et al.[cf.)[cf.)[i.e., trial with two neutral faces. Using this new measure of bias, Koster et al., found that individuals with anxiety have had difficulty in disengaging their attention from highly threatening pictures. This measure of bias has been used but other investigators to assess the specific componets of attentional bias in anxiety [et al.[et al.[When the participants who received the real treatment where asked to predict condition the absolute majority 81%) thought they had been randomized to the placebo group. One would think that such a low level of positive expectation would influence the trial. However, that cannot explain the lack of effect since the Amir et al. and Schml.[et al. papers r al.[cf.). Howevercf.)[cf.). Althougcf.)[cf.),51. The cf.)[cf.) proposed anxiety -55. The % thoughty [et al. or the Sl.[et al. studies.l.[et al.. It shoul.[et al.. PerhapsWe conclude that attention bias modification may need to be further investigated before dissemination for the Internet.Seven of the eight authors declare that there is no conflict of interest. However, Dr Amir has founded a company that market online anxiety relief products.All authors contributed to the design of this study. PC drafted the manuscript. All authors contributed to the further writing of the manuscript. All authors read and approved the final manuscript.


Laser-induced breakdown spectroscopy (LIBS) is typically performed at ambient Earth atmospheric conditions. However, interest in LIBS in other atmospheric conditions has increased in recent years, especially for use in space exploration  or to improve resolution for isotopic signatures. This review focuses on what has been reported about the performance of LIBS in reduced pressure environments as well as in various gases other than air. However, interest in LIBS under other atmospheric conditions has been a growing area of study both for fundamental knowledge and challenging applications.Laser-induced breakdown spectroscopy (LIBS) is a popular technique because of its speed, simplicity, and usually inexpensive hardware. Additionally, LIBS requires little or no sample preparation and can provide simultaneous multi-element analysis. Thus, it is not surprising that LIBS has been used for a wide variety of applications, such as material analysis , environ2, has been explored primarily for geological characterization [LIBS in pressures and atmospheric compositions other than Earth ambient has gained interest as LIBS has been promoted for space exploration applications –16. Chemrization –26. Currrization ,28, whicrization . Determirization –33. Besirization .2, Ar, and CO2, which may also report results incorporating pressure dependent experiments. Studies examining the effect of pressure conditions on LIBS of ambient gas have been investigated [While there have been several studies of LIBS under non-Earth ambient conditions, none of the studies currently available are comprehensive. Therefore, this review focuses on compiling an understanding of LIBS phenomena that have been gained through the various pressure dependence and atmospheric composition studies. The pressure studies have been divided into two regimes: >760 Torr and <760 Torr. The gas composition studies include comparisons of air, He, Nstigated , however2.2.1.Performing LIBS on a surface at reduced pressures (pressures below atmosphere) can result in enhanced spectra and improved ablation. Specifically, these enhancements are an increase in spectral intensity, spectra signal-to-noise (S/N), spectra resolution, increased ablation, and more uniformed ablation craters. These enhancements are generally seen when using both femtosecond and nanosecond lasers; however, the explanation for these enhancements vary slightly for the two laser’s pulsewidths.−5 Torr. Though the intensity of the LIBS spectrum taken at vacuum is less intense than the LIBS spectrum taken at atmospheric condition, it is clear that the LIBS spectrum at vacuum is of higher resolution [et al. [In demonstrating the efficacy of a fiber optic feed-through design for integration of LIBS in a vacuum environment, Cowpe and Pilkington  producedsolution ,38. This [et al. .et al. performed LIBS at varying reduced pressures on a hematite sample [et al. comparing LIBS at lunar simulated condition of 5 × 10−5 Pa (∼10−7 Torr) and atmospheric conditions [et al. also observe a significant decrease in spectral intensity of ionic species between 7 and 5 Torr, suggesting a rapid decrease in electron density [et al. analyzed the LIBS spectra using a non-gated spectrometer. It is well known that a gated spectrometer yields higher quality LIBS spectra if the time is optimized; however, Dreyer et al. chose a non-gated spectrometer to remove some bias introduced in gated schemes when timing is optimized for one condition and is then carried through for all other conditions, which is a conundrum that other researchers have observed and dealt with in various ways.Using a Nd:YLF laser with a 10 ns pulse duration, Dreyer e sample . Figure nditions . Dreyer  density . Dreyer et al. [−2, Fe showed a 2.2-fold increase in ablation rate in 0.75 Torr Ar compared to 750 Torr Ar atmosphere. A similar result was seen on ablation of Zn. With a laser fluence of approximately 3 J/cm−2, the Zn ablation rate was 3.7-fold greater in 0.75 Torr Ar compared to 750 Torr Ar atmosphere. It was also noted by Vadillo et al. that the crater rims were free of deposited material after ablation at 0.75 Torr, while ablation at 750 Torr left craters with a visible ring of deposited material [et al., the 498.17 nm emission line from Ti(I) was monitored during laser ablation studies [et al. [et al. noted reduced LIBS intensity after 10 to 20 shots at the same location [In addition to having an effect on the emission intensity and resolution, reduced pressures have also been shown to effect ablation from LIBS significantly. For example, in work performed by Vadillo et al. , the ablmaterial . In othe studies . It was  [et al.  made the [et al. –44. Dreylocation .et al. reported an S/N