Evidence for the brain mechanisms recruited when processing concrete versus abstract concepts has been largely derived from studies employing visual stimuli. The tasks and baseline contrasts used have also involved varying degrees of lexical processing. This study investigated the neural basis of the concreteness effect during spoken word recognition and employed a lexical decision task with a novel pseudoword condition.The participants were seventeen healthy young adults . The stimuli consisted of (a) concrete, high imageability nouns, (b) abstract, low imageability nouns and (c) opaque legal pseudowords presented in a pseudorandomised, event-related design. Activation for the concrete, abstract and pseudoword conditions was analysed using anatomical regions of interest derived from previous findings of concrete and abstract word processing.Behaviourally, lexical decision reaction times for the concrete condition were significantly faster than both abstract and pseudoword conditions and the abstract condition was significantly faster than the pseudoword condition (p < 0.05). The region of interest analysis showed significantly greater activity for concrete versus abstract conditions in the left dorsolateral prefrontal cortex, posterior cingulate and bilaterally in the angular gyrus. There were no significant differences between abstract and concrete conditions in the left superior temporal gyrus or inferior frontal gyrus.These findings confirm the involvement of the bilateral angular gyrus, left posterior cingulate and left dorsolateral prefrontal cortex in retrieving concrete versus abstract concepts during spoken word recognition. Significant activity was also elicited by concrete words relative to pseudowords in the left fusiform and left anterior middle temporal gyrus. These findings confirm the involvement of a widely distributed network of brain regions that are activated in response to the spoken recognition of concrete but not abstract words. Our findings are consistent with the proposal that distinct brain regions are engaged as convergence zones and enable the binding of supramodal input. How conceptual knowledge is represented and organised in the brain is a question that remains a point of contention. Previous language studies have attempted to unravel some of the complexities surrounding the organisation and access of semantic conceptual representations and have investigated processing differences between concrete and abstract words. Concrete words such as ‘hospital’ are grounded in sensory-motor experiential knowledge while abstract words such as ‘knowledge’ refer more to verbally encoded concepts. ImageabBehavioural evidence from healthy individuals has demonstrated that concrete items are processed faster and more accurately than abstract items, 5–7. ThTheories of semantic memory have been developed to explain concreteness effects. Two prominent and competing accounts are the dual coding theory and the In comparison, the context availability theory suggestsBoth theories acknowledge that abstract words are associated with a reduced set of semantic associations compared to concrete words either in terms of imagery or available context. HoweverMore recent theories of semantic memory have combined neuroimaging and behavioural data to explain conceptual processing. One such account by Binder and Desai describeas well as experiential knowledge[It is clear that embodiment theories can account for the representation of concrete concepts but the case for abstract concepts is less obvious. Recent studies have investigated the semantics associated with abstract words, 22, 23.nowledge, 23. Hownowledge, 23, 24.Increasingly, neurophysiological evidence has been used to buttress accounts of concreteness and define putative brain regions associated with concrete and abstract conceptual processing. Both auditory and visual modality paradigms have been employed but visual modality tasks have been the most utilized and have included visual recognition, 26, visRecent meta-analyses by Binder et al. and WangOne type of task commonly used to explore putative cognitive processing mechanisms associated with word recognition and semantic processing is a lexical decision task which is the focus of the present study. Lexical decisions have been shown to produce reliable semantic effects when used in a priming paradigm and haveFindings from auditory modality studies investigating lexicality have in some cases not observed activity in regions associated with phoneme to grapheme conversion, 51, furAs such, the aim of this study was to investigate the neural basis of the concreteness effect during spoken word recognition and employ a lexical decision task with a novel pseudoword condition. The method used in this study to generate the pseudowords is comparable to the Balota et al. study in that The stimuli consisted of 60 English real words and 60 phonologically legal pseudowords. The 60 real words comprised 30 concrete, high imageability polysyllabic nouns  and 30 abstract, low imageability polysyllabic nouns . Stimuli in both real word conditions were controlled for the following variables; (i) spoken word frequency (SpFreq), (ii) written word frequency (WFreq), (iii) phoneme length, (iv) phonological neighbourhood density (PND), (v) concreteness (Concr), (vi) imageability (Img) and (vii) number of syllables . After hearing each stimulus, subjects had 3.5 s to respond. During the response/sound window a black “+” was on the screen plus Yes/No indicating the correct orientation for response. Once a response had been received, a large blue cross (84 point font) appeared on the screen. After 1 s, this changed to a black cross (84 point font) which remained visible until the start of the next trial/event as detailed in Figure A long inter-trial interval  was employed to allow for the delayed hemodynamic response function observed with stroke patients, 62 and 3 resolution; TE/TR 2.4/1900 ms TI 900 ms).Structural and functional images were collected on a Siemens Trio  at the Royal Brisbane and Women’s Hospital. The design of the fMRI task was a pseudo-randomised event-related design consisting of two runs of approximately 14.4 minutes administered across one session. Gradient echo EPI images with BOLD sensitivity were acquired during each of the runs, resulting in 390 brain volumes, 36 × 3 mm slices, with a 0.3 mm gap, FOV 220x220mm, flip angle 90, matrix 64x64, TE 30 ms, TR 2210 ms. A high resolution 3D structural T1 weighted MP-RAGE image was also acquired ((0.9 mm)http://www.fil.ion.ucl.ac.uk/spm)) with MATLAB 2009a . To allow for steady state magnetization, five dummy scans were acquired at the start of each run and excluded from analysis. INRIAlign[1 image which had been acquired in the same scan session. The T1 image was segmented using New Segment[3).Images were processed and analysed using statistical parametric mapping software , incorrect trials were excluded from the time series. Realignment parameters in 6 degrees of freedom were included as regressors of no interest. Covariates were created to model the BOLD signal change associated with the different event trials and contrasts included concrete – abstract, concrete – pseudoword and abstract - pseudoword. The mean corrected reaction time was included as a parametric modulation per condition, similar to Binder et al.[A random effects analysis of the group was carried out on each condition. Differences in BOLD signal between the three conditions of concrete, abstract and pseudoword were evaluated using a regression analyses. The FWHM was calculated from the square root of the residuals  and useRegion of interest (ROI) analyses were carried out based on a priori hypotheses about hemispheric lateralization differences associated with the processing of concrete and abstract words and findings from the Binder et al. meta-analysis. As a rehttp://www.fil.ion.ucl.ac.uk/spm). Anterior ROIs (aSTG and aMTG) were subdivided based on the classification of Indefrey and Levelt[ROIs were created by using the IBASPM 116 Human Atlas in WFU PickAtlas, 69 in Sd Levelt of y < -d Levelt, a regiod Levelt were appd Levelt.F = 91.582, p < 0.0001). Participants responded fastest to concrete words followed by abstract words then pseudowords  = 18.248, p < 0.0001. Increased BOLD signal was observed for concrete (p < 0.0001) and abstract (p = 0.008) compared to pseudowords and concrete compared to abstract words (p < 0.0001). There was a main effect of condition for the right AG, F = 21.15, p < 0.0001 and increased BOLD signal for concrete (p < 0.0001) and abstract (p = 0.025) compared to pseudowords. Concrete words also elicited increased BOLD signal compared to abstract words (p = 0.007).Results from the ROI analyses are shown in Figure F = 8.812, p = 0.003. There was increased BOLD signal for concrete compared to abstract (p = 0.049) and pseudoword (p = 0.004). There was a main effect of condition for left MFG F = 9.897, p = 0.002 and increased BOLD signal for concrete compared to abstract (p = 0.008) and pseudoword (p < 0.0001). There was also a main effect of condition for left posterior cingulate F = 12.579, p = 0.001 with increased BOLD signal for concrete compared to abstract (p = 0.004) and pseudoword (p < 0.0001). A main effect of condition in the left fusiform F = 4.811, p = 0.03 was also seen with increased BOLD signal for concrete compared to pseudowords (p < 0.0001).A main effect of condition was observed for left SFG F = 7.49, p = 0.006), but with increased BOLD signal for concrete compared to pseudowords (p = 0.025).The left IFG , aSTG and aMTG ROIs were included to investigate association with abstract word processing. Of thesThe whole brain results for concrete greater than pseudoword, abstract greater than pseudoword and concrete greater than abstract are shown in Table , left MFG, left posterior cingulate. Significant signal differences were also observed in the left fusiform and left aMTG for concrete words but only for the concrete – pseudoword contrast. The AG was the sole brain region which elicited significant differences in activity between all three conditions, with concrete words eliciting greater activation than