The basic symptoms (BS) approach provides a valid instrument in predicting psychosis onset and represents moreover a significant heuristic framework for research. The term “basic symptoms” denotes subtle changes of cognition and perception in the earliest and prodromal stages of psychosis development. BS are thought to correspond to disturbances of neural information processing. Following the heuristic implications of the BS approach, the present paper aims at exploring disturbances of information processing, revealed by functional magnetic resonance imaging (fMRI) and electro-encephalographic as characteristics of the at-risk state of psychosis. Furthermore, since high-risk studies employing ultra-high-risk criteria revealed non-conversion rates commonly exceeding 50%, thus warranting approaches that increase specificity, the potential contribution of neural information processing disturbances to psychosis prediction is reviewed. In summary, the at-risk state seems to be associated with information processing disturbances. Moreover, fMRI investigations suggested that disturbances of language processing domains might be a characteristic of the prodromal state. Neurophysiological studies revealed that disturbances of sensory processing may assist psychosis prediction in allowing for a quantification of risk in terms of magnitude and time. The latter finding represents a significant advancement since an estimation of the time to event has not yet been achieved by clinical approaches. Some evidence suggests a close relationship between self-experienced BS and neural information processing. With regard to future research, the relationship between neural information processing disturbances and different clinical risk concepts warrants further investigations. Thereby, a possible time sequence in the prodromal phase might be of particular interest. Disturbances of information processing are a core feature of psychosis, particularly schizophrenia, with a significant impact on vulnerability and course , 2. HencIn the present review, we will consider three major areas of research, the basic disturbances concept and the event-related research employing either electro-/magnet-encephalographic (EEG/MEG), or functional magnetic resonance imaging (fMRI). Thereby results are emphasized that allow for a detection of those subjects clinically at high risk , who ind“Basic symptoms” (BS) are conceptualized as a phenomenological counterpart of neural pathological changes in brain functioning and have been demonstrated to represent core features of psychotic disorders –8. TheseIn line with the assumption of a close relationship to neurobiological changes, BS represent not only the earliest symptoms of psychosis development, but almost persistent phenomena that can be observed independently of positive and/or negative symptoms throughout large periods of the course of illness , 14, 15.Since BS are thought to mark the earliest stages of psychosis development, they have been employed to the aim of identifying subjects in presumably pre-psychotic stages of illness , 14–16. 14The currently most widely used clinical criteria of psychosis prediction are the so called ultra-high-risk (UHR) symptoms . AccordiFollowing the heuristic implications of the BS approach it can be assumed that the uncertainty of prediction results from an inherent ambiguity of (attenuated) psychotic symptoms. UHR criteria presumably represent disturbances of higher order integrative functions, reality testing, inner monitoring, and context evaluation possibly arising from various neural aberrations that do not necessarily comprise the specific processes leading to the development of, e.g., schizophrenia. The “prodrome,” however, is presumably characterized by specific disturbances of neural processing that are not mirrored by rather unspecific psychotic phenomena , 21–24. 21In the present paper, following the BS concept, we aim at exploring if and to what extent objective parameters of neural information processing allow for improving the prediction of psychosis onset. After an overview of the research on disturbances of neural information processing in the at-risk state, we will focus on findings indicating predictive capabilities.We used the following Medical Subject Heading (MeSH) categories: (fMRI OR EEG OR MEG) AND [UHR OR prodrome OR at-risk mental state (ARMS) OR clinical high risk ] AND . Particularly the functionality of the prefrontal cortex, contributing to executive and working memory functions, and moreover subserving emotion processing, reward, and social cognition, has been demonstrated to be impaired in schizophrenia .Investigations employing tasks related to attention control, verbal fluency, and working memory, respectively, suggested almost consistently a gradual decline in frontal and striatal activation from the clinical risk state to chronic psychosis , 31. FurA recent meta-analysis demonstrated a prefrontal dysfunction in the at-risk state across different paradigms . AdditioTwo studies yet compared fMRI measures in converters vs. non-converters –41. They39Taken together, functional disturbances in at-risk subjects have consistently been demonstrated, thereby pointing particularly to dysfunctions of frontal regions. However, definite conclusions should be deferred since the published fMRI studies vary markedly with regard to both, methodology and paradigms. Furthermore, the number of studies investigating differential deficits in converters and non-converters is yet sparse and none of the available studies reported discriminative statistics as required for predictor models.Contrary to the antecedent axiom that sensory brain regions simply relay neural representations of the environment to higher order networks, the very high complexity of early sensory information processing has to be appreciated: even at the earliest stages of sensory processing, incoming information is preconsciously filtered and digested via top-down and bottom-up loops . In focuHowever, typical paradigms point to event-related potentials  appearin4646Overall, sensory processing deficits in subjects displaying high-risk symptoms have been demonstrated to encompass the full range of processing, i.e., early as well as later stages. However, it is not yet clear how the observed disturbances differentially relate to the pathophysiology of psychosis development.One of the earliest steps in neural processing contributes to filtering of sensory information. In at-risk subjects, significant deficits of the respective correlates have been demonstrated in studies on sensory gating (P50/N100 components) and prepulse inhibition (PPI) paradigms , 49–54. 