Proposed in the early 1970’s, the Gaia hypothesis suggests that our planet earth has a self-regulating ability to maintain a stable condition for life. Tropical cyclone (TC) is one of the earth’s most hazardous disasters; it is intriguing to explore whether ‘Gaia-like’ processes may exist in nature to regulate TC activities. El Niño can shift the forming position of the Western Pacific typhoons away from land. This shift enables typhoons to travel longer distances over ocean and is known to be a positive process to promote TCs to achieve higher intensity. What is neglected, however, is that there co-exists a negative process. Here we show that during El Niño, typhoons intensify over region undergoing strong ocean subsurface shoaling where upper ocean heat content can drop by 20–50%. This ‘worsen’ ocean pre-condition can effectively reduce ocean’s energy supply for typhoon intensification during typhoon-ocean interaction. We find this an elegant, ‘Gaia-like’ process demonstrating nature’s self-regulating ability. Though during El Niño, typhoons can take advantage of the longer travelling distance over ocean to achieve higher intensity, nature is also providing a damper to partially cancel this positive impact. Without the damper, the situation could be even worse. Known as typhoons, each year these TCs impose threats to a billion population and mega volume , and 43 ms−1 (not as intense), respectively  position to the southeast , TCs can45678However, for long the above framework neglects the fact that ocean subsurface condition is also different under El Niño. In El Niño years, strong shoaling takes place in the subsurface of the WNPO1920212223As will be shown, we demonstrate that there is an additional process from subsurface ocean taken place in the El Niño-typhoon intensity relationship. In contrary to the commonly-known positive facilitator related to the longer travelling distance, the subsurface factor is a damper which acts as brake to restrain typhoons from over-intensifying. This is found not only in the strong 1997 El Niño year, but also in the other El Niño years.Ocean is the energy source for TC intensification, thus is a very important environmental factor for TCs192023252627921222324252627282930313215161718192021222324252627This is because these more favourable pre-TC ocean conditions will later limit the TC-induced ocean cooling effect (i.e. more prohibition of subsurface cold water to be mixed to the ocean surface by the TC wind), during TC-ocean interaction1920212223192023252627−2. As compared to the normal heat content of >100 kJ cm−2  is found  and Latent Heat Flux (LHF)] from ocean for TC’s intensification19202223272829CH and CE are the sensible and latent heat exchange coefficientsW is TC wind speed, TS and Ta are the during-intensification SST  is small, it means that ocean is less warm during TC-ocean interaction. The less-warm ocean thus supply less sensible heat flux (SHF) from ocean to TC because SHF is a function of the difference between atmosphere and ocean, i.e. TS-Ta (s-qa). As qs is a function of the during-TC SST (i.e. TS), smaller LHF and (qs-qa) corresponds. This reduction in the flux supply is found not only in the 1997 El Niño, consistent results are also found in the composites of other El Niño events  than the supply if there was no shoaling (~712 Wm−2), under the same atmospheric Ta and qa input. This can be a substantial reduction in energy supply for TC intensification 1920329 Wm−1 (712 Wm−2 × 3066 km). This is a very large supply, equivalent to ~197% as compared to the normal supply of 1.127 × 109 Wm−1  flux to be supplied for intensification1920229 Wm−1 . However, if there was no subsurface damper, the supply would be even higher, i.e. going up to 1.676 × 109 Wm−1   .This research shows that nature is more complex than originally perceived in the current research framework. It appears to be similar to the Gaia hypothesis which states nature’s self-regulating ability34456789Otherwise, if the ocean was to remain the same, then given the much-longer travelling distance during El Niño, the total distance-integrated flux supply would be extremely high  as the normal supply). However, because nature provides a ‘worsen’ ocean along TC’s intensification track, a co-existing negative (damping) effect can be effectively at work to cancel a large (though not the entire) part of the positive effect. As a result, during El Niño, typhoons could still be more intense than normal, since the residual distance-integrated flux supply after offset is still higher than normal  El Niño or the ‘Modoki’, Central-Pacific (CP) El Niño11121314There are interesting issues to be explored in the future research. For instance, whether other climate modes (e.g. the Indian Ocean Dipole (IOD)) may further modify the observed process would be interesting. Though the 1997 event was one of the strongest El Niño ever observed, IOD was also active in that yearOcean Reanalysis System 4 (ORAS4) data of the European Centre for Medium-Range Weather Forecasts (ECMWF) was used. This is a monthly, one-degree grid reanalysis ocean data set. Sea Surface Temperature (SST), depth of the 26 °C isotherm (D26), Upper Ocean Heat Content , and ocean depth-temperature profiles were all obtained or derived from this data set.http://www.aviso.oceanobs.com/) was used.Monthly, delayed-mode, gridded, multi-altimetry SSHA data from the AVISO  data base (The atmospheric data was based on the monthly data from the ECMWF (European Centre for Medium-Range Weather Forecasts)’s Interim Reanalysis database at each 1.5 degree grid.−1, category 2: 44–50 m s−1, category 3: 51–59 m s−1, category 4: 59–71 m s−1, category 5: > 71 m s−1. The long-term climatological mean genesis and intensity peak (i.e. life-time maximum of a TC) locations were calculated based on the average of all western North Pacific TCs (746 cases), from August to November of each year, between 1958 and 2010. The mean intensification track was obtained through a 2-D polynomial regression from the individual intensification track locations of each TC  of the 746 cases (details see supplementary). Similarly, the mean genesis location, mean peak location, and mean intensification track was obtained for the El Niño years.TC track and intensity data was from the US Joint Typhoon Warning Center (JTWC)’s Best Track data base . TC intensity categories are defined from the Saffir–Simpson TC scale (in 1-min maximum sustained wind speed): category 1: 34–43 m sas El Niño peaks towards winter, the corresponding TC season in this study is defined as from August to November (ASON).in situ ocean subsurface observations to be included in the reanalysis data. Only from the 80’s, there were more subsurface measurements, e.g., from the TAO  array (from mid 80’s) and Argo floats (2000).8 El Niño events since 1980s  are included in this study1112131411121314−1) of the western North Pacific TCsDuring-intensification SST cooling  was esti2321Ta and qa are from the ECMWF data (point 1c). The exchange coefficients were from the CBLAST field experimentas in the main text, SHF and LHF are calculated separately from the bulk aerodynamic formula. The SST input was from the output of the 3DPWP simulation, i.e. the SST under the corresponding TC wind e.g.  during TStatistical significance test was performed based on the two-sample T-test. Uncertainty in the mean is assessed based on the 95% confidence interval (CI). Details and results see supplementary.How to cite this article: Zheng, Z.-W. et al. A Long Neglected Damper in the El Niño —Typhoon Relationship: a ‘Gaia-Like’ Process. Sci. Rep.5, 11103; doi: 10.1038/srep11103 (2015).


The Gardens of Abel – looked at changes introduced by the radical psychiatrist Franco Basaglia in an asylum in the north-east of Italy (Gorizia). The article examines the content of this programme for the first time, questions some of the claims that have been made for it, and outlines the sources used by the director, Sergio Zavoli. The article argues that the film was as much an expression of Zavoli's vision and ideas as it was linked to those of Franco Basaglia himself. Finally, the article highlights the way that this programme has become part of historical discourse and popular memory.This article examines a celebrated documentary made for Italian state TV in 1968 and transmitted in 1969 to an audience of millions. The programme –  In the 1960s, a small group of radical psychiatrists in Italy began to transform mental health care. The focus of the early movement was the town of Gorizia, in the extreme north-east of the country, right on the border with Yugoslavia, where a psychiatrist called Franco Basaglia took over as Director of the Provincial Psychiatric Hospital in 1961. In the years that followed he revolutionized the institution of which he was in charge – altering the architecture of containment, freeing up the patients and empowering them. Daily general assemblies were introduced and the patients produced their own newspaper. Basaglia was influenced by a mixture of theories and practical examples of reform – from phenomenology to the Therapeutic Communities movement in the UK to his reading of Primo Levi's work on his time in Auschwitz. He often compared psychiatric hospitals to concentration camps  – in 1968 (Colucci and Di Vittorio I giardini di Abele (TheGardens of Abel).Basaglia (and the group he worked with) began to publicize their ideas and produced a best-selling collective book – Clausura 1958), which was translated into six languages, before moving to the TV sector. For the TV critic and historian Aldo Grasso, this was a documentary ‘which was heard all around the world’ , which covered Italy's annual cycling race, the Giro d'Italia  and had worked in radio for many years, producing award-winning sound-only documentaries such as a study of a covent , edited by Franco Basaglia and his wife Franca, were crucial to the content and structure of the film. The book would appear in print in 1969, after Zavoli's programme had gone out on air  were picked up and adapted. Thus far, however, despite frequent references to Zavoli's film in the secondary literature, no critic or historian has commented on or even noticed the links between Morire di classe and I giardini di Abele.This article shows that there were strong parallels and connections between the thrust of Zavoli's programme and that photobook. Quotations and arguments from One of my arguments in this article is that the documentary that emerged from Zavoli's time in Gorizia was a blend of Basaglia's ideas and Zavoli's interpretation of what he had seen and heard there. Basaglia had become one of the leaders of the critical or radical psychiatry movement at that time  – so much so that the movement was often described as ‘Basaglian’. While the film clearly supported the changes introduced by Basaglia in Gorizia, and gave ample space to Basaglia's philosophy in an interview with him included in the documentary, the overall feel of the film was also very much linked to Zavoli's religious and political outlook – and his desire to tell a ‘universal’ and ‘poetic’ story. This was not a story that Basaglia was particularly interested in, however. Zavoli used Basaglia for his own ends, while Basaglia – at the same time – was happy for Zavoli to publicize the changes happening in Gorizia and elsewhere. The differences between the two men in terms of their ideas and outlooks have rarely, if ever, aroused any comment.everyone – at a high, spiritual level. Zavoli was interested in a collective ‘conscience’ concerning these people (the ‘mad’) and the places where they were locked and hidden away. Basaglia, on the other hand, had a much more radical and non-spiritual approach to his work. He believed that a social and political analysis of the asylum system was required. Basaglia argued that contradictions should be ‘opened up’ within the system to help to ‘overturn’ the institution of which he was Director. Zavoli watered down Basaglia's ideas, and at one point made a clear statement in opposition to some of what Basaglia was saying. In addition, the contrast between ‘open’ and ‘closed’ hospital systems drawn by Zavoli was a moderate and reformist interpretation of what Basaglia thought he was doing. Thus, while the documentary gave space to Basaglia's ideas – and often reflected them directly – its overall feel was linked to Zavoli and his long dominant voiceover, the editing, and the heavy authorial feel imposed by the director. The radical ‘Basaglian’ terms ‘overturning’ and ‘negation’ were never used in the film, despite the existence of a best-selling book with that title. Zavoli's message was intended to be universal, while Basaglia's was political, specific and linked to daily practice and activity within the hospital. The 1969 programme can only be understood if we see it as a combination, a hybrid, of Basaglia's vision (and that of his team) and  Zavoli.Crucially, Zavoli wanted to draw attention to what he saw as the ways in which the existence of the mentally ill was a problem for seen it. It has certainly not been studied. Undoubtedly, the programme marked an important moment in the history of public television and is now often seen as a kind of historical document. But we need to be very careful here. Claims for the programme cannot simply be taken at face value. Evidence and analysis are required. We also need, finally, to look at the programme itself. I now look at the documentary in detail – taking in the voiceover (written and spoken by Zavoli), the images, and the sources for the film.This article now goes on to analyse the programme itself and the claims made for it over the years. I underline the fact that, while the programme is often cited, it has never been analysed or unpicked in a serious way. It is almost as if people have watched the documentary, but they haven't actually I giardini di Abele, ‘Gorizia. I am here because of the psychiatric hospital. The madhouse – if you will.’ A TV camera is moving down a long flat road, mounted on a car. The place is an anonymous one. It could be anywhere; any city. A voiceover starts in the background. This was a well-known voice in Italy – a household name – that of Sergio Zavoli. It was 1968, and he was in Gorizia. The voiceover continued:We tend to find the mentally ill on the edge of our cities, perhaps because their presence worries us. Here I am in Gorizia, at the extreme periphery of the city: one wall of the hospital marks the border between the Italian state and that of Yugoslavia. I wanted to get to know about what is going on in this hospital because the history of this place has attracted the attention of scientists and men and women of culture from across the world, but in Italy this experiment risks being associated merely with a piece of bad news.  gates towards the asylum itself Pitrelli , 74. SooMorire di classe and had already been seen on posters and in exhibitions. Cosimo Schinaia, a historian of photography, later referred to this photo as ‘the symbol of marginalization in asylums’ (fatto di cronaca’ (‘a piece of bad news’). A patient (Giovanni Miklus) on day release from Gorizia's asylum had murdered his wife with an axe, leading to national press coverage and a local backlash against the Gorizia experiment led by Basaglia, as well as judicial proceedings against Basaglia and others . The Miklus case had possibly led to the postponement of the screening of the film, which had been shot and edited in 1968, until early 1969.The next shot was from a photo by Carla Cerati of a man holding his head in his hands: a psychiatric patient from the asylum in Colorno. This image would be reproduced time and time again by the Basaglian movement in the years to come. It would appear soon afterwards in asylums’ , 41. TheThis 27-minute made-for-TV documentary, directed, written and narrated by Zavoli with the help of a large crew was notable for its direct interviews with patients and with Basaglia himself. It was shot in stark black and white, and used music, a voiceover, various fragments of archive film (none of which was sourced or cited in the credits) and other visual effects including close-ups and slow motion. This was a made-for-TV film with cinematic qualities and a (it seems) a large budget. It portrayed patients leaving the hospital to go to work or on walks, and carried discussions about the meaning of madness itself. It also showed patients knocking down their own fences  and another one between mental health treatment and wealth (which Zavoli accepted); the voice given to the patients, a sense of resistance to change which needed to be overcome and aspects of horror and torture as features of the ‘old’ asylums. But Zavoli's film also added other, original and different elements: visual depictions of Gorizia and the patients and their voices, the voice and face of Basaglia himself, and a specific ‘Zavolian’ social and quasi-religious reading of what was going on in Gorizia and the issues the experiment there had thrown up.***             One particular exchange from Zavoli's interview with Basaglia  is often repeated and cited , the historical background , the nurse's debate (which represented the idea of a struggle against the forces of conservatism), the interview with Basaglia, a voiceover analysis over film of patients in the park, interviews with the patients, and a final march towards redemption, or perhaps to nowhere . Throughout the film, a sharp distinction was drawn between what he called the ‘closed asylum’ and this ‘open asylum’.These images were taken in this hospital. They document the reality of a closed hospital, a traditional institution. In Gorizia, that period has been rejected and filed away as part of the past – its seems now to be far away.After entering the ‘open’, ‘Basaglian’ hospital, Zavoli outlined what life was like in other asylums, and how things had been in Gorizia in the past, accompanied by archive film of patients in the courtyards of Gorizia, from the time when the hospital was ‘closed’. These clips show patients sitting, nodding repeatedly and lying down on benches. Zavoli continued:of practising new forms of violence against these people by showing without any kind of filter those faces filled with pain which are usually hidden away because of the fear of stigma which accompanies those who live in this world of the excluded, within the walls of the hospital.Yet some sections of the film were undoubtedly voyeuristic: the close-ups in the interview section, the shots of patients in the final part, the focus on falling-apart shoes and twitching feet  and drew on comparisons with concentration camps but also on a social analysis of the issues at stake. This citation was very similar to that of French psychiatrists Le Guillart and Bonnafé (1952) used on both the cover of the first book to discuss the Gorizian experience edited by Basaglia (Che cos'è la psichiatria? (What is Psychiatry?), and as one of the epigraphs inside Morire di Classe  – which described the architecture and functioning of the asylum, with locks being shut, bars, grills, a patient being held down (accompanied by dramatic music) and placed in a strait jacket, barred windows, baths in the centre of rooms, hooks with numbers on them Goffman .6 Many o***             For Zavoli, throughout the film, the key issue at stake was not to do with ‘mental illness’, but with society. Everyone was responsible for what was going on. This was not a film aimed at psychiatrists, or patients, but at Italians in general. As Babini argues, the film was not merely a report on what was going on in Gorizia, it was also concerned with people's general attitudes to mental patients and to mental illness. It was a film about the ‘sane’ as much as it was about ‘the mad’. As Babini  was then contrasted, to the sound of more hopeful and upbeat music, with archive shots of the patients themselves knocking down the fences that held them in  and then original footage of people simply leaving the hospital altogether, for a walk, seemingly unaccompanied (apart from by Zavoli's camera). These were the same gates that Zavoli had entered, by car, at the start of the film. This new, reformed, hospital, it seemed, really was ‘open’, and the patients themselves had participated in this ‘opening’.s Babini , 8 has aIn November 1962 the équipe led by Franco Basaglia opened up the first wards in the Psychiatric Hospital and Italy saw its first therapeutic community. The life of the hospital was marked by ward and general meetings. The mentally ill were given, once again, a social and human role in life. They managed their own lives, thanks to ongoing communication with those who looked after them.Once the ‘prison-like nature’ of the institution had been ‘suppressed’ by Basaglia and his équipe ‘it was time to start to understand the kind of prejudice to which the mentally ill had been subjected’ (and this is another citation that seems to pick up from Le Guillart and Bonnafé). After this historical introduction, which could have been written by Basaglia himself, and was certainly fruit of discussions with Basaglia and the équipe in 1968, we were then plunged into a meeting of nurses, some of whom were clearly opposed to the changes that had taken place in the hospital.It was then time for Zavoli to tell the story of what had happened in Gorizia in more detail. He provided his viewers with a short history lesson:appear, on the surface, to demonstrate how the patients are governing their own lives – but everything is constructed, directed and pre-prepared. The same people - more or less - take part in the debates and make speeches - and act as spokespeople for the proposals of the Leader.The nurses ‘debate’ was a way of bringing these disputes out into the open.The contrast in this constructed and somewhat artificial ‘nurse's debate’ .But some nurses had been transformed by the experience, and one of these ‘Basaglian’ nurses spoke out during the filmed ‘debate’:Extracts from a general open patient meeting would not have fitted with the rest of the film. The tone of the entire documentary is controlled and elegiac, ‘poetic’ and a reflection of the directorial voice of its author, director and narrator: Zavoli himself. It is very much a film ‘by Sergio Zavoli’, heavily directed and measured, precise, authorial, sermon-like: and this creates a certain distance from many aspects of the spontaneous chaos of the Gorizian experiment. Zavoli never lost control of his own film.***             The third act of the documentary took us into the gardens of the asylum, gardens that had been cited in the opening prologue (and in the title of the documentary itself). Classical music accompanied the camera as it swooped and floated through the gardens, in slow motion, almost hiding amongst the trees.In these vast and hospitable gardens, with their ancient beauty, we can see much of the hypocrisy with which, usually, we protect ourselves from our consciences. In these thriving gardens, on the other side of the fence, we can observe the mentally ill living their free and serene lives, but in reality these are the gardens of our inconvenient brothers – the gardens of Abel. Zavoli , 241The slow motion and music was used to prick the emotions of the audience, as patients walked around the grounds of the hospital, sometimes accompanied by nurses. For Zavoli, the whole issue of mental illness and its treatment forced us to look deep into ourselves. His programme served, he thought, to highlight this contradiction, to underline the presence ‘at the edges of our cities’ of these ‘inconvenient brothers and sisters’. By observing these people, we were forced to observe ourselves.Next came the celebrated and oft-cited interview with Basaglia, who spoke as he walked quickly, up and down, in a small office. Basaglia was youthful with short hair, dressed in a jacket and tie, surrounded by scholarly tomes and papers. Zavoli remained in the background again: as just a voice, almost as if he was directing Basaglia's movements .They were part of the reason that places like this existed, after all. He appeared to tick his viewers off. Now the voiceover became a sermon. ‘We need to free ourselves from much scepticism and just as much laziness in order to give a proper reply to this question.’Another short but dense history lesson followed, against the background of patients wandering in the gardens, or shots of the spectacular vegetation there. There was a direct quote from the 1904 law which had set up many psychiatric hospitals and (until 1968) was still in force, and strong hints at a Foucaultian analysis with a phrase about how leper colonies later became mental asylums  conclusion/punchline to the story was similar to that of Basaglia earlier in the documentary: ‘the rich are not mad, at least from a giuridical point of view, while the poor are mad’  related his experiences as a patient, and his loss of voting rights as he was interned in the ’ Zavoli , 243. ThL'istituzione negata, in which she was described as ‘One of the best known and most listened-to patients in the hospital’  described how she had been treated in the ‘closed’ asylum. Hers was a story of institutionalized violence: ‘They tied me up, they beat me, they wanted to give me electro-shock treatment and I was terrified.’ This was ‘Carla’, an important figure in the Basaglian hospital, the secretary for many of the general meetings and an Auschwitz survivor who was interviewed early on in Basaglia , 24. CarCarla seemed both strong and fragile at the same time. She claimed that she ‘didn't care’ what people thought, but she also said that, in life, she was ‘all alone’. At one point she burst into tears, and her crying was captured and highlighted by the camera, as was her smoking  was perhaps the most interesting of all. Good looking, young, blonde, fresh-faced, he was, in one sense, and certainly physically, almost the opposite of popular ideas and stereotypes about the ‘mad’ and their physical appearance. But he also had a slightly strange smile, and he shook his foot continually, something that was picked up by the camera, which zoomed in on his foot  might also have been a visual trope – taken in part from the photography of Luciano D'Alessandro  and material in a previous documentary film, perhaps I am not ill’ .Basaglia's revolution had not resolved the problem of mental illness, or of the marginalization of the poor. There was much to be done. Outside, in the world of the ‘sane’, there was another kind of ‘prison’. These in-mates were an Then came the epilogue. We now saw this ‘motley crew’ of patients, volunteers and nurses walking along a river bank, outside of the hospital's grounds, with Mahler playing in the background. One of those walking was the first interviewee. The camera zoomed in on personal details, scuffed shoes, dirty clothing  in detail – looking at its visual features and its voiceover and dialogues. The links between the photobook Morire di Classe, which came out in 1969, have been noted and commented upon. Zavoli's film presented Basaglia's theories and examples of his practical work in Gorizia, but it also refracted and adapted those ideas, and that practical work, to Zavoli's own ideas and his own message. This was a film about Basaglia and Gorizia, but it was above all a film ‘by Sergio Zavoli’. This article has presented the first detailed analysis of this well-known and oft-cited film, and the first detailed reading of its contents, as well as its context. As for the effects of the film on popular opinion, this has often been assumed in the secondary literature, but in reality was almost certainly complicated and contradictory. In many ways, it is impossible to say what the real effects of this documentary were in 1969 and since. Given this, it is important to return to the document itself and analyse what is really there – not merely what has been picked out in a largely celebratory and superficial way by most of those who have written about I giardini di Abele.This article has examined Zavoli's celebrated documentary


We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that inhibition of ER α-glucosidases prevents release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages  in vitro and in vivo efficacy of the bicyclic iminosugar, celgosivir, which we demonstrate to lack capacity for inhibition of glycosphingolipid processing.Current treatment of dengue virus infection is supportive; however, iminosugars have been widely investigated as an antiviral strategy. The means by which these molecules are thought to exert their antiviral effects is through inhibition of host-resident glycoprotein processing enzymes, the endoplasmic reticulum-resident α-glucosidases, but many iminosugars are also capable of inhibiting host glycolipid processing and are utilized clinically for the treatment of lysosomal storage disorders such as Gaucher’s and Niemann-Pick type C diseases. The work presented here is the first to conclusively differentiate the antiviral properties of these two major mechanisms of action of iminosugars, and our data support the long-standing hypothesis that inhibition of glycoprotein processing is the essential antiviral property of iminosugars in the case of dengue virus infection. These results indicate that further development of iminosugars as dengue antivirals should focus on optimization of glycoprotein inhibition efficacy with reduction or elimination of glycolipid modulating properties to minimize off-target effects. These results are supported by the  Samples were incubated for approximately 4 hours , and the absorbance at 490 nm (A490) was measured on a SpectraMax M5 microplate reader (Molecular Devices). Absorbance was normalised to untreated controls whereby cytotoxicity resulted in decreased A490.Cytotoxicity of compounds was assessed by a cell proliferation assay for metabolic activity using a CellTiter 96 AQ2). For collection of infectious virus, supernatant was harvested and centrifuged for 5 minutes  to pellet any cells/debris, and supernatants were aliquoted and stored at -80°C until analysis by plaque assay. In the case of FOS and GSL assays where MDMΦ were uninfected, treatments were essentially as above except that no virus was added and cells were washed three times with sterile PBS after 24 or 48 hours as per sample time point. On the third wash, cells were removed from tissue culture plastic by mechanical disruption (scraping) and transferred to microcentrifuge tubes for centrifugation . Cells were lysed by three cycles of freeze-thawing  in deionised H2O and stored at –80°C for subsequent protein, FOS, or GSL assay.MDMΦ were infected with DENV2 16681 diluted to a multiplicity of infection (MOI) of 1 in X-VIVO10 without supplements for 90 minutes . Upon removal of virus, fresh MDMΦ growth medium without IL-4 but containing serial dilutions of drug or control was placed on the cells, and cells were incubated for 48 hours , and virus was incubated on cells for 90 minutes . Upon removal of virus, first-overlay containing nutrients and low-melting point tissue culture-grade agarose [2). A second-overlay containing nutrients, low-melting point tissue culture-grade agarose, and neutral red stain [2) after which plaques were counted by eye. The limit of detection for this assay is 33 plaque forming units per ml (pfu/ml).Virus titres for samples from human MDMΦ and C6/36 infections were obtained by LLC-MK2 plaque assays as per previous descriptions . Briefly agarose  was addeed stain  was adde5 cells/well) in α-MEM medium containing 5% HI-FBS for 3 hours . Medium was removed, log10 serial dilutions of virus in α-MEM with 2% HI-FBS were added, and cells were incubated for an additional 2 hours . Following incubation, 1.5 ml of α-MEM containing 5% (v/v) HI-FBS and 1% (w/v) low-melting point tissue culture grade agarose (Sigma) was added to each well and allowed to solidify at room temperature before further incubation  for 5 days. Plaques were visualised after fixation in 10% formaldehyde  and removal of the agarose plug by staining briefly (approximately 30 seconds) with 1% (w/v) crystal violet (Sigma) in 20% (v/v) ethanol . Concentrations were normalised to tissue mass (in grams) or serum volume (in ml) as appropriate.Virus titers of samples isolated from infected mice were obtained by BHK-21 plaque assays in accordance with previous protocols ,34. BHK-et al. [xxx) at 595 nm and 420 nm were measured, and the ratio of A595 to A420 was used to determine protein concentration in comparison to a serially diluted standard of bovine serum albumin.MDMΦ FOS were detected as previously described by Alonzi et al. . Cells wet al. . Briefly6 cells/ml as described above and treated with appropriate dilutions of iminosugars for 48 hours at 37°C, 5% CO2. Following incubation, supernatants were removed and cells were washed twice in pre-warmed PBS before mechanical removal by scraping. Flasks were washed once with warm PBS, and this wash was combined with the scraped cell fraction and pelleted by centrifugation . The MDMΦ pellet was washed once more in PBS before resuspension in 300 μl of water. Three cycles of freeze/thawing were executed to lyse samples, and an aliquot was removed for modified Bradford protein assay for normalisation. The remaining fraction was used to extract glycolipids by adding 400 μl chloroform and 800 μl methanol (MeOH) to 300 μl of aqueous lysate for lipid extraction using a modified Svennerholm and Fredman method [pH 5.0, containing 1 mg/ml sodium taurodeoxycholate). Oligosaccharides released from GSLs were brought to 30 μl with water and labelled with 2-AA as described for FOS. These labelled oligosaccharides were analysed by NP-HPLC as described above.MDMΦ glycosphingolipids (GSLs) were detected using NP-HPLC as previously described . BrieflyTaq polymerase at 95°C. PCR thermocycling with fluorescence detection was executed for 45 cycles of 95°C for 15 seconds followed by 60°C for 60 seconds and a fluorescence read step. Samples were read in technical duplicate and compared to a standard curve generated from high-titer viral RNA isolated from C6/36-grown DENV2. 95% confidence intervals were determined based on biological and technical variation and graphed using Prism 6 .DENV RNA in cell culture supernatants was isolated according to the manufacturer’s protocol for Direct-zol RNA MiniPrep Kit (Zymo Research) and assayed by reverse transcription-real time polymerase chain reaction (qRT-PCR) on an Applied Biosystems 7500 real-time PCR system (Life Technologies). Thermo-Start DNA Taq polymerase was used to enable Taq-mediated release of fluorescently labelled dyes from a DENV2 NS5-specific probe adapted from a previously published protocol . Probe ain vitro experiments. Rodent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control (Life Technologies #4308313) containing a VIC reporter dye was attached to a TAMRA (tetramethylrhodamine) quencher, and experimental DENV NS5-specific target was labelled with FAM (6-fluorescein amidite) reporter dyes with a TAMRA quencher. GAPDH primers were used at 10 μM and probe at 20 μM. In the case of DENV qRT-PCR, quantitation using a DENV2-specific primer/probe set directed to NS5 was conducted using all primers and probe at 10 μM final concentration [in vitro experiments. Viral RNA from tissues was quantified by ΔΔCt method, whereas plasma samples were quantified relative to a standard curve as for in vitro experiments. Samples were read in technical duplicate and 95% confidence intervals were determined based on biological and technical variation and graphed using Prism 6 .For animal experiments, multiplex qRT-PCR was conducted on all tissue isolates and plasma as described for ntration . Thermoc5 pfu of DENV2 D2S10 was injected intravenously. Beginning immediately following infection, mice were treated with 33.3 mg/kg celgosivir three times daily (t.i.d.) via oral gavage (p.o.). Treatment was continued for 80 hours, at which point mice were anaesthetised by isoflurane inhalation and euthanized after cardiac puncture.All experimental procedures were pre-approved by the UC Berkeley Animal Care and Use Committee and were performed according to the guidelines of the UC Berkeley Animal Care and Use Committee, the United States Public Health Service, and the USDA Animal Welfare Act. Mice of the 129/Sv background deficient in interferon (IFN)-α/β and -γ receptors (AG129 mice) were infected with DENV under antibody-enhanced conditions as previously described ,39. Miceg, 30 minutes, 4°C), and supernatant was collected. Cells were resuspended in 0.14 M ammonium chloride (Sigma), 17 mM Tris , pH 7.2 in H2O and incubated at 37°C for 5 minutes to lyse red blood cells (RBCs). RBC lysis