both abstract and pseudowords. Abstract words also produced greater activity than pseudowords in bilateral AG. Greater activity for abstract words compared to concrete words was not observed in any of the nine ROIs. Whole brain results showed bilateral activity for concrete and abstract words when compared to pseudowords. Bilateral activation was also seen for concrete words when directly compared to abstract words.There was a concreteness effect in terms of faster speed of response for concrete words compared to abstract words. Anatomical ROI analyses revealed significant increased signal for concrete words compared to both abstract and pseudowords in left and right AG, left SFGThe aim of this study was to investigate brain activity associated with recognising spoken concrete and abstract words. We predicted that concreteness effects would be reflected in reaction time data and accuracy of response. We also predicted that the use of an opaque pseudoword condition would provide a robust baseline against which to measure differential brain-related activity elicited directly in response to concrete and abstract spoken word recognition. We anticipated that concrete words would elicit increased brain activity in bilateral brain regions while abstract words would elicit greater activity in left-lateralised language processing networks. The present study revealed bilateral activity for concrete words, however we did not see a left-lateralised pattern of activity for abstract words. Our results confirm the involvement of the angular gyrus, posterior cingulate and dorsolateral prefrontal cortex in retrieving concrete versus abstract concepts during spoken word recognition, consistent with previous meta-analyses on concrete and abstract processing based primarily on visually presented words, 43. OveThe behavioural results showed that concrete items were responded to significantly faster than both abstract and pseudoword conditions and the abstract condition was significantly faster than the pseudoword condition. The observed increased efficiency for concrete over abstract words is in accordance with the two dominant theoretical accounts used to explain effects of concreteness; dual coding theory and contOf the nine ROIs selected to investigate concrete and abstract word processing, the right and left AG proved to be the sole brain region that showed significant differences in activation between all three contrasts with concrete words eliciting greater activity than both abstract words and pseudowords and abstract words eliciting stronger activation than pseudowords. Increased activation for concrete items in the right AG has previously been reported previously in visual word processing studies investigating concrete and abstract words, 39. TheThe ROI analysis also revealed greater activity for concrete versus abstract words in the left posterior cingulate, SFG and MFG. The posterior cingulate has previously been described as a connector hub, structurally linking other cortical and subcortical brain networks. Binder Significantly greater activity for concrete compared to abstract words in the left MFG and SFG also suggests a critical involvement of these frontal regions in the processing of concrete information. However, the specific role of these regions in semantic conceptual processing is not well understood and their activation in language-based tasks is commonly thought to be in response to more executive type task demands. The MFG and SFG have previously been associated with working memory and havePreviously, executive task-related engagement of frontal regions has been associated with increased effort and a corresponding increased response time, however the findings in this present study do not support this view as our behavioural results demonstrate clear involvement of these regions in concrete word processing without any associated increase in time on task. It is possible these frontal regions are recruited to monitor activation of the word candidate in the associated lexical-semantic network while a decision on lexicality is made, howeverROI analyses of both the left fusiform gyrus and aMTG elicited additional activation in the concrete greater than pseudoword contrast. The fusiform has been referred to as a basal temporal language area and our The aMTG was examined as an ROI to explore the aSTS, which was reliably activated by abstract words in the Binder et al. meta-analysis. In ordecharity might be associated with nonverbal, imaginal associative knowledge such as a collection tin or a red cross sign[Our results clearly demonstrate that lateral and ventral temporal regions were engaged in lexical-semantic processes but only for the concrete greater than pseudoword contrast. No differences were observed in these regions for either the concrete greater than abstract or abstract greater than pseudoword contrasts. As such, we tentatively propose that these regions may serve as lexical-semantic stores prior to subsequent processing and that the selective activation observed for concrete words when compared to pseudowords may be due to equivalent activation in these regions from processing nonverbal codes associated with abstract words and processing verbal codes associated with opaque pseudowords. To expand on the first point, we suggest that abstract words like concrete words are in fact associated with a set of nonverbal imaginal representations although they are just more weakly represented than the nonverbal representations associated with concrete words. For instance, a word such as oss sign and willSecondly, no significant differences in activation between abstract and pseudowords was observed in these lateral and ventral regions. Indeed, the lack of activation differences associated with abstract and pseudoword processing was a common finding in this study. Of the nine ROIs, only bilateral AG elicited a difference between abstract and pseudoword processing. We suggest that this finding may be due to the type of pseudowords employed in this study and the verbal codes attached to them. The opaque pseudowords shared features common to both the abstract and concrete words such as phonemes and syllables which enabled them to be processed in the same lexical networks as the real words. HoweverIn summary, our findings demonstrate that brain activity was reliably elicited during this auditory lexical decision paradigm in most of the regions previously showing activity for concrete word processing using visual word processing paradigms; bilateral AG, left posterior cingulate, SFG, MFG, fusiform and aMTG. In terms of the prominent theories regarding concrete and abstract word processing our findings for the recognition of spoken concrete words provide some support for both a dual representation and single system model. The selective activation of concrete words in three of the six ‘perceptual’ regions; left SFG, MFG and posterior cingulate, identified in the Binder et al. meta-analysis, provideHowever, we also observed quantitative activation differences for all three conditions in left and right AG suggesting that this brain region serves as a common zone which is differentially activated in response to both abstract and concrete terms compared to pseudowords. These findings provide support for the context availability theory which proposes that in a single system model, common regions will be implicated in real word processing but to differing degrees. Since concrete words have a richer set of contextual representations available to them than abstract words, they will elicit stronger activity than abstract words although activity will occur in common regions.Importantly, our findings provide support for the embodied abstraction theory proposed by Binder and Desai which suLastly, the ROI and whole brain results for abstract word processing in this present study are in agreement with both dual coding and context availability theory in that neither theory proposes that greater activity should be elicited by abstract compared to concrete words. However, our results also show minimal processing differences when abstract words were compared with pseudowords and this lack of activation difference was a pattern which was consistent in all ROIs except bilateral AG. This is an interesting finding and whilst we have provided a tentative explanation for the increased activity observed for concrete but not abstract words when compared to pseudowords, the present study cannot definitively say whether the this pattern of activity can be attributed to the type of stimuli, the task employed or modality of presentation. Future studies investigating spoken word recognition of concrete and abstract words will need to consider possible effects of the verbal and nonverbal, imaginal codes likely associated with abstract conceptual representations.Whilst this study has provided a number of findings regarding the processing of spoken concrete and abstract words during a lexical decision task, we acknowledge that there are some limitations with the stimuli selection employed. Firstly, we were unable to control for the first phoneme across the conditions and as such, this needs to be considered as a possible confound and the results interpreted accordingly. Secondly, the abstract stimuli selected for use in this study were also of very low imageability and it may be helpful in future studies to attempt to match for imageability across both concrete and abstract conditions although this is challenging.Findings from the ROI analyses in this study demonstrate that the recognition of spoken concrete words reliably activates a wide network of brain regions in the AG bilaterally, left posterior cingulate and left dorsolateral prefrontal cortex more than both abstract and pseudowords. Concrete words also activated regions in the left fusiform and left anterior MTG but this activation was only elicited for the concrete greater than pseudoword contrast. Our findings further define possible convergence zones proposed to bind supramodal input from modality-specific and heteromodal systems, and confirm the role of these regions in processes beyond initial modality specific lexical processing.