4949Later stages of sensory information processing occupy the interface between perceptual and cognitive systems. A commonly used paradigm involves neural ability to discriminate deviant stimuli in a series of predictable standards , 46, 48.59Investigations employing the P3 component, which is thought to reflect automatic processing of novelty and memory updating, have demonstrated significant deficits in at-risk subjects compared to healthy controls , 70–76. 70Nine studies have been identified that statistically compared neurophysiological measures in converters vs. non-converters , 71, 73 Ziermans and colleagues suggested a differential PPI deficit in converters and non-converters . SensoryPredictive models employing neurophysiological parameters were provided by two published investigations , 73. FolTaken together, correlates of sensory processing and pending higher order functions indicate significant disturbances of neural information processing in at-risk subjects. Future research has to elucidate the pathophysiological meaning of these disturbances and their impact with regard to different outcomes of the at-risk state. However, first steps have been made to identify neural markers of psychosis development.Impairments of neural information processing in the pre-psychotic state have been demonstrated by a rising number of investigations, thereby providing evidence for the heuristic implications of the BS concept. However, although studies employing fMRI measures are sparse compared to investigations focusing on neurophysiology, significant evidence for information processing deficits is provided across methods and paradigms.Functional magnetic resonance imaging studies most consistently demonstrated dysfunctions of regions differentially contributing to executive and memory functions, social cognition, emotional processing, and language processing, respectively , 31. Neu495972However, since most studies did not comprise converters in their high-risk samples. As a considerable part of theses samples may not proceed to psychosis or even remit  from the at-risk state , 80, 81,Although fMRI investigations have demonstrated significant differences between converters and non-converters , 42, no Regarding neurophysiology, two studies yet established predictive models , 73 baseHowever, since only one study investigated the P3 as a predictor of psychosis onset, this finding needs further corroboration.Mismatch negativity amplitude reductions may predict conversion to psychosis and enable the stratification of risk into different classes . The resSensory gating presumably refers to early pre-attentive processes, whereas P3 likely represents cognitive management of salient stimuli, and MMN may straddle bottom-up stimulus registration and top-down change detection. In synopsis of the currents literature, sensory gating deficits might predominantly indicate liability to psychotic experiences, P3 might be primarily susceptible to cognitive disturbances, and the MMN may be best suited to support the identification of future converters.The heuristic implications of the BS concept predict that disturbances of information processing can be observed largely independent of positive symptoms and are closely related to particular aberrations of brain functioning , 14, 15.Among the most predictive BS, two concern disturbances of language processing , 14, 15.The relationship between BS and particular brain regions has still to be investigated directly. However, the synopsis of the current literature provides an impetus for future research.Since all samples discussed in this review are preselected by clinical risk criteria, it remains unclear if objective measures would perform equally well as screening tools in non-selected, help-seeking samples. Furthermore, their predictive value beyond clinical criteria has still to be evaluated. However, different to any clinical approach, neurophysiological parameters provide an estimate of the remaining time until transition , 79, whiThe specificity of neurobiological parameters represents another major topic. Neural information processing deficits have been observed in non-psychotic disorders, too, as well as in healthy individuals, although some parameters may be more specific to psychosis , 84–87. 84The heuristic implications of the basic disturbances concept predict that particularly aberrations of neural information processing represent the objective counterparts of cognitive and perceptive BS. The current literature provides evidence that information processing deficits can discriminate at-risk subjects converting to psychosis from those who will not develop a psychotic disorder. In fMRI investigations, group differences have been demonstrated particularly with regard to brain regions involved in language processing. Event-related potentials may enable a quantification of risk in terms of magnitude and time. Particularly the latter finding represents a significant advancement compared to clinical approaches. In synopsis of the literature, some findings seem to support the assumed close relationship between self-experienced BS and neural information processing. Disturbances of language function have been identified as predictive symptoms in clinical BS studies and neural correlates have been demonstrated in both, fMRI and neurophysiological investigations. Even though the relationship between BS phenomenology and neural activity appears yet largely unknown in general, deficits in neurophysiological parameters seem to be more closely related to cognitive deficits than to psychotic symptoms.Taken together, the BS concept provides not only a valid instrument in predicting psychosis onset but represents moreover a significant heuristic framework for future research.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.