was repeated three times, and cells were washed in serum-free α-MEM (Gibco) prior to resuspension and storage in RNAlater (Qiagen). Spleen, liver, small intestine, kidney, and lymph node tissues were collected into tubes containing zirconia/silica beads (1.0 mm diameter) and protease inhibitor cocktail (Roche) in α-MEM. Tissues were homogenised using a Mini-Beadbeater-8 (BioSpec Products) and stored immediately at -80°C until future use. PBMCs and other tissue samples were thawed on ice in the presence of 40 mM dithiothreitol (DTT) and mixed with three volumes of buffer RLT (Qiagen) containing 40 mM DTT prior to isolation of cellular RNA by RNeasy Mini Kit (Qiagen) as per the manufacturer’s instructions. Plasma RNA  was isolated by QIAamp Viral RNA Mini Kit (Qiagen) in accordance with the manufacturer’s instructions.Cardiac bleeds (approximately 1 ml) were collected in citrate anti-coagulant tubes (Becton Dickinson) and stored on ice prior to isolation of PBMCs and plasma. To separate plasma, whole blood was centrifuged . The same protocol was applied to GSL assays. Statistically significant differences (α<0.05) in viral load for animal studies were assessed by non-parametric Mann-Whitney testing with Bonferroni correction for multiple comparisons.Antiviral ECNB-DNJ and NN-DNJ can reduce production of infectious DENV in this model [4 pfu/ml (mean +/- SD) of infectious DENV 48 hours after infection with MOI 1 DENV2, strain 16681, covering a >40-fold range from 2.3–97.5 x 104 pfu/ml. DNJ-derived compounds successfully reduced production of infectious virus with EC50s between 1.2 and 10.6 μM, as we have previously reported [NB-DGJ, but for NN-DGJ the maximum non-toxic dose was determined to be 31.6 μM by MTS assay.Cells of the myeloid lineage, including tissue-resident macrophages and dendritic cells as well as circulating monocytes, are instrumental in supporting early DENV infection and directing host immune responses –42. We his model . In ordein vitro. Furthermore, the antiviral properties of the pro-drug in comparison to castanospermine have not been addressed. In order to compare the optimization efforts of monocyclic and bicyclic iminosugars, we performed a titration of NN-DNJ and celgosivir as well as the “parent” compounds DNJ and castanospermine in our human MDMΦ model. Comparison of the two optimized candidates in MDMΦs reveals similar antiviral efficacy , and both drugs are at least 7-fold more potent than the parent compounds  where iminosugars with increasing alkyl chain length, again mimicking the natural substrate of the enzyme glucosylceramide synthase, were more potent inhibitors of the enzyme. A similar trend can be observed for both DNJ and DGJ iminosugars, suggesting that this effect is not specific to sugar stereochemistry.Given the diversity of functions of glycans hibition . Unsurprin vitro testing of isolated enzymes indicates that inhibition of either α-glucosidases or glucosyltransferases is likely the essential property of iminosugars required for antiviral efficacy. Furthermore, the exclusive ability of glucose-mimicking iminosugars to potently inhibit α-glucosidases provides considerable support for the hypothesis that α-glucosidase inhibition is the single class of target enzymes responsible for antiviral activity against DENV; however, it is equally possible that only DGJ-derived iminosugars fail to access glycolipid processing enzymes in cells. Thus, we next assessed whether these iminosugars could achieve inhibition of glycolipid processing in our human MDMΦ model. In order to address this question, MDMΦs were treated for 48 hours with the maximal non-toxic dose (MNTD) of drug used in antiviral studies as in d-galactose and one β-d-glucose head group, facilitates analysis by normal phase high performance liquid chromatography (NP-HPLC). As shown in NB-DNJ was therefore investigated in three donors over a titration of 1 to 100 μM of drug. 50 of NB-DNJ with respect to reduction of GM3 is approximately 10-fold lower than the antiviral EC50 . Although 90–95% inhibition of GM3 levels could be reached with 10 μM treatment of NB-DNJ, increasing concentrations up to 100 μM did not lead to further inhibition of GM3 levels, suggesting that this is the maximal effect possible for iminosugars. In summary, these data indicate that all of the monocyclic iminosugars tested could effectively inhibit glycolipid processing to equivalent levels at the concentrations tested in the antiviral assays presented in The NB-DNJ for 48 hours in the absence of DENV infection. As a consequence of inhibition of ER α-glucosidase II, a monoglucosylated glycoprotein is produced. After trimming of the glycan precursor by mannosidases and recognition of the glycoprotein as terminally misfolded, a Glc1Man4GlcNAc1 free oligosaccharide species is cleaved from the peptide during ER-associated degradation. In the case of inhibition of ER α-glucosidase I, a similar process produces a Glc3Man5GlcNAc1 species. Thus, the presence of each species of FOS can be correlated with successful inhibition of the respective cellular α-glucosidase [NB-DNJ led to accumulation of peaks representative of inhibition of both α-glucosidases  and are frequently implicated in disorders of glycolipid processing [Whereas both DNJ- and DGJ-derived iminosugars can effectively inhibit glycolipid processing in cell culture, the inhibition data from isolated enzymes suggests that only glucose-mimic iminosugars are likely to inhibit glycoprotein processing. Generation of FOS can be used as a marker of successful inhibition of ER-resident α-glucosidases , and thicosidase . Nearly osidases . Similarthe MNTD . The majinidases . These eocessing .NB-DNJ or celgosivir led to accumulation of Glc1Man4GlcNAc1 but not Glc3Man5GlcNAc1 in all donors tested , and this trend appeared to extend to infectious virus; however, results for the plaque assay were not statistically significant (α = 0.100) due to the considerable variation in the assay. Additional tissues including kidney, parenteral lymph nodes, liver, and small intestine showed similar trends in RNA levels; however, in the case of the spleen, celgosivir enhanced viral RNA levels by approximately two orders of magnitude . OverallIn this work, we tested a panel of iminosugars covering a range of specificities of enzyme inhibition profiles to address the role of glycoprotein and glycolipid inhibition on DENV antiviral activity. Coordinated analysis of isolated enzyme inhibition profiles, primary human cell culture antiviral, glycoprotein, and glycolipid inhibition profiles, and animal model efficacy allows us to present the most complete deconstruction of iminosugar antiviral properties necessary for efficacy against DENV to date. Prior to this publication, a large body of work existed in support of the hypothesis that iminosugar antiviral activity against DENV is a function of inhibition of endoplasmic reticulum resident α-glucosidases; however, exclusion of alternative mechanisms of action related to inhibition of additional sugar processing enzymes had not been undertaken. Knowledge of the precise mechanism(s) of action of iminosugars as DENV therapeutics is essential as these drugs enter and progress through clinical trials.NB-DNJ and celgosivir  was considerably more pronounced than the improvement of celgosivir in comparison to the parent castanospermine (7-fold). In a BHK-21 (hamster kidney) cell culture model, celgosivir exhibited ~100-fold enhancement of antiviral efficacy over castanospermine [Our results demonstrate that production of infectious DENV in a primary human macrophage model was controlled in a dose-dependent manner with DNJ-derivative iminosugar treatment, as previously reported , and varspermine ; howeverin vitro, our panel of closely related enzymes helps to answer the question of iminosugar specificity. Inhibition of isolated enzymes occurs in line with anticipated headgroup stereospecificity , but this specificity does not extend to glycolipid processing enzymes such as the glucosyltransferase of HL60 cells, where factors relating to alkyl chain structure and length are more likely to be relevant. It is worth noting that the inhibition of enzymes in vitro is less potent for celgosivir than for castanospermine, a result that is likely to be the product of interference of the butanoyl group with binding of the inhibitor. In cell culture and animal models, this group is cleaved so that the more potent inhibitor, castanospermine, is in abundance. Trends observed for isolated enzymes are replicated in cell culture; thus, the question of antiviral efficacy cannot be explained by differential uptake, a phenomenon much more likely to be mediated by differential alkylation. Rather, this work demonstrates that in the presence of maximal glycolipid processing inhibition (~95% reduction of GM3), DGJ-derived iminosugars fail to induce any accumulation of FOS related to ER α-glucosidase inhibition. Although equivalent glycolipid processing effects are observed for DNJ-derivatives, the cellular inhibition of ER α-glucosidases correlates remarkably well with observed antiviral efficacy of iminosugars. Whereas GM3 accumulation occurs at approximately 10-fold lower concentrations, α-glucosidase inhibition maps very well to antiviral efficacy. In particular, inhibition of α-glucosidase II appears to be of specific importance as can be observed by comparison of celgosivir and NB-DNJ titration curves which reveals a more potent inhibition of α-glucosidase II by the more potent antiviral, celgosivir. This trend can be extended even in the case where α-glucosidase I inhibition occurs at lower concentrations than α-glucosidase II inhibition. In the case of DNJ based iminosugars CM-10-18 and CM-9-78, α-glucosidase I inhibition occurs at 3- to 5-fold lower concentrations than α-glucosidase II concentrations. For these molecules, α-glucosidase II inhibition occurs with an EC50 of 2.6 and 1.6 μM for CM-9-78 and CM-10-18, respectively. Antiviral EC50s in the same A549 cell line are 1.5 and 1.1 μM while α-glucosidase I inhibition EC50s of 0.42 and 0.46 μM are noted, respectively [How does the difference in glucose and galactose stereochemistry confer such a striking difference to antiviral efficacy of iminosugars? Although it remains a technical challenge to produce sufficient quantities of pure human ER α-glucosidase to test this enzyme ectively . Thus, wIt has been suggested that iminosugars reduce infectivity of HIV ,60 but pin vitro macrophage data in in vivo mouse data in Lack of antiviral efficacy observed in the first human studies of iminosugars as DENV therapeutics is a challenge for the field ,62. Use In summary, this study provides confirmation of the long-standing dogma that iminosugar inhibition of DENV is accomplished via inhibition of ER α-glucosidases. Our data are in agreement with the mounting bank of evidence that α-glucosidase II is the glucosidase responsible for anti-DENV activity, and we suggest that any further optimisation of iminosugar candidates focuses on enhanced inhibition of this enzyme. For monocyclic iminosugars, the necessary specificity for glucose-processing enzymes is provided by the DNJ headgroup. Alkylation or modification of the ring nitrogen on the other hand constitute amenable targets for medicinal chemistry efforts. Modulation of the alkyl group in particular is promising as we have demonstrated that glycolipid inhibition, which may be altered or abolished with changes in the alkyl group is of no consequence to antiviral activity against DENV. At present, high-resolution structural data for the complete α-glucosidase II enzyme is unavailable; however, a crystal structure of a lectin receptor domain has been solved . AvailabS1 Fig(a) parenteral lymph nodes, (b) small intestine, (c) liver, (d) kidney, and (e) spleen.Tissues were collected from the mice treated in (DOCX)Click here for additional data file.S1 TableTitre of infectious dengue virus measured in the supernatant of primary human MDMΦs 2 days after infection with MOI 1 DENV2, strain 16681, as determined by plaque assay. Cells were treated with 25ng/ml recombinant human IL-4 for 3 days prior to infection with DENV, as described in Methods.(DOCX)Click here for additional data file.