Multiple growth factors are known to control several aspects of neuronal biology, consecutively acting as morphogens to diversify neuronal fates, as guidance cues for axonal growth, and as modulators of survival or death to regulate neuronal numbers. The multiplicity of neuronal types is permitted by the combinatorial usage of growth factor receptors, each of which is expressed in distinct and overlapping subsets of neurons, and by the multitasking role of growth factor receptors, which recruit multiple signalling cascades differentially required for distinct biological outcomes. We have explored signalling robustness in cells where a given receptor tyrosine kinase (RTK) elicits qualitatively distinct outcomes. As the HGF/Met system regulates several biological responses in motor neurons (MN) during neuromuscular development, we have investigated the signalling modalities through which the HGF/Met system impacts on MN biology, and the degree of robustness of each of these functions, when challenged with substitutions of signalling pathways.Pea3 expression domain in the spinal cord can be accomplished through several alternative signalling cascades, differentially sensitive to the Pea3 dosage. Finally, we show that the regulation of MN survival by HGF/Met can equally be achieved in vitro and in vivo by alternative signalling cascades involving either PI3K-Akt or Src and Mek pathways.Using a set of mouse lines carrying signalling mutations that change the Met phosphotyrosine binding preferences, we have asked whether distinct functions of Met in several MN subtypes require specific signalling pathways, and to which extent signalling plasticity allows a pleiotropic system to exert distinct developmental outcomes. The differential ability of signalling mutants to promote muscle migration versus axonal growth allowed us to uncouple an indirect effect of HGF/Met signalling on nerve growth through the regulation of muscle size from a direct regulation of motor growth via the PI3 kinase (PI3K), but not Src kinase, pathway. Furthermore, we found that HGF/Met-triggered expansion of Our findings distinguish MN survival and fate specification, as RTK-triggered responses allowing substitutions of the downstream signalling routes, from nerve growth patterning, which depends on a selective, non-substitutable pathway.The online version of this article (doi:10.1186/s12915-014-0056-6) contains supplementary material, which is available to authorized users. The assembly of locomotor circuits relies on the parallel development of spinal motor neurons (MNs) and their target muscles, and subsequently, on their coordinated functional wiring. This involves the stepwise acquisition of cell diversification programs in both cell types, each of which leads to the generation of distinct subtypes, the subsequent matching of each motor pool with its cognate target muscle through topographical arrangement of their axonal projections, and finally a process that allows numerical matching of MN pool size to the size of the target muscle ,2. Durinin vivo [An additional level of complexity appears when considering signalling modalities. The actions of each growth factor receptor upon ligand binding involve the activation of cytoplasmic signalling pathways . The disin vivo –12. The Among the plethora of factors known to influence MN biology, we chose to focus on hepatocyte growth factor (HGF), a multifunctional growth factor whose functions are mediated by the Met RTK. HGF influences MN biology by acting both on MNs and on muscles. Produced by the limb bud mesenchyme, HGF first acts on muscle migration by triggering the delamination of myoblast precursors from limb level somites . This evcutaneous maximus (CM)) [pectoralis minor muscle, whereas it is no longer required to support CM MNs [Using tissue-specific ablation of the Met receptor, we have recently shown that as development proceeds, HGF acts on distinct MN pools, in which it successively controls several specific biological pathways . On a suus (CM)) ,9,16. Afus (CM)) , we recet CM MNs . Thus, tmet, including: a) the metd signalling dead allele [met2P and met2S signalling mutant alleles, in which the multifunctional docking site is converted into optimal binding sites for phosphatidylinositol 3-kinase (PI3K) or Src, respectively [metLacZ allele [metFlox allele [Pea3 expression domain in the spinal cord [Pea3 dosage; 3) regulation of MN survival by HGF/Met can be equally achieved in vitro and in vivo by alternative signalling cascades involving either PI3K-Akt or Src and Mek pathways.In this study, we took advantage of an allelic series of d allele ; b) the ectively ; c) an aZ allele  and d) ax allele ,21. We anal cord , can be 1349VHVNATY1356VNV) is permissive to the binding of multiple signalling adaptors  or for Src (Y1349/1356EEI), the Met2P and Met2S receptors, respectively, were designed to determine the impact of imposing the cytoplasmic effector through which Met can signal, thus raising its contribution above generic signalling levels , modifymet or hgf loss-of-function mutants [in vivo demonstration of a direct requirement for Met in axon growth and path-finding is still currently lacking. We have previously shown that neural-specific Met excision with a Nestin-cre driver was only complete by the end of the NOCD period [HGF, initially provided by the limb mesenchyme and later by the muscles themselves ,27,28, i mutants ,17,18, r mutants . Thus, i mutants ,17,18 an mutants , formal D period , preventCM muscle [CM MN axons to their target does not reflect a direct function of HGF/Met in axon growth, as it is conditioned by an early specification program establishing Pea3 expression domain in a MN pool, which is under the control of Met as well (see later). To circumvent these caveats and examine signalling modalities of HGF/Met in a system where they exert direct control of axonal growth and guidance, we chose to investigate the motor nerve pattern in the dorsal hindlimb. While Met expression in brachial spinal cord segments is restricted to the Pea3-expressing MN pool, in which HGF/Met controls cell fate specification, Met is also expressed in pools of lumbar MNs, including medial and lateral LMC MN columns at sciatic levels L4 to L6, in which Pea3 is not co-expressed  compared to controls . In WT embryos, the dorsal shank is mainly innervated by the peroneal nerve branch emerging from the sciatic plexus  muscle on its proximal extremity, and continuing its distal progression towards the foot in the space between the EDL muscle and the tibialis anterior (TA) muscle  of approximately 120° on average. Furthermore, at the contact site between the nerve and each muscle, a few axonal side branches leave the nerve to enter the muscle where they will later establish synapses with muscle fibres . Although we found reduced amounts of Pea3-expressing MNs in the lumbar region  Pea3 is not co-expressed with met in peroneal MN pools at L4–L5 levels  showing a reduced mass but almost normal shape, while muscles of the anterior group (including EDL and TA muscles) were severely reduced in size, and the distal muscle mass was completely absent . Furthermore, they indicate that distinct signalling cascades, such as those triggered by these two receptor variants, are not equivalent in their capacity to execute nerve patterning by Met.We first established a cartography of developing muscles in t Figure B,C,G. Ths starts . While ePea3-expressing MN population at brachial levels is constituted of two main subgroups of MNs [Pea3 expression is initially induced by glial-derived neurotrophic factor (GDNF), produced by the target muscle [Pea3 expression in additional neurons, a feed-forward induction process that leads to lateral and anterior expansion of the Pea3 expression domain  embryos, which also lack limb muscles [Pea3 on E12.5 spinal cords revealed the Pea3 expression domain in met2P/2P and met2S/2S spinal cords had a normal shape, contrasting with the reduced shape displayed in metd/d mutants  no detectable changes in the amount of Ret-expressing MNs were found in E12.5 metd/d, met2P/2P and met2S/2S brachial spinal cords  . Since at the stage at which MNs are collected for these primary cultures (E12.5) most Met-expressing MNs are equally present in all genotypes, , or by y Figure D, but noy Figure D. IntereP Figure D. Togeths Figure D. This il Figure E.in vivo. In contrast to early stages, once MNs have become dependent on muscle-derived signals for survival, depletion of limb muscles in met-signalling mutants causes limb MN death [Pea3-expressing neurons  to demonstrate the direct control by HGF/Met of the in vivo survival of a MN pool in the C8-T1 region corresponding to the pectoralis minor muscle [Nestin-Cre-mediated Met ablation occurred only after the onset of NOCD, this strategy allowed us not only to preserve muscle development, hence maintaining an unaltered amount of muscle in the limb, but also to bypass the early function of Met in the specification of the Pea3 pools and in motor nerve growth . In contrast to Nes-Metd/flox mutants, the numbers of MNs measured in the Nes-Met2P/flox and Nes-Met2S/flox mutants were equivalent to WT, with only minor non-significant reductions at T1 level  of this response, compatible with a positive outcome. Considered together, all components of alternative/substitutable pathways capable of mediating a given response can constitute an equivalence group, where the lack of one component  can easily be compensated for by the network of available relationships between the others –41. ThisConversely, a pathway is considered non-permissive when it is not substitutable with another one, and when activation of an alternative pathway will not lead to the same output .A growing set of data supports the idea that signalling effectors are in charge of selective biological responses following RTK stimulation. While the view 15 years ago conveyed the idea that a given signalling cascade would be uniquely required for a given response, it has now emerged that several alternative pathways, although distinct from each other by their components, can substitute for one another to activate a given biological response. Therefore, the robustness of a biological outcome correlates with the capacity of cells not only to buffer variations of signal intensities , but alsWhether certain pathways are equivalent (permissive) or not depends, on one hand, on the cell type  and, on the other hand, on the nature of the biological outcome. Our results distinguish