enhancement at 4 Torr compared to atmospheric condition of 240-, 840-, and 629-fold for 0, 85, and 200 ns delays, respectively. It was also observed that a Mg(I) line at 383.8 nm, which was nearly unresolved at atmospheric condition, is easily resolved at 4 Torr. As pressure is reduced further, much of the enhancement seen at 4 Torr is lost, which can be explained by et al. examined 2D plasma images of a Cu plasma at 760, 1.79, 0.85, and 0.167 Torr. et al. explains that this expansion causes a decrease in collision excitation, resulting in a dimmer  plasma. The enhancement seen at 4 Torr compared to atmosphere is likely a result of reduced cooling of the plasma at low pressures. Yalcin and co-workers showed that the lifetime of a LIBS plasma created at 4 Torr is much greater than that of a LIBS plasma created at atmospheric condition. Because of this increased lifetime, the LIBS plasma emission is stronger for a longer period of time, which results in enhancement. There was no significant difference in ablation at 4 Torr and atmospheric pressure, which may be unique to LIBS plasma generated with femtosecond lasers [Yalcin and co-workers  investigd lasers .Whether a femtosecond or nanoscecond laser is employed in surface LIBS experiments, there are some advantages in lowering the surrounding pressure. These advantages are higher spectral resolution, greater S/N, and increased spectral intensity. For ablation, it appears that only lasers with nanosecond pulse lengths can take advantage of lower pressures. The majority of photons from femtosecond lasers reach the surface before the laser plasma develops, while with the nanosecond pulse lasers, a significant portion of the photons are able to interact with the expanding plume. At reduced pressures, a plasma generated with a nanosecond pulse is expanding in a less dense atmosphere, which results in a less dense shock wave. The reduced density in the shock wave results in reduced plasma shielding; thus, allowing more photons to reach the sample. Increasing the number of photon interacting with surface results in increased sample ablation, which can also lead to a more intense spectrum. Because there is little plasma shielding during LIBS using a femtosecond laser, another explanation must be considered for the spectral enhancement observed at reduced pressures. During LIBS plasma expansion, energy is lost to the surrounding atmosphere. This loss of energy reduces the lifetime of the laser plasma. Therefore, reducing the pressure increases the lifetime of the plasma, allowing for more light from the laser plasma to be collected. If pressures are too low (<∼7 Torr), there is a steep loss in LIBS spectral intensity. This loss in intensity is likely due to a disordered plasma, which results from the lack of sufficient atmosphere to provide adequate confinement.2.2.et al. investigated the effect of background high-pressures on LIBS of Basalt rock to examine the potential use of LIBS for future Venus missions [5 Torr, respectively). et al. [LIBS at high pressure has been primarily investigated for applications. For example, Arp missions . Figure . et al.  noted thet al. studied the influence of increasing atmospheric pressure (1 to 80 atm) of N2 and He on LIBS spectra of carbon [2 atmosphere at 1 atm. Vors et al. found that LIBS spectral intensity decreases with increasing pressures when either He or N2 are used. Interestingly, LIBS spectral intensity decreased more rapidly with increasing He atmospheric pressure than with increasing N2 pressure, which can be seen when comparing et al. argues that this more rapid decrease in LIBS spectral intensity is likely due to the greater thermal conductivity of He compared to N2. The higher the thermal conductivity, the more rapidly a LIBS plasma will cool, which leads to a shorter plasma lifetime.Vors f carbon . The higure 1 to 0 atm of et al. [In a study examining Al(II) at 281.6 nm and Al(I) at 308.2 nm lines from LIBS, Owens and Majidi  observedet al.  where th3.versus atmospheric composition can be seen in et al. are likely a result of a combination of lower plasma temperatures and lower electron densities. Iida also studied laser vaporization rates in He and Ar. Sensitive sample mass measurements, made during LIBS under atmospheres ranging from just under 10 to 1,000 Torr, showed a significant decrease in vaporization rates for Ar with increasing pressure. Helium experiments showed only a modest decrease in vaporization rates from 10 to 1,000 Torr. The above observations, explained by Iida, are primarily related to plasma shielding and how different gases effect plasma shielding. Laser plasmas developing rapidly with a sufficient amount of electrons can absorb a significant portion of the laser pulse through inverse Bremsstahlung. Ar is more easily ionized than He and the breakdown threshold in gas for He at 1 atm is ∼3 times greater than Ar and ∼5 times greater at 100 Torr [Iida  conducte100 Torr . Iida noet al. compared the effect that 1 atmosphere of Ar, He, and air had on plasma temperature and electron densities during LIBS of steel [et al. [et al. [Aguilera of steel . It was  [et al.  and Lee  [et al. , the reaet al. [et al. argue that plasma shielding is likely the dominant mechanism for the improvement seen in ablation using an atmosphere of He compared to Ar. The ionization potential for He and Ar are 24.4 and 15.8 eV, respectively. Under an Ar environment, it is likely that plasma shielding could be a factor because the ionization potential is lower and ionization cross section greater than the ionization potential and ionization cross section for He.The composition of the background pressure has been shown to greatly influence the rate of ablation. In a study by Mao et al.  involvinet al. . Ablatioet al. [versus measurement locations above the sample surface. In et al. argues that increased electron density results in an increase in line broadening observed in Ar. et al. also made similar measurements at 100 Torr where Ar also exhibited self-absorption. Lee et al. explains the results at 100 