Pedicle screw fixation allows purchase of all three spinal columns without encroaching into the spinal canal improving fracture fixation, as well as deformity correction. Fortunately, neurologic injury associated with pedicle screw malposition is rare.A 19-year-old boy was surgically treated for severe right thoracic scoliosis associated with a Chiari Type 1 malformation and a C6 to T7 syringomyelia. Six months after the initial surgery, the patient was referred to our institution after three weeks of gait disturbances and repeated falls. Imaging showed the gross misplacement of the left T5 pedicle screw, which crossed the center of the vertebral canal. The initial surgery used a freehand technique of pedicle screw insertion, with anteroposterior and lateral postoperative X-ray control. During the surgery, no SEP modifications were noted during pedicle screw placement. However, after insertion of the second rod and scoliosis correction by posterior translation technique, SEP responses decreased considerably. Revision surgery was performed to remove the misplaced screw. During the first three months after screw removal, repeated clinical examinations showed progressive recovery of the neurological deficits. Gait and bladder functions were normal six months after screw removal, and clinical signs of spasticity disappeared. SEP explorations performed at final follow-up showed similar responses to those performed before the initial surgery for scoliosis correctionNeurologic injury associated with pedicle screw malposition is rare. In early or delayed neurological status worsening, intraoperative or postoperative imaging must be done to detect pedicle screw misplacement. In the current case, thanks to cobalt-chromium and titanium use, MRI and CT scan allowed good visualization of the spinal canal and spinal cord. Experimental studies have shown that neurophysiological monitoring of the spinal cord does not detect moderate compression. In that way, neurophysiological monitoring is an all-or-nothing technique which can misdiagnose early stage of spinal cord injuries. Major penetration of the spinal canal by pedicle screw may conduct to hardware removal.In early or delayed neurological status worsening, intraoperative or postoperative imaging must be done to detect pedicle screw misplacement. In the current case, thanks to cobalt-chromium and titanium use, MRI and CT scan allowed good visualization of the spinal canal and spinal cord. Major penetration of the spinal canal by pedicle screw may conduct to hardware removal. The patient was operated on by posterior approach on a Hall frame and with permanent sensory-evoked potential (SEP) monitoring. SEP monitoring protocol consisted in a continuous SEP monitoring with bilateral posterior tibial nerve stimulation and cortical and cervical detection. SEP amplitudes and latencies were recorded in a continuous fashion from patient positioning under general anaesthesia to the end of the surgical procedure.A 19-year-old boy was surgically treated for severe right thoracic scoliosis associated with a Chiari Type 1 malformation and a C6 to T7 syringomyelia. The Chiari Type 1 malformation was first operated on at the age of 14; the result was considered satisfactory, and the syringomyelia was considered stable between ages 14 and 19. Because Cobb angle increased to 95°  of the right psoas, the right flexor digitorum profundus, and the right triceps. There was a right Babinsky sign with increased deep tendon reflexes in both lower limbs. Romberg sign was positive. The patient also presented severe dysuria with bladder distention requiring urinary catheter. There was no recent history trauma, no fever and no remaining biological sign of infection. Axial, sagittal and coronal planes of a thoracolumbar CT scan confirmed the gross misplacement of the pedicle screw, which crossed the center of the vertebral canal (Fig. /5 of theRevision surgery was performed to remove the misplaced screw. No spinal canal exploration was performed at this time. No cerebrospinal fluid effusion was noted during screw removal, and no meningocele occurred postoperatively. During the first 3 months after screw removal, repeated clinical examinations showed progressive recovery of the neurological deficits. Gait and bladder functions were normal 6 months after screw removal, and clinical signs of spasticity disappeared. The patient reported no motor or sensory deficit at final follow-up, 18 months after screw removal. SEP explorations performed at final follow-up showed similar responses to those performed before the initial surgery for scoliosis correction Fig. , and finPedicle screw fixation allows purchase of all three spinal columns without encroaching into the spinal canal. This theoretical advantage has been translated to superior clinical results in fracture fixation, as well as in deformity correction (Dickman et al. Fortunately, neurologic injury associated with pedicle screw malposition is rare. One author evaluated 3204 screws and reported no vascular, neurologic or visceral injuries (Kim et al. Routine MEP after insertion of each pedicle screw in the thoracic spine, as well as triggered EMG stimulation of each pedicle screw to rule out breaching once all screws have been inserted, could improve safety in challenging cases (Calancie et al. As patient safety becomes more and more of a concern, the use of intraoperative CT scanning, with real CT imaging or CT fluoroscopy of pedicle screws post-insertion, may become a standard of care in the future (Flynn and Sakai Some technical tricks, such as the funnel technique Gaines , allow dThis uncommon case of completely misplaced pedicle screw crossing the spinal canal draw the reader’s attention to the risk of pedicle screw placement in severe thoracic curves. In case of early or delayed neurological status worsening, intraoperative or postoperative imaging must be done to detect pedicle screw misplacement. In the current case, thanks to cobalt-chromium and titanium use, MRI and CT scan allowed good visualization of the spinal canal and spinal cord. Major penetration of the spinal canal by pedicle screw may conduct to hardware removal.


Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed. For maintenance of cellular homeostasis, it is not only essential that cell components are synthesized and assembled when required but also that these components are removed and degraded when they are aberrantly synthesized, become damaged or when they are functionally redundant. When these processes are not carried out properly, the cell may either die or turn into a tumor cell in which cell growth proceeds unrestrained.The major protein degradation systems include the ubiquitin–proteasome pathway  (Ciechanover The process of macroautophagy (hereafter referred to as “autophagy”) has gained tremendous scientific interest in recent years. This is not only because of the partial unraveling of the protein and lipid machinery participating in this complicated cell biological process Ohsumi  but alsoAccording to current opinion, the primary function of autophagy is to allow the cell to survive under stress conditions rather than to function as a cell death mechanism , the proteins ATG13, FIP200 (yeast Atg17), ATG101 and many other components , or by displacement of Beclin1 from its complex with Bcl-2 by other BH3-containing proteins such as the proapoptotic protein BAD and the proautophagic protein BNIP3  protein and can also associate with the anti-apoptotic proteins Bcl-2/Bcl-xL which contain a BH3-binding groove. This means that association of Beclin1 with these anti-apoptotic proteins is inhibitory for autophagy and their dissociation is essential to allow Beclin1 to bind to PIK3C3 and to initiate autophagy. The dissociation of the inhibitory Beclin1–Bcl-2 complex is promoted by either JNK-1-mediated phosphorylation of Bcl-2, by phosphorylation of Beclin1 mediated by the tumor suppressor DAPK P3 and PIP2, the products of PIK3C1, are inhibitors of autophagy, possibly because these lipids compete with PI(3)P  at near-physiological concentrations could mimic the inhibitory effect on autophagic proteolysis of a complete mixture of all amino acids; either alanine or leucine alone had no effect  the phosphorylation of a protein that was identified as ribosomal protein S6, with rapid kinetics . The stimulation of S6 phosphorylation by amino acids was not due to the inhibition of a protein phosphatase acting on S6  was obtained by the Amsterdam group in a study carried out with Pi-labeled whole cells or from the pool associated with the lysosomal membrane exhibited constitutive [32P]GTP charging that was unaltered by amino acid withdrawal. In addition, in cells with mutant Rag which was unable to bind GTP, activation of MTOR by amino acids still occurred  which are predominantly produced in the mitochondria  activates autophagy as a protective mechanism, and this occurs in a manner that is sensitive to antioxidants  but, in addition, is also a potent inhibitor of GDH P2 is able to bind to RAPTOR where it serves to recruit MTOR downstream targets and/or to bring MTORC1 to the proper location in the cell P, actually stimulate the production of PIPp62/SQSTM1 may also be compartmented because only a small part of the total cellular p62 is bound to the MTORC1 complex through its association with the Rag GTPases   amino acid availability by mammalian cells  for acetyl CoA, acts as a sensor of cytosolic acetyl CoA which translates increases in cytosolic acetyl CoA into inhibition of autophagy P2 and PIP3 is also MTORC1 independent, and thus rapamycin insensitive.Our original discoveries of amino acid-stimulated, MTOR-mediated, signaling and its role in the control of autophagy, the regulation of these two pathways by the energy sensor AMPK and the role of phosphatidylinositol 3-phosphate in autophagy have opened new perspectives in the understanding of the regulation of cell metabolism. Given the enormous impact of autophagy on cell function, and its regulation by amino acid signaling, it is not surprising that research in these fields has expanded exponentially over the last two decades Klionsky . Proper


The pullout strength of pedicle screws is influenced by many factors, including diameter of the screws, implant design, and augmentation with bone cement such as PMMA. In the present study, the pullout strength of an innovative fenestrated screw augmented with PMMA was investigated and was compared to unaugmented fenestrated, standard and dual outer diameter screw.Twenty four thoracolumbar vertebrae  from three cadavers were implanted with the four different pedicle screws. Twelve screws of each type were instrumented into either left or right pedicle with standard screw paired with unaugmented and dual outer diameter screw paired with augmented fenestrated screw in any given vertebra. Axial pullout testing was conducted at a rate of 5 mm/min. Force to failure (Newtons) for each pedicle screw was recorded.The augmented fenestrated screws had the highest pullout strength, which represented an average increase of 149%, 141%, and 78% in comparison to unaugmented, standard, and dual outer diameter screws, respectively. Pullout strength of unaugmented screws was comparable to that of standard screws, however it was significantly lower than dual outer diameter screws.Fenestrated screws augmented with PMMA improve the fixation strength and result in significantly higher pullout strength compared to dual outer diameter, standard and unaugmented fenestrated screws. Screws with dual outer diameter provided enhanced bone-screw purchase and may be considered as an alternative technique to increase the bone-screw interface in cases where augmentation using bone cement is not feasible. Unaugmented screws can be left in the pedicle even without cement and provide similar pullout strength to standard screws. Pedicle screw fixation is one of the most commonly used forms of stabilization in the thoracic and lumbar spine for trauma, correction of deformity or instability, and fixation in oncologic and fusion procedures ,2. HowevFixation of the screw into the vertebral body is traditionally evaluated by determining the axial pullout strength of implanted screws. In the present study, the two most frequently used methods, augmentation with PMMA and large diameter pedicle screws, were tested. Previous biomechanical studies have demonstrated that pedicle screw augmentation using PMMA markedly increases the strength of the bone-screw interface -4,8-10.However, few biomechanical studies have compared the pullout strengths of these two frequently used methods for improving the bone-screw interface strength ,10. For Twenty-four thoracolumbar vertebral bodies (T10-L5) from three cadavers  free of metastatic disease or primary bone disease were used in the study. All cadavers were donators at the Institute of Anatomy at LSU in New Orleans (USA) and were provided for the purpose of this study according to the regulations of the ethics committee. Lateral and anterior radiographs were taken for each specimen to rule out any pathological fractures or other bone lesions. The vertebrae were freed from all muscular attachments. All vertebrae were tested for osteoporosis via dual energy X-ray absorptiometry (DXA). The limit for osteoporosis was set at <0,8 g/cm2 .Twelve screws of each type were inserted in the 48 pedicles of 24 vertebral bodies. A pedicle awl was used to perforate the cortex, and a pedicle probe to open the pedicle pathway. A ball-tip probe was used to ensure the pedicle was intact. A 4.5 mm tap was used prior to screw insertion. Pedicle screws were inserted straight into the pedicle, with as minimum convergence as possible ,13. In eIn the case of fenestrated screws augmented with PMMA, 2 to 3 ml of bone cement  was injected according to the recommended technique -17. CemeAfter 24 hours, specimens were mounted on a custom-designed fixture attached to the actuator of the test machine . The fixture allowed for rotation in the YZ-plane and adjustment of the XY-plane to ensure vertical pullout alignment Figure . Axial pFailure was defined as the point at which the load peaked and then decreased sharply with increasing displacement. The mean pullout strengths for various screws were calculated. The vertebrae were disarticulated and the pedicles were examined after the completion of the mechanical testing in an attempt to determine the site of failure and any gross physical differences among the four screws were noted.t test to determine whether axial pullout strengths differed among different screws. Statistical evaluation included paired t test between FSP screw versus DOD screws and unaugmented FS screws versus SS screws. For all the other comparisons, unpaired t test was used. Significance was set at p < 0.05.In two specimens, the T10 pedicle size was too small resulting in breakage of pedicles. In two other specimens, the L5 vertebra was damaged during pullout testing. These four vertebrae were therefore removed from the study. Therefore, ten screws of each type were used for data analysis. The configuration of each type of screw in each specimen is shown in Table The unaugmented fenestrated, standard and dual outer diameter screws failed at the bone-screw interface due to bone fracture. For the fenestrated screws augmented with PMMA, entire pedicle was pulled off of the posterior aspect in a few of the specimens, while others failed at the cement-bone interface, where a void was created inside the vertebral body after the removal of the screw/cement section.2 that indicated osteoporosis [Overall the mean pullout strengths for each screw type for the 3 specimens tested were: standard screw 610 N ±264), unaugmented fenestrated screw 591 N (±238), dual outer diameter screw 827 N (±274), and fenestrated screws with PMMA 1470 N (±218). These values showed that augmentation with PMMA significantly improved the pullout strength to 149%, 141%, and 78% in compare to the unaugmented fenestrated screw , standard screw  and dual outer diameter screw , respectively. No statistical significance was seen between pullout strengths of the standard and fenestrated screws . The dual outer diameter screw significantly improved pullout strength compared to the unaugmented fenestrated screw . No statistical significance was seen between pullout strengths of the standard and dual outer diameter screw . The means and standard deviations for mean pullout strengths for different screws are presented in Figure , unaugmeUse of standard pedicle screws in the osteoporotic spine remains a challenge due to the mechanical instability of the bone-screw interface. Numerous techniques have been proposed to increase the bone-screw interface strength including bicortical purchase, pedicle undertapping and offset laminar hooks . FurtherIn the present study, pedicle screws were inserted at a straight angle as opposed to the standard angled screw insertion. Sterba et al. (2007) have demonstrated that straight screw insertion results in a more stable pedicle screw construct as opposed to angled screw insertion technique . The autAugmentation using PMMA resulted in highest pullout strength in the three specimens tested. Over all, pedicle screw augmentation with PMMA increased the pullout strength to 241% compared to that of standard screws. Other biomechanical studies have demonstrated that pedicle screws augmented with PMMA improve initial fixation strength and fatigue strength of instrumentation in the osteoporotic spine ,8,9. CooA commercially available radiopaque PMMA was used to strengthen fenestrated screws. PMMA is typically used to increase bony purchase ,9. One dIn each vertebral body, the standard screw was always paired with a fenestrated screw and the dual outer diameter screw was paired with a fenestrated screw augmented with PMMA on the contralateral side, to minimize the potential differences in bone mineral density and be able to compare the pullout strengths of the two frequently used methods of fixations. In addition all the vertebrae used had a bone mineral density less than 0,8 g/cm2, it means in effect they were all osteoporotic . Dual ouFurthermore there was no significant difference between the pullout strength of the unaugmented fenestrated screws and the standard screws. Previous studies  have shoLimitations resulted from the experimental setup. Factors as muscle strength, body weight and height were not taken under consideration. Moreover this study did not analyze the age- and sex-specific differences. The use of cadaver specimens also brings restrictions. Despite rapid action and optimal humidification during the experiment, there was a certain degree of autolysis that cannot be prevented. Furthermore the stabilizing effect of the intervertebral discs, ligaments and muscle surrounding the spine in vivo was removed before the experiment. As a result changes in biomechanical properties cannot be excluded. Other limitations arise from the assumption that the measured bone density of vertebral body is equivalent to the bone density of the pedicle . AccordiThe novel fenestrated screws augmented with polymethylmethacrylate resulted in a significant increase in the axial pullout strength compared to dual outer diameter, standard and unaugmented fenestrated screws. This suggests that the novel fenestrated screws augmented with PMMA may be useful for pedicle fixation in patients with poor bone quality. The dual outer diameter screws improved the pullout strength compared to the standard and fenestrated screw and may be considered as an alternative technique to increase the bone-screw interface, in cases where augmentation using bone cements is not feasible. The novel fenestrated screw without cement augmentation demonstrated pullout strengths comparable to the standard pedicle screw.