three types of biological outcomes, which are discussed in the following paragraphs:2P and Met2S, with respect to axonal growth and intramuscular branching, are important for two main reasons:Pea3 MN pools). The amount of muscle mass present in the limbs due to limited myoblast migration varies from embryo to embryo, although to a similar extent in met2P/2P and met2S/2S embryos  result in the absence of nerve branches exiting the main peroneal nerve, even in a small subset of met2P/2P embryos. This reflects the fact that muscles are necessary for sending a branching signal. However, in the presence of equivalent amounts of muscle mass, such branches are unequivocally detected in met2P/2P but not in met2S/2S embryos, where the nerve pattern is indistinguishable from that seen in metd/d mutants. This indicates that Met signalling is essential for the decision of motor axons to exit the main nerve and enter the muscle, provided that the competent signalling effectors are mobilised. According to these results, the muscle-derived signal may be either HGF itself or a protein necessary for HGF to bind/activate its receptor, such as enzymes involved in HGF precursor cleavage and/or in its extracellular diffusion [They allow unambiguous uncoupling of Met requirements for axonal growth from muscle development, and they also allow distinguishing of a nerve pattern defect from the consequences of an altered MN specification process . These results apparently contrast with other ligand/receptor systems in which Src family kinases have been reported to be required for axon guidance but not for survival of MNs [Drosophila Src homologue causes aberrant midline crossing indicating that Src is required to inhibit crossing of longitudinal axons [2S may have turned an attractive/positive response into a negative/repulsive one, for example by mobilising a cascade normally involved in a negative response to another signal. In such a context, Src activation would therefore not be dispensable, but rather would antagonise the normal function of Met. In contrast, the PI3K pathway appears as a permissive pathway, which may serve as a switch to activate responsiveness or as a rheostat to modulate the intensity of a unique one-sided response of axonal growth [The complete inability of the Metl of MNs . In thisl of MNs , a decisl of MNs ,47–49. Sal axons . A possial axons ,52, but al axons . Thus, fl growth . What del growth –57. An aWe showed that switching Met binding preferences towards Src family kinases precludes axonal branching to target muscles by HGF/Met, while accommodating PI3K recruitment ensures correct peroneal nerve patterning in the hindlimb Figure . These d2S receptor to execute axon guidance decisions, the cascade of events allowing expansion of the Pea3 expression domain in the brachial motor columns is efficiently executed by both Met2P and Met2S receptors, the two pathways being, however, differentially sensitive to Pea3 gene dosage  and Etv5 expression in sensory neurons by target-derived neurotrophic factors such as NGF [2S receptor for survival also involved the ERK pathway through Mek activation. It is therefore tempting to speculate the existence of a Src-Mek-Pea3 circuit available downstream of HGF/Met in MNs. Consistent with this hypothesis, Pea3 was shown to be involved downstream of v-src signalling to activate transcription of collagenase 1/MMP1 [In contrast to the clear incapacity of signalling by the Mete Figure . Pea3 exhial MNs . We have embryos  and with embryos . The difgnalling –62. A reh as NGF . Our owne 1/MMP1 .Pea3 loss-of-function disrupts Src activation and cell mobility in Pea3-null fibroblasts, indicating that Src can also be activated and required downstream of Pea3 [Pea3 expression does not impair propagation of Pea3 expression in a WT Met signalling context, but becomes critical in the context of a signalling-switch mutant of Met predominantly relying on Src activation. In contrast, the fact that signalling by the Met2P receptor is not sensitive to reduced Pea3 dosage indicates that production of the signal mediation expansion of Pea3 expression can also be induced by other transcription factor(s). Thus, the robustness of the cell fate specification function triggered by the Met RTK relies on a complex signalling circuit possibly composed of a combination of distinct transcription factors, the activity of which is modulated qualitatively and/or quantitatively by upstream signalling cascades.Moreover,  of Pea3 . Such a 2P or Met2S receptors were equally capable of substituting for the WT pathways in regulating MN survival. This is supported by primary MN culture experiments and by in vivo studies on Met-dependent pectoralis minor MN pool survival [In vitro, survival by HGF in met2P/2P MNs is selectively mediated by PI3K, but not by Src or Mek, whereas in met2S/2S MNs, survival requires Src and Mek, but not PI3K. Neuronal survival by numerous neurotrophic factors, including HGF in MNs [2S implies either a parallel action of Src and Mek-ERKs pathways, or the existence of a circuit involving activation of Mek-ERKs by Src. A previous report on chick MNs has indicated that Mek signalling is dispensable for their survival [in vivo), the level of activation of each individual pathway may not be sufficient to promote survival independently of the others. This scenario coincides with the signalling requirements for survival we have evidenced in vivo, in the context of our neuronal-restricted Met specificity-switch mutants.Finally, we found that signalling pathways mobilised by either the Metsurvival  using neF in MNs , involveF in MNs ,67. BesiF in MNs . For exaF in MNs ,35,69–71survival . However2P and Met2S receptors are adequate for survival of MNs, but not of embryonic hepatocytes both in vitro and in vivo [Intriguingly, the signalling pathways initiated by Met in vivo –25. Such in vivo . It is iin vitro. This contrasts with the fact that Src is dispensable downstream of Met2P and PI3K is dispensable downstream of Met2S. A possible explanation of this apparent discrepancy could be that in MNs, Met specificity-switch receptors behave as mild gain-of-function mutants for some pathways, while being incapable of activating the others. This increased level of activation of individual signalling routes downstream of Met2P or Met2S would raise their activity above a threshold necessary to activate effectors of survival, whereas this threshold is only reached by the combined activity of PI3K, Src and MAPK pathways downstream of WT Met. According to this scenario, such increased levels above the threshold could alleviate the simultaneous requirement for the other pathways to induce a survival response successfully in MNs. As mentioned above, this contrasts with hepatocytes, in which signalling initiated by Met2P or Met2S receptors is insufficient to ensure a survival response to HGF [The high plasticity of signalling networks controlling MN survival is particularly intriguing, when taking into account the unexpected discovery that PI3K, Src and Mek-ERK pathways are all necessary downstream of the WT Met receptor, each of them being, however, insufficient on its own to provide a robust survival response in the absence of the other pathways e to HGF –25, thusHow can we interpret the specificity of signalling pathways necessary for axonal growth compared to the plasticity allowed when executing RTK-induced MN survival or specification responses? Axon guidance is a complex example of a biological response regulated by several non-equivalent signalling components. Their activation leads to choices between multiple qualitatively different outcomes, such as stop, go, turn, exit, etc., thereby requiring intercalation of multiple signalling systems to gate the decision-making process at the level of the growth cone. Such a context implies that some signalling cascades need to be specifically associated with selective outcomes rather than being permissive, for the proper integration of multiple instructive axon guidance signals. In contrast, MN survival and specification are dichotomic choices at the cell level, integration of which at the tissue level involves the quantitative assessment of individual neuron responses to the limited amount of neurotrophic factors produced by target tissues. Adaptation of the MN numbers in each pool to the size of its cognate muscle target is achieved by a mechanism capable of measuring accurately the amount of target-derived signal available, and of adjusting this amount with respect to an intrinsic threshold within each cell, to block the apoptotic cascade or to activate the specification program. Likewise, raising RTK signalling levels above endogenous thresholds can enhance the neuroprotective effect and limit susceptibility to MN diseases . The plamet gene used in this study  has been described previously [+ alleles, which contained a neo selection cassette flanked by LoxP sites, were referred to as met2Pneo and met2Sneo. Alleles without neo were obtained after excision of the neo cassette by crossing mutant mice with the Deleter-Cre transgenics [metLacZ allele is an alternative knockout/knock-in allele of met in which the LacZ reporter gene also reflects expression of the endogenous met gene [LacZ expression is conditioned by the removal of a Lox-stop-Lox cassette [Pea3-lacZ mice were used with the permission of Arber and Jessell, and genotyped as previously described [The generation of the different signalling alleles of the eviously ,15. The nsgenics . Genotypnsgenics ,12. The met gene . In the cassette . In the cassette . Pea3-laescribed . Animalsmet, MyoD (obtained from Ponzetto), Pea3, (from Jessell), Ret  and ChAT . Spinal cords were flat mounted as open-book preparations with MNs on the upper side, and imaged using a Zeiss Axiophot  or Leica stereomicroscope . Whole-mount embryos were partially cleared in 50% glycerol and imaged using a stereomicroscope.Embryos were collected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA). ISHs were performed with the relevant RNA probe on either whole-mount embryos, dissected limbs or spinal cords, according to previously published procedures . Whole spea3LacZ allele, we employed the traditional protocol using X-Gal substrate in combination with ferric and ferrous ions. Briefly, spinal cords were dissected from freshly collected embryos, fixed in 4% PFA in PBS (40 min), rinsed in PBS, and incubated in X-Gal in combination with potassium ferricyanide and ferrocyanide (FeCN). To detect β-galactosidase