Torr by suggesting that the atmosphere acts to dampen free expansion of propelled atoms from the sample surface. Lee et al. also notes that, due to the greater thermal conductivity, He will cool a plasma more quickly through collisions than Ar. Though not mentioned by Lee et al., it may be possible that LIBS surface plasmas generated in He atmosphere or in low pressure Ar atmosphere are not as optically thin as plasmas generated at 1 atm. Compared to Ar and atmospheric conditions, an expanding LIBS plasma is less likely to breakdown the surrounding low pressure or He atmosphere. This reduced breakdown may result in a less confined plasma, and thus a larger plasma than a plasma generated in 1 atm Ar.Lee et al.  studied 2 environment [et al. [2. From 2 atmosphere, LIBS spectra exhibit improved resolution and an increase S/N for the stronger lines in a 7 Torr CO2 atmosphere. For example, when using a gated delay spectral lines from Al (396.15 nm) and Ca (393.37 nm) both have an improvement of around 10-fold in S/N and Si (390.56 nm) has an improvement of nearly 40-fold in S/N in a 7 Torr CO2 atmosphere. The intensity is weaker in 7 Torr CO2 and some of the weaker lines seen in the atmospheric conditions are undetectable in a 7 Torr CO2 atmosphere. Some of these undetectable lines are Ti (398.97 nm) and Mn (403.31 nm). Also, the S/N for Fe (404.59 nm) at 7 Torr CO2 was about half of the S/N at atmospheric conditions. Salle et al. measured the full width at half maximum of several lines between 390 to 410 nm and the majority of the lines in this region either had no or only a modest improvement in resolutions. The scope of this study was to compare calibration curves from soil and clays at atmospheric conditions (585 Torr), Mars conditions (7 Torr CO2) and Moon/asteroid conditions (50 mTorr air). It was found that the best repeatability and linear regression was achieved at 7 Torr CO2.The potential use of LIBS on Mars has commenced many studies involving LIBS in primarily a COironment ,18–24. I [et al.  compareset al. [2 resulted in an improvement when compared with atmospheric conditions. Brennetot et al. also studied the effect of LIBS at varying CO2 pressures; at very low pressures (<3 Torr) the plasma plume was highly expanded and difficult to fully image on to fiber optic. At pressures between 3 and 15 Torr, the lifetime of Cu (515.324 nm) decreased gradually with increasing pressures. This reduction in lifetime with increasing pressure is likely do to the increase in collisions at higher pressures, resulting in more rapid cooling of the plasma. There was no mention of whether the increase in collisions at the higher pressures resulted in line broadening; however, spectra at atmospheric conditions did exhibit significant self-adsorption compared to spectra in 7 Torr CO2.In another study involving Mars atmosphere simulation, Brennetot et al.  found thet al. [2 environment. It was found that the optimum delays for LIBS at atmospheric conditions and 6 Torr CO2 were slightly different and that the intensity was always higher at atmospheric conditions. Although the maximum intensity is twice as intense at atmospheric conditions compared to 6 Torr CO2 at the respective optimum gate widths and delays, the S/N is not significantly different. This result differs from Brennetot et al. [2 compared to atmospheric conditions . This difference highlights the difficultly in comparing different LIBS experiments. Both authors note that changes in LIBS plume geometry could affect the collection of emission.Colao et al.  studied t et al. , where eet al. [238U and 235U, which are only 0.025 nm apart, in an enriched U sample. Pietsch et al. noted that to achieve lines narrow enough to separate U isotopes, LIBS must be conducted at low pressures to reduce the effects Stark broadening.LIBS requires little to no sample preparation, consumes minute quantities of material, and can be used in standoff applications. These attributes make it well suited for measurements of radioactive material. Measurements of radioactive isotope shifts require high-resolution spectroscopy; thus, LIBS is not typically used. However; Pietsch et al.  took advet al. [239Pu/240Pu isotope ratio of 49/51%, respectively. Here the expected isotope shift of 239Pu and 240Pu is 0.355 cm−1 (0.0125 nm), and is fairly well resolved in the LIBS spectrum. Smith et al. used a much higher atmospheric pressure (∼100 Torr) compared to the conditions Pietsch et al. used (∼0.02 Torr) and explained that the higher pressure should cool the plasma more quickly leading to reduced Doppler and Stark broadening. Due to the high ionization potential of He, a pressure of 100 Torr would likely not have the same adverse effects associated with other gases, such as N2 or Ar, which would breakdown more easily from the expanding plasma shock wave and shield the sample.In another study examining LIBS isotope measurements, Smith et al.  used a 14.et al. [et al. [et al. [et al. [Not all of the reviewed studies corroborate well with each other, in fact. the cited work by Vadillo et al.  seems toet al. ,39,45,49 [et al.  noted th [et al.  and Cola [et al. . Also, t [et al. –44.Although there is no definitive study that fully accounts for all of the phenomena that occur in LIBS as the pressure and atmospheric compositions are changed, the work of Iida  does proBased on this review, it is clear that reduced pressures (<760 Torr) tend to improve LIBS spectra by increasing the S/N and improving resolution. The observed improvement is primarily due to the reduced plasma shielding, resulting in more ablation, and less Stark broadening. However, if the pressures are reduced too much (<10 Torr), then LIBS spectra tend to degrade, primarily because of lack of plasma confinement. Due to its high ionization potential, He may be useful in improving LIBS spectra when pressures cannot be reduced. The more the nuances of what occurs in the LIBS plasma are understood, the easier it will be to optimize LIBS for extreme environments and applications requiring high resolution and S/N.