A number of CTD-binding proteins have been identified and the crystal structures of several protein-CTD complexes have demonstrated considerable conformational flexibility of the heptad repeats in those interactions. Furthermore, phosphorylation of the CTD at tyrosine, serine and threonine residues can regulate the CTD-protein interactions. Although the interactions of CTD with specific proteins have been elucidated at the atomic level, the capacity and specificity of the CTD-interactome in mammalian cells is not yet determined.Eukaryotic RNA polymerase II contains a C-terminal repeated domain (CTD) consisting of 52 consensus heptad repeats of Y2-phosphorylated but not pY1pS2-doubly phosphorylated CTD-peptides. However, we also found proteins that were preferentially pulled-down by pY1pS2- or pY1pS5-doubly phosphorylated CTD-peptides. We prepared an antibody against tyrosine phosphorylated CTD and showed that ionizing radiation (IR) induced a transient increase in CTD tyrosine phosphorylation by immunoblotting. Combining SILAC and IMAC purification of phospho-peptides, we found that IR regulated the phosphorylation at four CTD tyrosine sites in different ways.A proteomic study was conducted to examine the mammalian CTD-interactome. We utilized six synthetic peptides each consisting of four consensus CTD-repeats with different combinations of serine and tyrosine phosphorylation as affinity-probes to pull-down nuclear proteins from HeLa cells. The pull-down fractions were then analyzed by MUDPIT mass spectrometry, which identified 100 proteins with the majority from the phospho-CTD pull-downs. Proteins pulled-down by serine-phosphorylated CTD-peptides included those containing the previously defined CTD-interacting domain (CID). Using SILAC mass spectrometry, we showed that the in vivo interaction of RNA polymerase II with the mammalian CID-containing RPRD1B is disrupted by CID mutation. We also showed that the CID from four mammalian proteins interacted with pSUpon phosphorylation, the 52 repeats of the CTD have the capacity to generate a large number of binding sites for cellular proteins. This study confirms previous findings that serine phosphorylation stimulates whereas tyrosine phosphorylation inhibits the protein-binding activity of the CTD. However, tyrosine phosphorylation of the CTD can also stimulate other CTD-protein interactions. The CTD-peptide affinity pull-down method described here can be adopted to survey the mammalian CTD-interactome in various cell types and under different biological conditions. Toing Fig. b. ImmunoD1B Fig. b. We alsD1B Fig. c. FollowTo test if tyrosine phosphorylation of the CTD could disrupt the interaction between CID and CTD, HEK293T cells were co-transfected with GST (lane 1), GST-CTD (lane 2), Myc-RPRD1B WT (lane 3), Myc-RPRD1B MT lane 4) or combinations with an constitutively activated ABL kinase (AblPPn) lanes 5–10), and total cell lysates (input) were analyzed via immunoblottings to detect the transfected proteins  between non-irradiated and irradiated cells at 2 h, but reduced ratio 0.5) when the comparison was made between cells irradiated for 30 min or 2 h, suggesting that IR caused a transient reduction in pY-1874 and pY-1881 at 30 min with a return to un-irradiated level by 2 h (Table  when the2-CTD consistent with a recently published report that CTD-tyrosine phosphorylation inhibits RNAPII interaction with the Pcf11 transcription termination factor [Phosphorylation of the CTD generates “codes” for the selective binding of cellular proteins to regulate RNA processing and chromatin structure during transcription elongation . Becausen factor . HoweverWhile the CTD peptide-pull down method cannot distinguish between direct or indirect binding to alternatively phosphorylated CTD-repeats, it provides a way to survey the proteomic landscape associated with specified combinations of CTD repeat sequences and phosphorylation. This method can also be used to identify proteins that associate with regions of the CTD that contain non-consensus heptad repeats.http://massive.ucsd.edu/ProteoSAFe/status.jsp?task=dfa10c6566dc4bfca6362abc761b74bcAll supporting data have been deposited to the MassIVE repository developed by the NIH-funded UCSD Center for Computational Mass Spectrometry;


Aspergillus niger (AnPEP) has been identified as being able to accelerate breakdown of gluten in food using an in vitro digestion system. Such digests have been analysed in terms of coeliac disease t-cell epitope levels[Coeliac disease and IgE-mediated allergy to wheat are immune-mediated conditions which are thought to be triggered by digestion-resistant wheat proteins. Recently, a prolyl endopeptidase from pe levels but havein vitro batch gastric digestion model to break down a bread matrix with different amounts of AnPEP. SDS PAGE and immunoblots have been used to monitor protein digestion, and mass spectrometry has allowed for epitopes important to IgE-mediated wheat allergy and coeliac disease to be tracked. Sera were obtained from a Spanish patient panel with allergy to wheat induced by exercise and/or NSAIDs and used for additional immunoblots.We have used an SDS PAGE and immunoblotting with anti-gluten antibodies show more effective breakdown of gliadins and LMW glutenins in the presence of AnPEP. The impact of digestion on the IgE-reactivity of gluten by immunoblotting has been correlated with resistant proteins and peptides mapped by mass spectrometry. Use of 200 mg AnPEP/ g substrate showed the largest difference in digestion kinetics of gluten proteins however increased breakdown was still visible using 20 mg AnPEP/ g substrate.AnPEP alters the patterns and pathways of gluten in a simulated gastroduodenal digestion system. The potential application of such enzymes to modify the allergenic potential of cereals is discussed.


Background. To evaluate the correlation of fundus autofluorescence (FAF) with indocyanine green angiography (ICGA) in patients with various posterior uveitis disorders. Methods. Interventional case series including 23 eyes of 15 patients with diagnosis of a specific type of retinochoroiditis, such as acute posterior multifocal placoid pigment epitheliopathy (APMPPE), serpiginous-like choroiditis, multifocal choroiditis (MFC), Harada disease, and syphilitic retinochoroiditis. Also, some cases with undefined retinochoroiditis were included. FAF and ICGA were performed and correlated at baseline and during follow-up after treatment. Results. In ICGA, early hypofluorescence was found to be the hallmark of acute choroidal inflammation, resolving in later stages and remaining in the late phase in areas with retinal pigment epithelium (RPE) damage. Poorly defined hyperautofluorescent areas correlated with acute choroidal lesions. Hypoautofluorescent delineation suggested the initiation of RPE healing processes, correlating well with the late phase of ICGA and delineating the RPE damage. Early hyperautofluorescence with late hypofluorescence in ICGA indicated the presence of primary RPE involvement. Conclusion. FAF contributes to the interpretation of RPE disease and may be a useful tool for the follow-up of progressive inflammatory disorders. Comparative evaluation of FAF and ICGA allows a characterization of the sequence of inflammatory events and the level of tissue affected. Posterior uveitis is a general classification term, which includes various inflammatory disorders, affecting primarily the retina, the choroid, or both. In cases of multilevel involvement, discrimination of the initially or exclusively affected tissue allows a more precise classification into retinitis, choroiditis, or retinochoroiditis. Diagnosis and follow-up of posterior uveitis patients have evolved significantly with the advances in imaging technology, such as indocyanine green angiography (ICGA) and fundus autofluorescence (FAF). These imaging tools are complementary to the standard evaluation with fundoscopy and fluorescein angiography (FA) , 2.Retinal inflammatory diseases, manifesting mainly as vasculitis or necrotic retinitis, such as Behcet uveitis, ocular toxoplasmosis, and herpes-associated acute retinal necrosis, can be easily suspected or even detected during fundoscopic examination. In cases of suspected retinal vasculitis or secondary induced ischemia, FA is a very useful tool for the clinical diagnosis –7. HowevIn the last years, noninvasive FAF imaging has been introduced into the ophthalmological clinical practice, providing for the first time a qualitative measure of the status of RPE layer in terms of function and structure , 13. ThiIn an attempt to optimize diagnostic procedures, there is a need to identify the most informative and least invasive clinical protocol for the evaluation of posterior uveitis. To our knowledge, there is no published study to date exploring the correlation between ICGA and FAF in patients with different forms of posterior uveitis. This analysis may provide a better understanding of the diagnostic potential of noninvasive FAF imaging. In the current study, our aim was to investigate if the combined-mode imaging (ICGA+FAF) could provide useful additional information contributing to the diagnosis, follow-up, and treatment optimization in posterior uveitis.This study is an interventional case series conducted at the Department of Ocular Immunology and Inflammation of “G. Gennimatas” General Hospital in Athens, Greece. Informed consent was obtained from all participants. All procedures were done in adherence to the tenets of Declaration of Helsinki. The study was approved by the institutional ethics committee.All patients included in the study either were new referrals, with symptoms or signs of posterior segment inflammation, or were already diagnosed with uveitis, with a follow-up performed in our department. At baseline, all patients had a complete ophthalmological examination, including biomicroscopy and fundoscopy. In addition, FAF exam, ICGA, and FA were performed. A standardized ICGA protocol for posterior uveitis was followed , using tPharmacological treatment was prescribed when necessary. During the follow-up, FAF and ICGA examinations were repeated when the signs of acute inflammation subsided as a result of treatment or due to a self-limiting clinical course. Direct comparison was performed between baseline and follow-up images obtained with both FAF and ICGA to investigate the presence of clinically relevant correlations of the results obtained with the two imaging technologies. Likewise, the level of affected tissue at baseline and during follow-up was evaluated. The clinical course of each disease and its anatomic and functional impact on the eye structures were also documented.23 eyes of 15 patients with various types of posterior uveitis were included. Specific diagnosis was done based on the results of FAF, ICGA, OCT, and FA imaging in most of cases . All casDiscrete contiguous hypofluorescent areas of the posterior pole are characteristic of acute disease in ICGA. In our series, two different angiographic patterns were observed in different areas. In the first one, profoundly dark hypofluorescent patches appeared in the early phase of the ICGA exam and persisted throughout the complete sequence of the exam. In the other pattern, hypofluorescent areas appeared mainly in the late phase Figures .FAF imaging revealed well-defined lesions with stippled autofluorescence in the core and hyperautofluorescent (hyper-AF) borders, which were partially surrounded by hypoautofluorescent (hypo-AF) halos . These FIn the convalescent stage, the early phase hypofluorescent areas (seen acutely) seemed to have faded away in ICGA. On the other hand, late phase hypofluorescence remained unchanged. Correlation between FAF and late phase ICGA seemed to be much stronger in the convalescent compared to the acute stage in all cases. Figures .Four eyes of 4 patients with serpiginous-like choroiditis, in the presence of a positive Mantoux and Quantiferon gold test, were included. In the acute stage, ICGA revealed profoundly dark hypofluorescent areas during the complete sequence that surrounded areas of hyperfluorescence in a serpiginous or centrifugal pattern Figures . FAF imaIn the convalescent stage, the majority of early-acute hypofluorescent areas became invisible in ICGA after treatment application. There was an agreement between the late phase of ICGA and FAF images. Furthermore, a clearly defined hypo-AF rim delineated the periphery of the whole AF lesions, which seemed to have undergone a peripheral expansion compared to baseline Figures .In MFC, ICGA revealed multiple hypofluorescent spots which became more obvious and numerous in the late phase. These