in the spinal cords of embryos carrying the metLacZ allele, we used an alternative method based on Salmon-Gal  in combination with tetrazolium salt [2, 1 mg/ml Salmon-Gal (stock solution 40 mg/ml in dimethyl sulfoxide (DMSO)) and 0.33 mg/ml NBT (stock solution 75 mg/ml in 70% DMF)), for about 30 to 40 min at 37°C. In all cases , staining was terminated by rinsing in PBS, and post-fixing in 4% PFA. Spinal cords were flat mounted in 1 volume glycerol/1 volume 4% PFA for imaging.Staining for β-galactosidase activity was performed using two alternative methods. For spinal cords of embryos carrying the ium salt . BrieflyE12.5 mouse embryos were collected in Hibernate (E) medium (Life Technology) with B-27 supplement (Life Technology) and kept on ice until the dissection, while the genotype was determined by PCR. Ventral spinal cords from an equal number of WT and mutant embryos were dissected. MNs were isolated from either brachial + lumbar, or from cervical + thoracic + sacral segments, by a previously described method  involvin2O2 in Dent’s solution and further rehydrated through a progressive methanol series. Antibody incubations were performed in 80% newborn calf serum, 20% DMSO, 0.5% triton with thimerosal. Antibodies used were the following: anti-neurofilament antibody (2H3 and DSHB) and HRP-conjugated goat anti-mouse antibody (Sigma). Staining was developed using DAB Tablets . Embryos were cleared for imaging in BABB .Embryos were collected in cold PBS, fixed for 2 h in 4% PFA in PBS, and post-fixed overnight in Dent’s fixative  at 4°C. Whole-mount anti-neurofilament immunohistochemistry was performed as previously described . BrieflyQuantification of signal intensity was performed using the ImageJ software. Briefly, images of ISH were first converted to a grey scale and inverted to a negative scale . Signal intensity was measured along a horizontal line of a given pixel length (matching half a flat-mounted spinal cord, always positioned where indicated with dotted lines on Figure t-test when data were showing a normal distribution, and by the Mann–Whitney test otherwise. * indicates P < 0.05 and ** indicates P < 0.001.Results were expressed as mean ± standard error of the mean. Statistically significant differences were assessed by unpaired Student’s


Ventilator-associated pneumonia (VAP) can have a clear onset or may be a result of the gradual appearance of symptoms and signs of VAP . The aim of this paper is to describe the VAP development process with the intention of discriminating between those pneumonias with a clear beginning and those that are diagnosed after a period of maturation. In addition, we evaluate the effect of the starting time of antibiotic treatment in both situations.Consecutive ventilated patients fulfilling VAP criteria were included. The patients were monitored for clinical, microbiological, and inflammatory signs. Patients with VAP were classified into two groups: (1) nongradual VAP  and (2) gradual VAP (progressive appearance of signs and symptoms throughout the pre-VAP period [<96 h to >24 h before VAP diagnosis]).n = 38, 88 %) had late-onset VAP. Antibiotic treatment was given to 34 (79 %) patients with gradual VAP in the pre-VAP period, and empirical antibiotic treatment was appropriate in 22 patients (51 %). The patients with an appropriate empirical treatment had a higher percentage of early clinical response to treatment . An attempt was made to find a diagnostic test capable of identifying the infectious process underway, but clinical scales and biomarkers of inflammation helped us to achieve acceptable results.A total of 71 patients with VAP were identified, of whom 43 (61 %) had gradual VAP, most of whom (Gradual emergence of VAP, mainly of late onset, is a common condition. Clinicians should be aware of this gradual onset of the infection to establish an early antibiotic treatment, even before the classic diagnostic criteria for VAP are applied. However, ICU mortality and 28-day mortality were not influenced by the use of appropriate treatment in the pre-VAP period.Appropriate antibiotic treatment introduced in the pre-VAP period in the gradual VAP group was associated with a higher rate of early clinical response . The AUCs for CRP and PCT were 0.678 and 0.505, respectively. CRP and PCT were ineffective in identifying gradual VAP (Table Recognized cutoffs for antibiotic guidance in serum PCT (≥0.5 ng/ml and >1 ng/ml) and modified mCPIS (>5 points) were assessed to establish their capacity to identify an ongoing infection. Better rates were achieved with mCPIS  LH+ 6.67, LH− 0.60, AUC 0.670, 95 % CI 0.545–0.796; To our knowledge, we are the first to describe the concept of gradual VAP. Establishment of gradual VAP is a common process, mainly in the case of late pneumonia. More importantly, our data support the need to initiate proper antibiotic treatment in the early stages of infection without waiting for all the diagnostic criteria for VAP to be met. However, in this study, we were unable to identify effective diagnostic tools for gradual VAP, apart from the usual clinical criteria.ventilator-associated tracheobronchitis in their study), and VAP. During this developing period, the patient’s immune system will attempt to prevent the spread of the organism, and signs of this confrontation will be observable in the form of fever, leukocytosis, and purulent bronchial secretions. This process is typical in late-onset VAP , and its absence is probably justified by the presence of a sudden and large bacterial inoculum into the lung .In line with the idea proposed by Craven and Hjalmarson , our stuOur results do not support a nosological separation between VAP and VAT. As described in the literature, VAP and VAT are distinguishable only by the presence of visible lung infiltrate on a chest x-ray. However, the discriminative ability of a portable chest x-ray is more than doubtful. In fact, even in community-acquired pneumonia, plain radiography has shown a diagnostic inaccuracy of 17 % compared with chest computed tomography . NonetheIn our study, appropriate antibiotic treatment introduced in the developing period in gradual VAP was associated with a higher rate of early clinical response. Therefore, identifying the right moment to start antibiotics in this continuum between colonization and VAP seems to be a challenge. PCT may be a valuable tool in this context. Stolz et al. showed a safety algorithm for antibiotic discontinuation based on serum PCT after 72 h of antibiotic treatment in 101 patients with VAP . In our According to our results, in the event of suspected VAP, a mCPIS score higher than 5 could indicate antibiotic initiation. However, the lack of sensitivity of mCPIS in our study precludes a safe use of this tool to rule out the existence of an ongoing infectious process. Using a different methodological approach, Singh et al. demonstrated the usefulness of CPIS to safely withdraw antibiotics after 72 h of treatment in patients with an initial but incomplete diagnosis of VAP (CPIS ≤6 points at inclusion) .Our study has several limitations. The sample size is not large and could be responsible for the observed negative results. However, our sample size is not too far removed from that used in studies by Stolz et al.  and SingNot all pneumonias are developed similarly, which is probably due to differences in the pathogenic mechanism. But what is really important is the identification of the infectious process in place, in order to start appropriate antimicrobial therapy. According to our results, antibiotic treatment should not be delayed, even if the patient does not meet the prescriptive diagnostic criteria for VAP.2/FiO2, ratio of partial pressure of arterial oxygen to fraction of inspired oxygen; PCT, procalcitonin; SOFA, Sepsis-related Organ Failure Assessment; TBAS, tracheobronchial aspirates; VAP, ventilator-associated pneumonia; VAT, ventilator-associated tracheobronchitis.APACHE II, Acute Physiology and Chronic Health Evaluation II; BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; CPIS, Clinical Pulmonary Infection Score; CRP, C-reactive protein; GNB, gram-negative bacteria; ICU, intensive care unit; mCPIS, modified Clinical Pulmonary Infection Score; MV, mechanical ventilation; PaO


HIV infected patients have a higher risk of developing cancer than the general population. Kaposi's sarcoma, non-Hodgkin's lymphoma, primary CNS lymphoma and invasive cervical cancers are considered as AIDS defining [1]. An increased incidence in recent years has been reported also for other malignancies after the introduction of cART .We performed a retrospective multicentric evaluation of all HIV infected patients with both AIDS and non-AIDS defining neoplasms at six Infectious Disease Units spread throughout Italy since 1991 through 2013. Cases were compared with equal number of controls without neoplasia followed at the same institutions, matched for length of HIV infection.Since 1991, 339 consecutive cases of malignancy were collected from the six convening centres, including approximately an equal proportion of AIDS (51.2%) and non-AIDS defining tumours. Mean prevalence of tumours among centres was 8.3% (r. 6.1%–9.6%). Mean age at tumour diagnosis was significantly lower than in controls . As to risk factors for HIV infection, approximately 1/4 (26.1%) of patients were drug abusers, in equal proportion as in controls. A remarkable higher proportion of cancer patients had CD4 T-cell counts <200 c/mmc at time of diagnosis . Seventy percent of tumours occurred in males; 52.8% of tumour patients were diagnosed with AIDS before and 19.0% at the time of tumour diagnosis. Ninety (28.1%) tumour patients were dead at the time of data collection, a much higher proportion than among cases . Deaths among non-AIDS (20.8%) and AIDS defining tumour patients (35.0%) were significantly different (p=0.005). Predictors of AIDS defining tumours at the time of data collection were: male sex , CD4 T-cell counts <200 c/mmc , whereas being cART treated at the time of tumour diagnosis was protective . In the final multivariate model of logistic regression, male sex  and not being cART treated  held as independent predictors.Our retrospection revealed a considerably high proportion of non-AIDS defining tumours, apparently at rise in recent years. We registered high prevalence of tumours in each centre. Absence of cART seemed related with AIDS defining tumours: once more prevention of late presentation appeared the way to avoid worse prognosis in this setting.