Papio hamadryas), geladas (Theropithecus gelada), snub-nosed monkeys (Rhinopithecus spp.), and proboscis monkeys  , the different functional units of the society are clearly segregated in multilevel species (Swedell and Plummer . et al.  attempt  (et al.  rely on  (et al.  rely on n et al.  describeet al. Given the tremendous variety of habitats in which multilevel sociality has evolved, pinning it down to a universal environmental driver is utopian. Environmental features can constrain the evolution of modularity (Grueter and van Schaik cf. McGrew et al. (Studying the nuances of multilevel sociality in primates also has potentially important implications for hominin behavioral evolution. Chimpanzees may well be an apt referential model that helps us to characterize the social system of the last common ancestor (w et al.  attempt et al. Mandrillus leucophaeus: Astaras et al. Pygathrix spp.: Rawson Trachypithecus geei: Mukherjee and Saha Although hamadryas baboons, which are famous for their multilevel system, were among the first primate species to be studied in the wild Kummer , 1995, tet al. et al. Band formation in some multilevel species, e.g., colobines, has been argued to represent an adaptation to enhanced threat from conspecifics (Grueter and van Schaik et al. et al. et al. It is obvious that our knowledge of these intriguing societies is still limited. Unfortunately, most species showing multilevel sociality are endangered, and pristine conditions allowing the complexities of multilevel systems to unfold unhampered are becoming rare. Bands of snub-nosed monkeys often occur in fragmented habitats with limited or absent connectivity with neighboring bands (Grueter


Compulsive drug use and a persistent vulnerability to relapse are key features of addiction. Imaging studies have suggested that these features may result from deficits in prefrontal cortical structure and function, and thereby impaired top-down inhibitory control over limbic–striatal mechanisms of drug-seeking behaviour. We tested the hypothesis that selective damage to distinct subregions of the prefrontal cortex, or to the amygdala, after a short history of cocaine taking would: (i) result in compulsive cocaine seeking at a time when it would not usually be displayed; or (ii) facilitate relapse to drug seeking after abstinence. Rats with selective, bilateral excitotoxic lesions of the basolateral amygdala or anterior cingulate, prelimbic, infralimbic, orbitofrontal or anterior insular cortices were trained to self-administer cocaine under a seeking–taking chained schedule. Intermittent mild footshock punishment of the cocaine-seeking response was then introduced. No prefrontal cortical lesion affected the ability of rats to withhold their seeking responses. However, rats with lesions to the basolateral amygdala increased their cocaine-seeking responses under punishment and were impaired in their acquisition of conditioned fear. Following a 7-day abstinence period, rats were re-exposed to the drug-seeking environment for assessment of relapse in the absence of punishment or cocaine. Rats with prelimbic cortex lesions showed decreased seeking responses during relapse, whereas those with anterior insular cortex lesions showed an increase. Combined, these results show that acute impairment of prefrontal cortical function does not result in compulsive cocaine seeking after a short history of self-administering cocaine, but further implicates subregions of the prefrontal cortex in relapse. Following surgery, rats were housed individually, and fed 20 g of food each day within 1 h of testing. Water was always freely available in the home cage. The experimental procedures were conducted in accordance with the United Kingdom 1986 Animals (Scientific Procedures) Act (project licence PPL 80/2234).m quinolinic acid  in 0.1 m phosphate buffer (pH 7.2–7.4) via a 30-gauge stainless steel cannula connected by fine-bore polythene tubing  to a Hamilton precision microsyringe mounted in an infusion pump . The stereotaxic coordinates for each targeted area are shown in Table S1. All dorsal–ventral coordinates were taken from dura, and anterior–posterior coordinates were measured from bregma. Each infusion of 0.4–0.6 μL, depending on the structure targeted (Table S1), was made over 3 min, and the cannulae were left in place for a further 3 min. Rats receiving sham surgery were injected identically with phosphate buffer vehicle.The rats were anaesthetized with Avertin . All rats were implanted with a catheter  in the right jugular vein targeting the left vena cava. The mesh end of the catheter was sutured subcutaneously on the dorsum between the scapulae. Rats were then placed in a stereotaxic frame  with the incisor bar set at 3.3 mm below the interaural line  to prevent infection  equipped with two 4-cm-wide retractable levers that were mounted in one sidewall 12 cm apart and 8 cm from the grid floor. Above each lever there was a cue light , and a house light  was located on the opposite wall. A dipper delivered 0.04 mL of a 20% (w/v) sucrose solution to a recessed magazine (3.8 cm high and 3.8 cm wide and 5.5 cm from the grid floor) situated between the levers. Entry into this magazine was detected by the interruption of an infrared photo-beam. The floor of the chamber was covered with a metal grid with bars separated by 1 cm and connected to a shock generator and scrambler (Med Associates), which delivered 0.5-mA footshocks. The grid was located 8 cm above an empty tray. The testing chamber was placed within a sound-attenuating and light-attenuating enclosure equipped with a ventilation fan that also screened external noise. Silastic tubing shielded with a metal spring extended from each rat's intravenous catheter to a liquid swivel  mounted on an arm fixed outside the operant conditioning chamber. Tygon tubing extended from the swivel to a Razel infusion pump  located outside the outer enclosure. The operant conditioning chambers were controlled by software written in C++ with the Whisker control system . Responding was reinforced under a fixed ratio (FR) 1 schedule. Each lever press produced a 0.25-mg cocaine infusion (0.1 mL/5 s) accompanied by the withdrawal of the taking lever, the extinction of the house light, and the illumination of the stimulus light above the lever for 20 s. The sessions terminated after 30 cocaine infusions or 2 h, depending on which criterion was met first. Training of the taking response continued for five to seven sessions.Following acquisition of the taking schedule, the seeking–taking chain schedule was introduced, each cycle beginning with insertion of the drug-seeking lever with the taking lever