findings in the late phase of ICGA correlated well with hypo-AF lesions on FAF Figures . On furtIn the active/acute stage of the disease, ICGA revealed the presence of small hypofluorescent areas in the early phase that changed to a diffuse mottled hypofluorescence in the late phase. The latter largely corresponded with the diffuse hyper-AF area in the posterior pole as seen on FAF. SD-OCT showed a disruption of RPE and outer retina Figures .After finishing the appropriate treatment, a resolution of hypofluorescence in ICGA and hyper-AF in FAF was observed that was accompanied with the disappearance of inflammatory signs and gains in visual acuity Figures .In the acute stage of the disease, ICGA showed hypofluorescent dots in the early phase that were converted into hyperfluorescent spots in the mid and late phase. Large circular or oval areas of hypofluorescence  were found to be surrounded by hotspots. Figures . FAF imaTwo cases were classified as full-thickness inflammation of choroid and retina in the macular area. Their common characteristic was a significant macular hypofluorescence in ICGA that was compatible with choroidal ischemia Figures . FAF shoAs previously commented, there are specific diagnostic tools available in clinical practice, to evaluate the inflammatory disease of the posterior segment of the eye. These tools include standard methods, such as FA and ICGA, OCT, and the novel technology FAF, which is being increasingly utilized as a noninvasive imaging procedure in the field of uveitis. FAF imaging is based on the special fluorescence properties of various molecules found in ocular fundus collectively called “fluorophores.” These molecules can be excited by electromagnetic energy (light) to a higher energy level and then return to their previous state by emitting light of higher wavelength. The major source of autofluorescence in human fundus is lipofuscin. This term describes a heterogeneous group of fluorophores which are found in the RPE cells and are the end product of incomplete lysosomal digestion of phagocytosed photoreceptor outer segments. Increased fundus autofluorescence is observed with aging but also in pathologic conditions whereas decreased autofluorescence is noted in RPE atrophy and/or due to signal attenuation from other pigments , 29. In Most of cases included in our series were classified under the general term WDS that represents a wide spectrum of diseases, such as APMPEE, serpiginous choroiditis, and MCP. These conditions, despite the variability of the clinical manifestations, may share common pathophysiologic mechanisms. Recently, choriocapillaris inflammation and secondary ischemia have been proposed as the initial events in WDS. However, it remains controversial if the inflammatory sequence originates in other neighboring structures such as the RPE, as was initially hypothesized . A compaIn our APMPPE cases, ICGA provided better imaging of the disturbances occurring in the choroidal circulation. Acute inflammation of choriocapillaris layer with secondary ischemia is the initial event followed by a reperfusion state at a later, convalescent stage. ICGA allowed us to obtain a precise mapping of these stages that coexisted in discrete geographic areas at the posterior pole, in the form of multiple, polymorphous, and asynchronous patches. The analysis of the correlation between ICGA and FAF allowed us to obtain an accurate evaluation of the inflammatory impact occurring at the RPE level, in terms of time and extent. Well-defined FAF findings were apparent only in areas of reperfused choriocapillaris, suggesting an established RPE damage. In contrast, minimal, ill-defined, hyper-AF spots with fluffy margins correlated with areas of acute choriocapillaris inflammation, which imply that there was not remarkable initial RPE involvement at the time of primary disturbance in the choroidal circulation.During the convalescent stage, lesions in the late phase of ICGA and FAF presented a remarkable similarity in size and shape. According to these outcomes, both FAF and late phase ICGA seemed to delineate the whole area of functionally impaired RPE, remaining after the resolution of acute inflammation. FAF imaging had the additional advantage of providing more information on the evolution and age of lesions. Nevertheless, OCT imaging, used in a complementary fashion, can further detect functional and structural alterations of the outer retina induced by the ischemia of underlying choriocapillaris. Overall, the area of initial choriocapillaris damage seemed to be much more extensive than the final RPE involvement, considering that not all acute lesions observed in ICGA were transformed into an abnormal FAF signal in the subacute or convalescent stage. Obviously, the size of the acutely ischemic areas at the choriocapillaris level may be the critical factor for the conversion of ischemia into an established RPE damage. Relatively small ischemic lesions were found to fade away in the follow-up, not affecting the overlying RPE Figures .Acute choriocapillaris inflammation may be the initial site of the pathological process in tubercular serpiginous-like choroiditis as well. In our series, FAF clearly identified the area of established RPE damage that corresponded with the reperfused choriocapillaris layer underneath. Likewise, hyper-AF margins were shown to be an indicator of an active underlying choroidal inflammation. Similar evidence has been recently reported by Gupta et al. in a series of serpiginous-like cases with associated tubercular infection . In the Regarding MCP, the analysis of the correlation between the two imaging modalities, ICGA and FAF, did not show any significant discrepancy in the findings at the inner-choroid and RPE level, as observed in the acute stage of the previous entities evaluated. In this condition, FAF was found to provide a better and more precise imaging of the RPE involvement than ICGA or fundoscopy in the form of hypo-AF spots scattered in the posterior pole. As choroidal structures seemed to be minimally affected, ICGA did not reveal significant findings in the early phase. ICGA was demonstrated to be inferior to FAF in the imaging of defects, even in its late phase. Indeed, the superiority of FAF over ICGA was observed to be even more significant in cases with exacerbations. In these cases, new hyper-AF spots in sites of previously normal background AF were confirmed to be indicative of a progression of the disease . AccordiIn ASPPC, the primary inflammation in the posterior pole has been postulated to be of infectious origin at the RPE/outer retina with a placoid pattern . In our In the group of disorders classified as stromal choroiditis, a granulomatous inflammation within the choroidal stroma targeting the choroidal melanocytes has been proposed to be the underlying pathophysiologic mechanism . Harada In conclusion, the evaluation of the correlation of FAF and ICGA can provide clinically relevant and complementary information about the timing and extent of RPE involvement compared to inner-choroid structures in disorders of the choroid-RPE complex.


Over the past 20 years, motor response inhibition and interference control have received considerable scientific effort and attention, due to their important role in behavior and the development of neuropsychiatric disorders. Results of neuroimaging studies indicate that motor response inhibition and interference control are dependent on cortical–striatal–thalamic–cortical (CSTC) circuits. Structural and functional abnormalities within the CSTC circuits have been reported for many neuropsychiatric disorders, including obsessive–compulsive disorder (OCD) and related disorders, such as attention-deficit hyperactivity disorder, Tourette’s syndrome, and trichotillomania. These disorders also share impairments in motor response inhibition and interference control, which may underlie some of their behavioral and cognitive symptoms. Results of task-related neuroimaging studies on inhibitory functions in these disorders show that impaired task performance is related to altered recruitment of the CSTC circuits. Previous research has shown that inhibitory performance is dependent upon dopamine, noradrenaline, and serotonin signaling, neurotransmitters that have been implicated in the pathophysiology of these disorders. In this narrative review, we discuss the common and disorder-specific pathophysiological mechanisms of inhibition-related dysfunction in OCD and related disorders. Response inhibition, the ability to suppress pre-potent behavior that is inappropriate or no longer required, is critical for goal-directed behavior in everyday life  and action cancelation  and pre-supplementary motor area (pre-SMA) are key components , dorsolateral prefrontal cortex (DLPFC), pre-SMA, and parietal regions  seems to be primarily mediated by serotonin . Seroto2/3-receptor availability in the striatum is associated with better Stop-signal task performance, i.e., shorter stop-signal reaction time (SSRT), and correlates positively with inhibition-related activation of the dorsal caudate and putamen , associated with reduced functional activity  and monoamine oxidase A (MAO-A) genes, coding for enzymes playing a role in neurotransmitter metabolism, have been associated with normal variations in inhibition-related activity as well. COMT is involved in the degradation of dopamine and noradrenalin and MAO-A is involved in degradation of dopamine, noradrenalin, and serotonin. A polymorphism of COMT with decreased function   that decrease the rate of re-uptake from the synaptic cleft, were associated with decreased interference inhibition  patients and in patients with related disorders.1- and D2-receptors in the striatum . We performed psychophysiological interaction (PPI) analyses and DCM and found abnormal connectivity between the IFG and amygdala in patients and their siblings, suggesting that this pattern of connectivity is an endophenotype. Limbic activation may interfere with CSTC circuit activation in OCD. We did not find evidence for altered connectivity between the IFG, pre-SMA, and striatum during inhibition. These results warrant replication in other samples.Two studies have investigated the effects of pharmacological treatment for OCD on response inhibition. Treatment with SSRI’s increased task-relevant brain activation during performance of an interference control task along with symptom improvement , trichotillomania (TTM), and attention-deficit hyperactivity disorder (ADHD), enabling the disorder specificity of these cognitive dysfunctions and enabling comparison of these inhibition deficits across these disorders.Gilles de la Tourette’s syndrome, also known as Tourette’s syndrome, is a neurodevelopmental disorder characterized by motor tics and vocal tics  and subcortical areas  . With increasing task demand , these compensational mechanisms fail and patients start to show behavioral impairments and decreased inhibition-related neural circuit activity. This phenomenon has also been observed in healthy subjects  or even natural aging  electrophysiological anomalies, neurotransmitter dysfunction, genetic variance, etc. A prime candidate for the cause of the shift might by dysfunction of dopamine signaling. Dopamine is the major neuromodulator in the CSTC circuits and can either facilitate or inhibit their activation depending on the activation of their different receptor subtypes and dopamine concentrations  and highly complex interactions with other neurotransmitter systems, including the serotonin, noradrenalin, and glutamate system, and even hormones, such as estrogens , while studies in TS have provided mixed results. To our knowledge, no study has yet been published on the neural correlates of response inhibition in TTM. Dopamine and serotonin signaling seems to be important for response inhibition and dysfunction of these neurotransmitters has been frequently observed in the obsessive–compulsive and related disorders. Nevertheless, almost all imaging studies on neurotransmitters have been performed in patients that received (chronic) pharmacotherapy, which may have influenced the scan directly, due to competition of the drug with a radioligand for a specific binding site, or indirectly because the brain adapts to the pharmacological effects (Wang et al., All authors were involved in design of this review; Laura S. van Velzen and Chris Vriend drafted the first version of the manuscript; all authors contributed to the interpretation of the literature, revised earlier drafts of this article and have approved the final version of the manuscript and its submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.