Although AIDS-related deaths have had significant economic and social impact following an increased disease burden internationally, few studies have evaluated the cause of AIDS-related deaths among patients with AIDS on combination anti-retroviral therapy (cART) in China. This study examines the causes of death among AIDS-patients in China and uses a methodology to increase data accuracy compared to the previous studies on AIDS-related mortality in China, that have taken the reported cause of death in the National HIV Registry at face-value.Death certificates/medical records were examined and a cross-sectional survey was conducted in three provinces to verify the causes of death among AIDS patients who died between January 1, 2010 and June 30, 2011. Chi-square analysis was conducted to examine the categorical variables by causes of death and by ART status. Univariate and multivariate logistic regression were used to evaluate factors associated with AIDS-related death versus non-AIDS related death.3 at the time of cART initiation , ART naïve  and age <39 years .This study used a sample of 1,109 subjects. The average age at death was 44.5 years. AIDS-related deaths were significantly higher than non-AIDS and injury-related deaths. In the sample, 41.9% (465/1109) were deceased within a year of HIV diagnosis and 52.7% (584/1109) of the deceased AIDS patients were not on cART. For AIDS-related deaths (n = 798), statistically significant factors included CD4 count <200 cells/mmFor the AIDS patients that were deceased, only those who initiated cART while at a CD4 count ≥200 cells/mm3 were less likely to die from AIDS-related causes compared to those who didn’t initiate ART at all. The AIDS epidemic is a serious global public health issue . AIDS-re3, and this threshold for eligibility was further increased to <350 cells/mm3 in 2012 [China’s national cART program began in 1999, and in 2003 the "Four Frees and One Care" policy led to improved access to cART provided free-of charge . In 2008 in 2012 .The positive impact of cART on reducing AIDS related deaths is well known. However, relatively few studies have examined AIDS-related deaths among HIV patients on cART in China. Furthermore, the existing studies have used data from the Chinese National HIV Registry. This data may suffer from misreporting, particularly in regard to cause of death. This study explored whether or not cART made a difference in the proportion of causes of death that were AIDS-related as compared to non-AIDS related, and also explored the factors related to dying from these causes using a sample including all Chinese HIV positive individuals who died in three geographical areas, spread over three Chinese provinces between January 1, 2010 to June 30, 2011. These areas were chosen to represent key drivers of the epidemic in China historically–injection drug use (IDU), blood plasma donation, and sexual transmission–and to investigate if there were any systematic differences among these highly differentiated demographics. Before 1995, the key driver of the epidemic was IDU. This was followed by former plasma donors (FPDs), commonly located in rural areas in central China, infected at mass as a result of clinical malpractices by private actors on the poorly regulated blood product market in the mid 1990’s . This grIn order to evaluate if there are any systematic differences in the causes of death between deceased AIDS patients from areas with different key drivers of the epidemic in recent years, all reported deceased AIDS cases  in Dehong Prefecture in Yunnan Province, Zhumadian City in Henan Province, and the entire Jiangsu Province between January 1, 2010 and June 30, 2011 were included in the sample. These areas were chosen based on the understanding that the HIV epidemic in these areas have been primarily driven by a certain mode of transmission . Even though FPD as a mode of transmission is negligible for newly infected cases in recent years, a significant proportion of people living with HIV in China were infected through plasma donation, and it is therefore of interest to investigate whether the causes of death differs for this subpopulation. The FPDs have been living with HIV a long time, many of them without access to treatment for years after being infected, and a significant proportion of these cases are in a stage of clinical AIDS.The National Center for AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease Control and Prevention (China CDC) National Surveillance HIV/AIDS Reporting System (NSHRS) is a comprehensive internet-based database first used in 2005 that covers more than 3,000 counties. The lists of all deceased AIDS cases between January 1, 2010 and June 30, 2011 in the specified regions were downloaded from the database. The data included demographic information, history of HIV related risk behaviors, date of cART initiation, date of death, date of HIV diagnosis and CD4 count at cART initiation.All diagnosed and confirmed HIV-infected cases in China are required by law to be registered in the National HIV Registry within 24 hours of detection, and all HIV positive individuals are required to go through regular follow-up medical examinations ..A detailed twenty nine-item questionnaire was developed specifically for this study in order to gather more complete information on the cause of death for the subjects. Additional questions included demographics, history of high-risk behaviors, and co-existing co-morbidities before time of death First, investigators completed a review of the death certificates for the subjects in the sample. Out of the 103 subjects  where the investigators could access the death certificates, only 35 were judged to be complete enough to accurately determine cause of death. For the remaining cases, investigators went on to review medical records to determine the cause of death. Out of 374 medical records that could be found for the remaining subjects without confirmed cause of death , 150 were judged to be complete enough to accurately determine the cause of death. For the remaining 935 , personnel involved in the study proceeded to complete the household survey .Health care workers conducted semi-structured face-to-face interviews, where they were instructed to follow the format of the survey for the interviews, with key informant family members, close friends, former partners or the village doctor in a private room after obtaining oral informed consent. The completed questionnaires were then used to determine cause of death. For the sample used for analysis , death certificates were used for 3.2% , medical records 13.5%  and informant data 83.3%  of the cases to determine cause of death.All collection of medical information and household surveys were conducted by certified health professionals, who had provided healthcare services to the deceased AIDS patients and were familiar with the patient’s family members. These health professionals also spoke the local language in the regions where they conducted the survey.3 confirmed with western blot testing or presence of opportunistic infections, in accordance to the Chinese Ministry of Health HIV Infection Diagnostic Criteria.In order to streamline the coding for cause of death in the subjects where questionnaires were used, ICD-10 coding with the Chinese Ministry of Health HIV Infection Diagnostic Criteria  and the This coding included three main categories with 49 sub-categories: AIDS related death (30 sub-categories), non-AIDS related death (11 sub-categories), and injury related death (8 sub-categories). AIDS-related death was defined as opportunistic and parasitic infections, AIDS-related malignancy, and other AIDS specified disease syndromes, including encephalopathy, lymphoid interstitial pneumonia, generalized lymphadenopathy, or AIDS wasting syndrome as main cause of death. Non-AIDS related death was, in accordance with the ICD-10 coding, defined as digestive diseases (including hepatitis B or C), cardio-cerebrovascular disease and disease of respiratory, urogenital, endocrine, hematological, and central nerve systems. Deaths from drug overdose, poisoning, suicide, homicide, or fracture was categorized as injury related death. For the analysis in this paper only the main categories  were used, but disaggregated figures can be found in The final decision for cause of death for each AIDS subject was confirmed by members of investigation group who were trained in the standardized coding procedure and supervised by experts from NCAIDS. All causes of death were adjudicated by NCAIDS.All survey data was entered and verified at the local CDC level in each of the three geographical areas and validated using Epidata 3.1 . Further3, and ART initiated at CD4 count ≥200 cells/mm3. Third, stepwise univariate and multivariate logistic regression with significance criterion p<0.05 were used to construct the most robust correlates of cause of death among the subjects. A multivariate model where the outcome of AIDS-related death as compared to non-AIDS related death was constructed. Kaplan-Meier curves were constructed as a visual tool to show the time of death stratified by cART status.The causes of death were investigated using three distinct methods. First,Chi-square was used to examine any differences in the proportion of cause of death  in the three regions. Second, Chi-square was used to examine any differences in the proportion of death by ART category: ART naïve (never received ART), ART initiated at CD4 count <200 cells/mmIn order to complete the questionnaires, trained national NCAIDS CDC staff invited health care workers from both local hospitals and community clinics to provide preliminary information, and when needed, families and neighbors of the deceased were asked to provide supplementary information that could help determine cause of death.Further assistance was obtained from specialists at the China CDC National Cause of Death Surveillance Department in Beijing and China CDC specialists on ART and AIDS epidemiology. To ensure follow-up and completion, staff from local CDCs in the three areas regularly checked the collected information on a daily to weekly basis to ensure progress and compliance. Data was first obtained on the local level and controlled for completeness and accuracy by NCAIDS CDC Staff in Beijing.Data downloading was in compliance with the Chinese national regulatory standards of surveillance data, and the research protocol was reviewed and approved under the supervision of the Institutional Review Board of National Centre for AIDS/STD Prevention and Control, China CDC . All personnel involved in the study were allowed to access data from the National HIV Registry using standard China CDC