retracted; the first press on the seeking lever initiated a random interval (RI) schedule on the seeking lever. The RI parameter was progressively increased from 2 to 120 s. The first lever press after the RI had elapsed terminated the first link of the chain, resulting in the retraction of the seeking lever and insertion of the taking lever to initiate the second link. One press on the taking lever was followed by the drug infusion accompanied by the same stimulus events as during the training of the taking response. There was then a time-out (TO) period, which was progressively increased across sessions from 20 s to 10 min after each cocaine infusion. The seeking lever was then reinserted to start the next ‘cycle’ of the schedule. Consequently, at the end of five training sessions, the rats were responding on a heterogeneous chained  FR 1 TO schedule, allowing a maximum of 11 cocaine infusions.To investigate whether the incentive value of cocaine was affected by lesions of the different PFC subregions or the BLA, we assessed responding on the taking lever under a progressive ratio schedule. The ratio requirement of the taking response was increased after each reinforcer according to the following progression: 1, 3, 6, 9, 12, 17, 24, 32, 42, 56, 73, 95, 124, 161, 208, 268, 346, 445, 573, and 737  delivered under a VI schedule, which progressively increased (from 2 and 15 s) to 60 s on the third day. This last day was considered to be the first day of baseline. Responding for sucrose allowed for the assessment of general suppression of appetitive behaviour by, and thus the specificity of the effects of, the subsequent introduction of intermittent punishment of the cocaine-seeking response.et al., Three further sessions of training under the seeking–taking chain with concurrent sucrose reinforcement established a baseline in which performance during the seeking link of the chain is monotonically related to the cocaine dose under the TO conditions of the session , a clicker was presented for 1 min, which ended with the delivery of a single footshock . One minute later, the rat was returned to its home cage. After 24 h, the rat was returned to the conditioning chamber for a 20-min test session, during which the clicker was alternately switched on (1 min) and off (1 min). Freezing was scored every 5 s during the test session.m phosphate-buffered saline followed by 4% (w/v) paraformaldehyde in phosphate-buffered saline. Their brains were removed and postfixed in paraformaldehyde before being dehydrated in 20% (w/v) sucrose; they were then sectioned coronally at a thickness of 60 μm on a freezing microtome, and every third section was mounted and allowed to dry. Sections were passed through a series of ethanol solutions of descending concentration , and stained for 5 min with Cresyl Violet . After staining, sections were rinsed in water and 70% ethanol before being differentiated in 95% ethanol. Finally, they were dehydrated and delipidified in 100% ethanol and Histoclear  before being cover-slipped and allowed to dry. The sections were used to verify lesion placement and assess the extent of quinolinate-induced neuronal loss and gliosis.At the end of the experiment, rats were deeply anaesthetized with intraperitoneal Euthatal  and perfused transcardially with 0.01 anovas were conducted for the progressive ratio, extinction probe test, and baseline seeking–taking motivational measures. Seeking under punishment was analysed with a mixed two-way anova with group as the between-subjects measure and session as the within-subject measure. Seeking at reinstatement was assessed with a between-groups one-way anova. Significance was set at α = 0.05. Significant interactions were analysed further with protected Fisher's LSD post hoc analyses.Between-subjects F5,133 < 2.1).After training on the seeking–taking task, and before introduction of the punishment contingency, the effects of the PFC and BLA lesions on the motivation to respond for cocaine were measured. The multiple analysis of variance performed on the break points under the progressive ratio schedule , on seeking performance during the non-reinforced seeking probe test  or during baseline responding  and actually increased their cocaine-seeking responses during the punishment sessions, thereby showing evidence of compulsive behaviour (P < 0.001) , whereas BLA-lesioned rats showed increased responding (P < 0.05) after both cocaine and shock as compared with that seen during the first cycle, and thus no sign of suppression across the session .All rats with ACC, prelimbic cortex (PLC), infralimbic cortex (ILC), OFC or anterior IC (AIC) prefrontal lesions suppressed their seeking responses to the same extent as controls, i.e. abstained from responding. In contrast, rats with lesions to the BLA both maintained  for 2–7 days (P < 0.05), but this soon returned to baseline. However, BLA-lesioned and ILC-lesioned rats differed from their respective sham controls  by maintaining stable responding for sucrose across baseline and the early phase of the intermittent punishment contingency .The majority of rats showed an initial decrease in concomitant responding for sucrose following introduction of the punishment contingency  , and there was a similar trend for ACC-lesioned rats (P = 0.16). In contrast, PLC-lesioned rats decreased their seeking responses (P = 0.03), similar to the trend observed in rats with OFC or ILC lesions (P > 0.23).Prefrontal cortical lesions differentially affected the reinstatement of drug-seeking responses after the return of rats to the test environment  .Given the marked increase in cocaine seeking under punishment in the BLA-lesioned rats, they and their respective sham controls were subjected to fear conditioning to assess the extent to which reduction in fear evoked by shock was a factor in the persistent seeking of cocaine. BLA-lesioned rats showed a reduction in conditioned fear, as expressed by a significant decrease in freezing in response to the conditioned stimulus previously associated with footshock as compared with sham controls (Student's Histological assessment was performed after completion of the experiment, and was conducted blind to the behavioural results. The sites and extent of lesions are illustrated in Fig. et al., et al., et al., et al., et al., et al., et al., et al., In this study, we tested the hypothesis that prefrontal cortical or limbic cortical loss of function plays a causal role in the development of compulsive cocaine seeking, as suggested by correlations between reduced grey matter volume or reduced metabolic activity and addiction to cocaine in human populations (Volkow The results showed that pre-cocaine exposure lesions of the ACC, PLC, ILC, OFC or AIC did not result in compulsive cocaine seeking, as measured by its resistance to punishment. Lesions of the BLA, however, resulted in both resistance to punishment-induced suppression and an increase in cocaine seeking, but the characteristics of this apparent compulsive cocaine seeking were somewhat different from that seen in the subpopulation of intact rats in which this behaviour has emerged over a long cocaine self-administration history. By contrast, there was evidence of opposing influences of the AIC and PLC on the reinstatement of cocaine seeking after abstinence; rats with AIC lesions showed an enhanced propensity to relapse, whereas those with PLC lesions showed a reduced propensity to relapse. The results are consistent with the notion of top-down inhibitory control by the PFC of maladaptive drug-seeking behaviour during relapse.et al., et al., et al., et al., Lesions of the IC are known to reduce aversive (Bermudez-Rattoni & McGaugh, et al., et al., Lesions of the PLC, in contrast, decreased the propensity to relapse when abstinent rats were returned to the drug-seeking environment. These results therefore indicate opposing influences of the PLC and AIC on relapse following punishment-induced abstinence. Opposing effects of ILC and ACC lesions on relapse following extinction have also been demonstrated, with functional inactivation of the ILC favouring, and inactivation of the ACC preventing, reinstatement (Peters et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al.,The lack of effect of PFC lesions on the tendency to maintain or even increase cocaine seeking under intermittent punishment conditions is perhaps surprising, particularly in the case of OFC lesions. In rats, pre-training lesions of the PFC similar to those used here result in perseverative responding, a form of compulsive behaviour, in a reversal learning task (Schoenbaum et al., et al., et al., et al., et al., The lack of effect of the various prefrontal lesions on compulsive cocaine seeking clearly demonstrates that acute loss of function of the PFC is not, in itself, sufficient to result in compulsive cocaine seeking. This could reflect the fact that acute, pre-cocaine self-administration lesions do not capture the subtle nature of either pre-existing reductions in grey matter volume reflecting a vulnerable endophenotype (Ersche et al., et al., et al., et al., et al., In addition, decreased dopaminergic and serotoninergic activity in the PFC has been reported in drug-addicted individuals Wilson,  and in met al., et al., Using identical behavioural procedures to those used here, we have previously shown that the noradrenaline reuptake inhibitor atomoxetine also differentially affects responding under intermittent, unpredictable punishment and responding during relapse. Whereas acute systemic atomoxetine administration prevented relapse after voluntary abstinence (Economidou et al., et al., et al., et al. (et al., et al., et al., Rats with lesions of the BLA not only continued to respond despite the punishment contingency, but actually showed a marked increase in cocaine seeking above the pre-punishment baseline. This increase is unlikely to be attributable to baseline drift, as performance stabilized across the four baseline sessions, and previous data have clearly shown that non-punished animals do not increase their performance over time (Pelloux , et al.  and empl et al., . Howeveret al., et al., et al., et al., However, although these data suggest that the persistence of cocaine seeking under punishment may reflect amygdala dysfunction, a general loss of fear does not provide an explanation for the compulsive cocaine seeking that emerges over a long period of cocaine self-administration, as we have shown that this can occur without any deficit in fear conditioning (Vanderschuren & Everitt, In conclusion, the results of these experiments show that lesions of the BLA, but not of prefrontal cortical areas, can markedly increase the propensity to seek cocaine under punishment after a short cocaine-taking history, but that this effect does not capture the mechanisms underlying the emergence of compulsive cocaine seeking after a prolonged or escalated cocaine-taking history. Prefrontal cortical lesions that were without effect on compulsive cocaine seeking nevertheless influenced relapse after abstinence, with PLC lesions preventing and AIC lesions increasing relapse. These findings further indicate inhibitory control mechanisms that are dissociable from those underlying relapse in extinction–reinstatement models of relapse behaviour.


Dear Editor,I read with great interest the contribution “Effect of Gabapentin on Morphine Consumption and Pain after Surgical Debridement of Burn Wounds: A Double-Blind Randomized Clinical Trial Study” by Raziet al. . Carryinrandomized controlled trials (RCT), the design and description should adhere to the Consolidated Standards of Reporting Clinical Trials’ (CONSORT) statement. Lastly, we would also like to draw the authors’ attention to a similar study addressing the efficacy of Gabapentin in post-surgical patients conducted by Dierking et al. (Firstly, the authors have not mentioned how they randomized their samples. Moreover, the process of blinding was not explained in detail other than mentioning the envelope method. In this regard, it would be advised that for g et al.  publishe


Giardia duodenalis is a flagellate parasite which has been considered the most common protozoa infecting human worldwide. Molecular characterization of G. duodenalis isolates have revealed the existence of eight groups (Assemblage A to H) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals.G. duodenalis among Orang Asli in Malaysia. Stool samples were collected from 611 individuals aged between 2 and 74 years old of whom 266 were males and 345 were females. Socioeconomic data were collected through a pre-tested questionnaire. All stool samples were processed with formalin-ether sedimentation and Wheatley’s trichrome staining techniques for the primary identification of G. duodenalis. Molecular identification was carried out by the amplification of a triosephosphate isomerase gene using nested-PCR assay.This cross-sectional study was conducted to identify assemblage’s related risk factors of i.e. dogs and cats  was found to be significant predictor for assemblage A. On the other hand, there were three significant risk factors caused by assemblage B: (i) children ≤15 years old , (ii) consuming raw vegetables  and (iii) the presence of other family members infected with giardiasis .Sixty-two samples (10.2%) were identified as assemblage