procedures. Oral informed consent was obtained from all informants during the household survey, and a field supervisor was present in the field and required to sign the questionnaire if oral informed consent was given. The procedure for obtaining oral consent was also approved by the Institutional Review Board of National Centre for AIDS/STD Prevention and Control, China CDC. All records with personal information were eliminated and de-identified before data analysis.3 and initiated cART therapy at CD4 counts ≥200 cells/mm3), all socio-demographic and behavior-related variables for the sample  were found to be statistically significant using Chi-squared tests (p<0.001)  .3, and 32.2% (169/525) had CD4 cell count ≥200 cells/mm3 at time of ART initiation. Even with ART initiation, a significant proportion of deaths occurred within three month from ART initiation .Of the full sample used, a majority of those deceased (52.7%) never initiated cART . Of the The cause of death determined by the methodology in this study differed greatly from the data in the National HIV Registry. Only 69.1% of the study population had a registered cause of death that was consistent in the registry and the data obtained for this study through death certificate/medical record review and household survey . AIDS related deaths accounted for 72.0% (n = 798), of which 57.6% (460/798) were classified as AIDS-related infections, 27.7% (221/798) as other AIDS specified diseases or syndromes, 7.3% (58/798) as AIDS related malignancy and 7.3% (59/798) could not be sub-classified. Non-AIDS related deaths accounted for 19.5% , of which 19.2% (63/216) were sub-classified as hepatitis B or C, 24.1% (52/216) other digestive diseases, and 24.1% (52/216) cardio-cerebrovascular disease. Injury related deaths accounted for 8.6%  of AIDS patients deceased during the sample period, of which the largest subcategory was physical injury  (S2 Table).A total of 1,109 subjects were successfully assigned a cause of death using the methodology of this study , and the lowest in Jiangsu Province  . There w3, those initiating cART at CD4 counts ≥200 cells/mm3 exhibited a significantly lower proportion of AIDS-related deaths , but no statistically significant difference in injury-related deaths .Compared to subjects who were either cART naïve and those who initiated cART with CD4 count <200 cells/mm3 (p<0.0001). This was also true for non-AIDS related deaths  among cause of death  (p = 0.371) . Among cd deaths .The average time to death from HIV diagnosis was 37.3 months for ART-naïve subjects, 52.8 months for those who initiated ART with CD4 count <200 and 65.5 months for those who initiated ART with CD4 count ≥200.Given that we did not find a significant difference between the proportions for cause of death across the three geographical regions , ART naïve AOR 1.69  and age <39 with AOR 2.96   .In this study, we demonstrated that in for the subsample of deceased AIDS patients in three Chinese provinces, only those who initiated cART while at a CD4 count ≥200 cells/mm3 were less likely to die from AIDS-related causes compared to those who didn’t initiate ART at all. However, this could partly be due to the fact that the group of cART naïve subjects might have had a wide range of CD4 counts. Previous studies have shown that cART reduces HIV-associated mortality. Chinana et al. showed that the proportion of total deaths caused by AIDS in Malawi fell from 42% to 17% due to cART , and Bri3, compared to the subjects that initiated cART at CD4 count <200 cells/mm3 and those that were cART naïve. These results highlight the importance for further efforts to increase the proportion of early-diagnosed cases. This study was conducted before the cut-off level for cART eligibility was increased to CD4 count ≥350 cells/mm3 in 2008 but ought to still be relevant given that the antiviral drugs and protocol used in China have not been changed after this policy change, and that late diagnosis still is a salient issue in many parts of the country [Despite attempts to provide free cART to those eligible in China, this study shows that for this sample, 52.7% of deceased AIDS patients were not on cART, and 57.4% of those passed away within one year of being diagnosed with HIV. The corresponding figure for the full sample was 41.9%. Kaplan Meier curves showed that time to death occurred later for the subpopulation where cART was initiated at CD4 count ≥200 cells/mm country .The proportion of injury-related deaths in the sample is approximately at the same level as for the general Chinese population . For allDespite differences in the main mode of HIV transmission for the three geographical regions, we found uniformly high AIDS-related deaths and no significant difference in outcomes between the three regions or modes of transmission.AIDS-related deaths, as compared to non-AIDS related deaths, were more common for individuals aged 39 or below. This is may be due to the choice of sample and the behavioral differences these groups are likely to exhibit.Many of the older subjects have been diagnosed for a long time (33.9% aged >39 and 26.1% aged ≤39 were diagnosed at least 5 years ago), making this a subsample of individuals who have been living with HIV for an extended period of time. It is therefore likely that they lead distinctively different lives from the subjects who have been diagnosed more recently, and this older subsample are also likely exhibit higher rates of cART adherence. A large proportion of the older individuals in the sample were also from the FPD group. This group has been thoroughly screened, and thus, those living with HIV/AIDS are more likely to be on cART . Additionally, since older age is more commonly associated with all causes of death, many non-AIDS related causes of death are more common for this age group.Lastly, this study has given indications that a large proportion of causes of death may be misreported in the National HIV Registry. If this is the case not only in these isolated geographical areas, it could lead to biased results in studies on AIDS-related mortality in China, particularly if the misreporting were systematic in any way . Against this background, it may be of relevance to provide further training for local CDC staff to improve the mortality data quality.First, as some causes of death were not available in medical records, we commonly had to rely on information reported by family members or neighbors. Although recall bias or incomplete information could be an issue, trained CDC staff made efforts to follow-up with physicians or health care workers to confirm records, and we find no reason why this should bias the study in any particular way either by geographic region or by cause of death. We also confirmed accounts in the field and reconfirmed with available sources to obtain maximum accuracy. Second, even though medical records were thoroughly reviewed, some records might have been incomplete or inaccurate and some factors that could have influenced the cause of death might not have been recorded and thus omitted in this study. Third, patients included in the study that were reported to be on cART might have exhibited varying levels of medication compliance and been on different cART regimens that might have influenced mortality outcomes –30. HoweS1 Table14 patients had age <15 years.(DOCX)Click here for additional data file.S2 TableDigestive Disease Statistics do not include subcategories of hepatitis B & C.(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Social media has reshaped individual and institutional communication. The unrestricted access to spontaneous views and opinions of society can enrich the evaluation of healthcare interventions. Antimicrobial resistance has been identified as a global threat to health requiring collaboration between clinicians and healthcare users. We sought to explore events and individuals influencing the discourse about antibiotics on Twitter.www.topsy.com) was used to detect daily occurrences of the word ‘antibiotic’ from 24 September 2012 to 23 September 2013 in worldwide Tweets. Activity peaks (message frequency over three times that of baseline) were analysed to identify events leading to the increase.A web-based tool  referred to ‘antibiotic’. The greatest activity increases appeared after: (i) the UK Chief Medical Officer's (CMO's) declaration of antimicrobial resistance as a national risk (January 2013 and March 2013); (ii) the release of the US CDC's report on antimicrobial resistance (September 2013); and (iii) the US FDA announcement on azithromycin safety concerns (March 2013). The CMO report in March reached an estimated worldwide audience of 20 million users in a single day. However, the frequency of antibiotic Tweets returned to basal levels within 48 h of all four peaks in activity.Institutional events can rapidly amplify antibiotic discussions on social media, but their short lifespan may hinder their public impact. Multipronged strategies may be required to prolong responses. Developing methods to refine social media monitoring to evaluate the impact and sustainability of societal engagement in the antimicrobial resistance agenda remains essential. For example, public health commissioners and practitioners have been able to find feedback that has traditionally been difficult to obtain from populations where policies or interventions have been implemented.4 The use of social media also allows unrestricted access to the opinions and sentiments generated spontaneously by those same populations.The development of social media and social networks has provided unprecedented communication opportunities between individuals, companies and organizations. It is estimated that almost 60% of all Internet users engage with a social media platform.5 Generally, users will post about themselves to share information6 and around half of users in the USA use Twitter as a news source.7 Some users can have large numbers of followers (hundreds of thousands to millions of people), such as celebrities and news organizations. This direct access helps to explain the popularity of the platform. The use of web analytic measures to rank users according to their online social influence,9 although debated in terms of accuracy, does allow for the identification of many key influencers within the crowd of individuals and organizations, a useful feature that could be used to maximize the impact of a given health campaign or message.Twitter (https://twitter.com) is one of the many different social networks developed over the last decade. This free service allows the mass submission of messages of up to 140 characters, pictures and links (‘Tweets’). These messages are time-stamped and, if enabled, pinpoint the geographical location of the user at the time of posting. Users can be ‘followed’, which means that their messages will be delivered automatically to all their ‘followers’. Followers can endorse messages by ‘favouriting’ or disseminating them (‘Retweeting’) to their own followers  coauthored a short book explaining the tangible dangers posed by resistant bacteria.22Amongst current global public health problems, antimicrobial resistance has been cited by the WHO as one of the top three threats to human health.23 Using Twitter as a model platform, we set out to investigate patterns of discussions about antibiotics and to identify drivers that may increase the volume of discussions.Within this context of societal engagement there is potential for social media platforms to be used to raise awareness of growing antimicrobial resistance, to monitor the impact of campaigns and to seek better understanding of the perspectives of individuals on the use of antibiotics. To date, there has been limited analysis of how and when people talk about antibiotics on social media.www.topsy.com) was used to identify daily occurrences of the word ‘antibiotic’ from 24 September 2012 to 23 September 2013 in public Tweets and Retweets sent from any location worldwide. Topsy is one of the few companies with access to all historic Tweets sent since the launch of Twitter in 2006. Next, we calculated the daily average of antibiotic Tweets and used that average as the baseline of activity. Single day peaks, defined as those with a frequency over three times that of baseline, were identified from a graphical representation of activity generated using Topsy. The individual Tweets contributing to these peaks were analysed to identify the presence of particular events leading to the increase. In addition, information on the geographical distribution of users sending antibiotic Tweets on single day peaks, and on basal days, was sought from the analysis tools within Topsy.A web-based tool  contained the word ‘antibiotic’. Of these, 203 000 (84%) were original Tweets, whilst 39 000 (16%) were Retweets. On average, 556 Tweets about antibiotics were posted per day. Geographically, the majority of Tweets were submitted from users located in the USA , UK , Canada , Malaysia  and Indonesia .Peak 1, in January 2013, following the recommendation of the UK CMO to include antibiotic resistance on the national risk register (https://twitter.com/guardian/status/294218197928906752).Peak 2, in March 2013, after publication of the UK CMO's annual medical report, which expanded further on the threat of resistant bacteria .Peak 3, in March 2013, as a result of the US FDA's announcement on concerns about the safety profile of azithromycin .Peak 4, in September 2013, occurred after the release of the US CDC's antimicrobial resistance threat report (https://twitter.com/marynmck/status/379986025860186112).According to Topsy estimates, the CMO's March announcement reached a potential audience of >20 million users in a single day. Such reach was achieved via traditional news outlets such as the British Broadcasting Corporation (www.bbc.co.uk/news) or the Huffington Post (www.huffingtonpost.com). Only two individuals featured amongst the top 10 disseminators. For all the peaks identified, and the potential audience reached, the daily number of antibiotic Tweets returned to the baseline frequency within 24–48 h.The daily occurrences of antibiotic Tweets during the study period are shown in Figure Using a web-based tool, we have been able to produce a rapid, brief analysis exploring the presence of the term ‘antibiotic’ on a social media platform with >500 million registered users worldwide. Although messages predominantly originated from the USA and the UK, users in many other countries sent Tweets about antibiotics. The vast majority of these Tweets were original messages, rather than reposts of Tweets from other people, which required users to engage with the content of the message. The daily global discussion about antibiotics did not remain homogeneous in content or volume during the analysis period and we found clearly defined peaks in activity relating to antibiotics. Two of these peaks followed activities by the UK CMO concerning the threat of antimicrobial resistance, which stimulated discussions that reached a large global audience. It has not been possible to tell from our analysis what led to this particular impact compared with other announcements or events.24 Other studies suggest that Tweets related to time-sensitive topics or breaking news are more likely to be Retweeted,25 highlighting the need for concerted efforts from healthcare organizations to frame the use of Twitter within a cohesive communications strategy and even employing social media managers.In all four activity peaks, we found that Tweet activity reached maximal activity within 24 h and returned to the basal levels prior to the peak within 48 h. Thus, although messages about antibiotics spread rapidly in response to events, they do not lead to continued discussions on the Twitter platform at present. It has been suggested that for a certain Tweet, 50% of the total Retweet activity takes place between 4 min and 3 h after the update, suggesting that whilst Tweets can have a short-term impact, a more sustained response would require a multipronged strategy with several Tweets, each with their own large impact.26Interestingly, no other antibiotic resistance awareness campaigns resulted in peaks of activity comparable to those seen following the four news announcements discussed above. This was despite the fact that some, such as ‘Get Smart about Antibiotics’ in the USA and the European Antibiotic Awareness campaigns, originated from countries that accounted for the majority of global antibiotic-related messages on Twitter. This would suggest that until now such campaigns have not had a significant impact on this social media platform, which may be due to a lack of engagement with the most influential organizations or individuals on Twitter. Identifying key opinion leaders and gaining their support are essential steps in any public health intervention and appear to be even more crucial when using a network-based communication platform, as people who are more closely connected and interact more often are likely to have greater influence.15 Such engagement could create a strong peer-led approach to antibiotic overuse, e.g. encouraging individuals to Tweet when they used recommended self-management approaches rather than asking their healthcare provider for antibiotics. There is some evidence, however, that different types of users generate distinctive discourses on social media and therefore appealing to each audience would need adequate tailoring of messages; organizations and stakeholders in breast cancer, for example, would focus their Twitter updates on fundraising, screening and diagnosis, whilst the public would be significantly more likely to emphasize communal activities and events.27 In view of these differences, it is imperative that healthcare organizations evaluate the effectiveness of health promotion and communication theories in different social media platforms.28 On the other hand, the prominence of the activity peaks described may reflect the important role that negative sentiments (i.e. unpleasant emotions or attitudes) play when deciding whether to Retweet a message.29As three of the four activity peaks were related to antibiotic resistance, there appears to be a general receptiveness to messages on this topic and future campaigns should take this into consideration. Further, campaigns could look at ways to harness this willingness to discuss antimicrobial resistance into a more sustained dialogue.As far as we are aware, this is the first study that attempts to characterize events and occurrences driving people to discuss antibiotics on a social media platform. Our study has several limitations. First, by focusing on only one search term (‘antibiotic’) we will have under-reported the total volume of Tweets related to antibiotics, as some will have used other terms such as ‘antimicrobial’ or made direct reference to named antibiotics. We used ‘antibiotic’ as we felt this would be the word most likely to be used by the general population on this subject; indeed, we conducted the same search on Topsy using ‘antimicrobial’ and produced very few results over the study period. Second, our study was limited to terms in the English language only. Third, Topsy uses algorithms to determine whether the message is original or a Retweet and we cannot access these for verification. Fourth, the geographical location of individual Tweets is defined by the location of the user at the time of sending the Tweet and does not necessarily represent their habitual location. Finally, as highlighted in the Introduction, some demographic groups are under-represented amongst Twitter users, so care is required when considering extending our conclusions to other social media platforms and to the general population.In conclusion, people across the globe talk about antibiotics on social media and free tools can be used rapidly to gain initial insight into discussion topics and triggers for changes in the volume of conversations. Activity increases are global and not localized to the origin of the events. There is a need to understand the contribution and impact of social media tools on public health campaigns and institutional organizations must consider social media within their communications strategy. More evidence is still needed regarding the optimal mix of communication interventions, including the timing of message posting and the individuals best suited to post messages. The development of tools capable of deeper analysis across a broader range of social media platforms may aid assessment of the impact of antibiotic awareness campaigns and promote understanding of opportunities to influence individuals' thoughts about antibiotic resistance.UK National Institute for Health Research (NIHR) Biomedical Research Centre Funding Scheme at Imperial College (funding number not applicable) and the National Centre for Infection Prevention and Management funded by the UK Clinical Research Council (grant number UKCRC G0800777). A. H. is affiliated with the Imperial Centre for Patient Safety and Service Quality funded by the UK NIHR.This work was supported by the None to declare.O. J. D. undertook data acquisition and analysis, drafted and revised the paper and is the guarantor. E. C.-S. drafted and revised the paper. A. H. H. revised the paper. All authors contributed to the development of the concept for the paper and approved the final version.