A and 36 (5.9%) were assemblage B. Risk analysis based on the detected assemblages using univariate and logistic regression analyses identified subjects who have close contact with household pets G. duodenalis infection among Orang Asli was caused by both assemblages with significant high prevalence of assemblage A. Therefore, taking precaution after having contact with household pets and their stool, screening and treating infected individuals, awareness on the importance of good health practices and washing vegetables are the practical intervention ways in preventing giardiasis in Orang Asli community.The present study highlighted that  Giardia is a genus of intestinal flagellates that infects a wide range of vertebrate hosts. The genus currently comprises six species, namely Giardia agilis in amphibians, Giardia ardeae and Giardia psittaci in birds, Giardia microti and Giardia muris in rodents and Giardia duodenalis in mammals. These species are distinguished on the basis of the morphology and ultrastructure of their trophozoite . Subjects who participated in this study comprised 266 (43.5%) males and 345 (56.5%) females.More than half (68%) of the parents have low level of education i.e., less than 6 years of formal education. The majority of the parents did odd jobs such as selling forest products without any stable income. Some were daily wage earners working in rubber or palm oil plantations, unskilled labourers in factories or construction sites. Therefore, 51.6% of the households belonged to people who earned less than RM500 per month (≤US$156.02), the poverty income threshold in Malaysia  which is inadequate to maintain a good living standard. Although 61.9% if the houses have provision of basic infrastructure such as treated water supply and 71.4% have pour flush toilet, at least 38.1% are still using untreated water originating from a nearby river for their domestic needs and 28.6% still defecate indiscriminately in the river or bush. More than half of the households (55.8%) kept dogs and cats as their pets. Most of these pets are left to roam freely. The villagers have very close contact with the dogs and cats. Occasionally, these pets also slept, defecated indoors and accompanied the villagers into the forest to harvest forest products.G. duodenalis assemblage A and assemblage B, respectively. The prevalence of G. duodenalis assemblages A and B infections were not significantly associated with gender. However, the prevalence of G. duodenalis assemblage B infection was significantly higher in the younger age group of less than 15 years (P = 0.021).Table G. dudoenalis assemblages A and B infections and sociodemographic characteristics are shown in Table P = 0.042) and close contact with household pets  were significantly associated with G. duodenalis assemblage A infection. On the other hand, G. duodenalis assemblage B infection was associated with five factors which include children less than 15 years old , consuming raw vegetables , eating fresh fruits , non working mother  and the presence of other family members infected with giardiasis .The association of P = 0.002) more likely to be infected with G. duodenalis assemblage A as compared to those who do not keep dogs and cats as their pets. In addition, children less than 15 years old , those being a consumer of raw vegetables  and presence of other family members infected with giardiasis  were more likely to be infected with G. duodenalis assemblage B , AF069557 (assemblage A), L02120 (assemblage A) and U57897 (assemblage A)] in one cluster with high bootstrap support. Phylogenetic analysis confirmed the monophyletic group of assemblage B (bootstrap = 100%)  were successfully amplified based on analysis targeting Giardia duodenalis from human and various animals are morphologically similar, distinct host-adapted genotypes have been demonstrated within G. duodenalis[G. duodenalis known as assemblages A and B. Both assemblages are found associated with human infection globally and have also been detected in various animals. In this study, the results showed that all G. duodenalis infections in Orang Asli are due to assemblage A and assemblage B. This confirmed the results of a several local studies performed elsewhere [Molecular tools have been recently used to characterize the epidemiology of human giardiasis. Although isolates of uodenalis,30. Humalsewhere ,19.G. duodenalis assemblages or in Giardia species). In the course of this study, triosephosphate isomerase (tpi) gene was specially chosen because of the high genetic heterogeneity displayed by Giardia species at this locus, as depicted by Thompson and Monis [et al.[tpi gene achieved the highest percentage of amplicons produced (70%), followed by glutamate dehydrogenase (gdh) (45%) and beta-giardin (bg) (33%). Similar occurrences were also reported in previous studies [et al.[tpi gene is a good phylogenetic marker for analysis of the molecular evolutionary and taxanomic relationship of G. duodenalis.At present, various molecular methods are available to distinguish these assemblages, mainly by nested-PCR followed by DNA sequencing or restriction fragment length polymorphism (RFLP), or by real-time PCR . The majnd Monis . A recens [et al. reported studies ,33. Furts [et al., the tpiG. duodenalis assemblages varied in different geographical areas. In the present study, sequences analysis of the 98 samples recovered from Orang Asli revealed 10.2% (62/611) G. duodenalis assemblage A and 5.9% (36/611) assemblage B, which were differs from previous local studies carried out by Mohammed Mahdy et al.[et al.[et al.[et al.[et al.[The distribution of dy et al. and Hueyl.[et al.. In theil.[et al. which oband 5.9% /611 assel.[et al.. Similarl.[et al.. Souza el.[et al. indicatel.[et al. observedet al.[et al.[et al.[et al.[et al.[G. duodenalis assemblage B infecting humans in the city of Rio de Janeiro, Brazil. Results from each of these studies are not strictly comparable since amplifications were done on different G. duodenalis genes. Differences of G. duodenalis assemblage among the studied populations could be due to different modes of transmission in each area, comprising human-to-human, foodborne, waterborne or zoonotic transmissions.In contrast, a high prevalence rate of assemblage B was reported by Hatam-Nahavandi et al.. Likewisl.[et al. in Thaill.[et al. observedl.[et al. indicatel.[et al. did not G. duodenalis assemblages A and B infections between genders. Similar findings were observed by Gelanew et al.[et al.[et al.[Results of this study indicate a non-significant difference in the prevalence of ew et al. and Anthl.[et al. which fol.[et al. demonstret al.[et al.[G. duodenalis assemblages show age-specific pattern. Compared to previous studies carried out in Ethiopia [Interestingly, this study showed that children age less than 15 years were at higher risk of being infected with assemblage B. This finding was in agreement with Mohammed Mahdy et al. and Sadel.[et al. that addl.[et al.,34,50. TEthiopia , PhilippEthiopia , it seemG. duodenalis assemblage B in the present study. The most common vegetables consumed in Orang Asli were tapioca shoots, wild fern shoots and locally planted leaves. It was believed that this association was due to eating these vegetables with contaminated hands or hands that were insufficiently washed. Contaminated hands have been implicated to play a major role in the faecal-oral transmission of the communicable faecal-oral transmitted diseases in developing countries [Giardia cysts [Foodborne transmission of giardiasis was suggested in 1920s ,53 and aountries ,56. Otheountries ,58. Vegeia cysts ,60, althia cysts .G. duodenalis assemblage B was predominantly human and to a much lesser extent dog and wildlife. The present finding suggests that humans are the major source of assemblage B and it indicates the possibility of infected family members as the source of infection and direct transmission occurring within the household. It has also been demonstrated in Bangkok where human-to-human transmission of assemblage B was found among humans in temple communities [et al.[G. duodenalis assemblage B was high in children younger than 15 years old. Furthermore, the prevalence of G. duodenalis assemblage B which indicates an anthroponotic transmission cycle has been seen in other countries in Asia [The host distribution of munities . Likewiss [et al. suggeste in Asia  and the  in Asia .G. duodenalis is still under debate and despite increasing knowledge of the molecular identification of Giardia from different host species; the zoonotic potential of G. duodenalis is not clear [G. duodenalis assemblage A. We considered household pets as dogs and cats that were kept contained in residence area (house and/or yard) for at least 12 h a day and allowed in the streets part of the day, either alone or accompanied by their owners. The role of dogs and cats as a definitive G. duodenalis host has been widely studied and recognized as being a public health problem, especially in developing countries and communities that were socioeconomically disadvantages as the one used in this study. In these communities, poor levels of hygiene and overcrowding, together with a lack of veterinary attention and zoonotic awareness, exacerbates the risk of giardiasis transmission [Giardia in dogs and humans living within the same household, although it would appear that the risk of dog-human transmission was low.Zoonotic transmission of ot clear ,67. Howesmission . FurtherGiardia positive samples collected from dogs in urban areas, 60% were infected with zoonotic Giardia from assemblage A, 12% with dog specific assemblages C and D and the remaining 28% harboured mixed infections [et al.[Giardia, six infected with assemblage A and 11 with assemblage F (the cat genotype). Based on these findings, we believe that there are two transmission cycles in dogs; (i) the normal cycles between dogs which involves transmission of G. duodenalis cysts that belong to assemblages C or D and (ii) the other cycler includes cross transmission of Giardia from humans belonging to assemblage A [G. duodenalis reservoir and transmit cysts in at least two ways which are from dog-to-dog and from humans-to-dogs and perhaps from dogs-to-humans.In a recent report from Germany, it was found that of 60 fections . Few stus [et al. examinedmblage A  that groG. duodenalis assemblages A and B were not detected in the current study. Similarly, Bertrand et al.[Giardia cysts of different genetic profiles or subsequent infection of an infected host by genetically different Giardia cysts. This was especially common in areas where giardiasis was endemic [It is interesting to note that mixed infections with nd et al. also didnd et al., Italy [nd et al., India [nd et al. and Unitnd et al.. The per endemic ,76,77.i.e. lipids, haemoglobin, bile salts and polysaccharides from mucus, bacteria and food degradation product) which can affect the result of amplification. For this reason, some extraction and amplification methods have been improved to develop more sensitive assays to identify gene. In some studies, specific DNA was detected at all target concentrations, demonstrating that QIAamp DNA kit extraction method could effectively remove PCR inhibitory substances [G. duodenalis up to sub-assemblages [G. duodenalis assemblage A isolates have been further grouped into sub-assemblages I and II, whereby the assemblage B isolates have been divided into sub-assemblages III and IV [Giardia isolate since different methods can group isolates into different assemblages and the resolution of sub-assemblages is dependent on the selected method.The present study however has several limitations. Firstly, direct amplification of cysts DNA from stool samples help to sole important questions such as presence of mixed infections, association between assemblages and host (pathogenicity) and selection for irrelevant genotypes during cultivation ,78,79. Bbstances ,81. Secoemblages . The G. I and IV ,84. ThisG. duodenalis assemblage is a useful way to understand the dynamic transmission of Giardia infection in Orang Asli. In the base of our findings, an anthroponotic origin of the infection route is suggested and underscored the fact that human are the main source of infection for assemblage B while close contact with domestic animals played a major role in the transmission for assemblage A. Because of the possibility of zoonotic transmission and the potential of household pets for hosting the parasite suggested by some researchers, further studies with a variety species of animal stool samples are recommended. Further studies using additional, more highly variable loci will provide more definitive evidence of both anthroponotic and zoonotic transmission in this community.In conclusion, determination of the The authors hereby declare that they have no competing interests.TSA was involved in all phases of the study, including study design, data collection, data analysis and write up of the manuscript; NM supervised the study, and revised the manuscript; NM was involved in the statistical analysis of data; FMS and SNA were involved in the collection and laboratory examination of samples. All authors read and approved the final manuscript. TSA and NM are the guarantors of the paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/78/prepub