Human polyomavirus JC is the causative agent of a deadly form of sudden onset dementia, progressive multifocal leukocoencephalopathy (PML). PML is highly prevalent in immunodeficient populations, specially those undergoing chemotherapy, immunosuppressive treatments for autoimmune conditions, and HIV-1/AIDS patients. In fact, before the highly active antiretroviral therapy (HAART) regimens became available, PML was a leading cause of death in HIV-1 seropositive individuals. However, patients under HAART show increased survival times with better prognoses. In this report we described the main differences between PML before and after the HAART era; highlighting the new patterns of presentation, the neurotropism of other human polyomaviruses, and the increased prevalence of immune reconstitution inflammatory syndrome (IRIS), as a complication of PML in patients under HAART. Lastly, we propose a revised classification of human poliomavirus-associated cerebral disorders that may reflect more accurately what clinicians encounter in their everyday practice. Since the introduction of highly active antiretroviral therapy (HAART), morbidity and mortality secondary to opportunistic disorders in patients infected with HIV-1 have significantly decreased . MoreoveHuman polyomaviruses JC and BK  rarely cause disease, except for patients with an underlying immunocompromise. JC virus (JCV) is the causative agent of a fatal demyelinating disorder termed progressive multifocal leukoencephalopathy (PML) . PML is Before HAART became widely available, there was no treatment for polyomavirus-associated disorders, and death from PML occurred as early as 3 months after diagnosis . The onemultifocal versus unifocal), and the nature of the clinical presentation—classic  versus variant (involvement of the cerebellar peduncles and/or brainstem)  , which mFurthermore, we have noticed a growing number of cases reporting worsening of PML after introduction of HAART –21. ThisLastly, we would like to comment about the newly discovered neurotropism of BKV . AlthougIn conclusion, all the aforementioned data suggest that human polyomaviruses are playing a major role in the post-HAART era, and nothing but a multidisciplinary study of their biology and the pathogenesis of their associated diseases will lead to more efficacious interventions.


Combined metagenomic and metatranscriptomic datasets make it possible to study the molecular evolution of diverse microbial species recovered from their native habitats. The link between gene expression level and sequence conservation was examined using shotgun pyrosequencing of microbial community DNA and RNA from diverse marine environments, and from forest soil.Across all samples, expressed genes with transcripts in the RNA sample were significantly more conserved than non-expressed gene sets relative to best matches in reference databases. This discrepancy, observed for many diverse individual genomes and across entire communities, coincided with a shift in amino acid usage between these gene fractions. Expressed genes trended toward GC-enriched amino acids, consistent with a hypothesis of higher levels of functional constraint in this gene pool. Highly expressed genes were significantly more likely to fall within an orthologous gene set shared between closely related taxa (core genes). However, non-core genes, when expressed above the level of detection, were, on average, significantly more highly expressed than core genes based on transcript abundance normalized to gene abundance. Finally, expressed genes showed broad similarities in function across samples, being relatively enriched in genes of energy metabolism and underrepresented by genes of cell growth.These patterns support the hypothesis, predicated on studies of model organisms, that gene expression level is a primary correlate of evolutionary rate across diverse microbial taxa from natural environments. Despite their complexity, meta-omic datasets can reveal broad evolutionary patterns across taxonomically, functionally, and environmentally diverse communities. Variation in the rate and pattern of amino acid substitution is a fundamental property of protein evolution. Understanding this variation is intrinsic to core topics in evolutionary analysis, including phylogenetic reconstruction, quantification of selection pressure, and identification of proteins critical to cellular function ,2. A divDeep-coverage sequencing of microbial community DNA and RNA (metagenomes and metatranscriptomes) provides an unprecedented opportunity to explore protein-coding genes across diverse organisms from natural populations. Such studies have yielded valuable insight into the genetic potential and functional activity of natural communities -19, but Specifically, we use coupled microbial (Bacteria and Archaea) metagenomic and metatranscriptomic datasets to explore the hypothesis that highly expressed genes are more conserved than minimally expressed genes. In lieu of conservation estimates based on alignments of orthologous genes, which are not feasible using fragmentary shotgun data containing tens of thousands of genes, sequence conservation was estimated based on amino acid identity relative to top matches in a reference database. Our results indicate a strong inverse relationship between evolutionary rate and gene expression level in natural microbial communities, measured here by proxy using transcript abundance. Furthermore, these results demonstrate broad consistencies in protein-coding gene expression, amino acid usage, and metabolic function across ecologically and taxonomically diverse microorganisms from different environments. This study illustrates the utility of environmental meta-omic datasets for informing theoretical predictions based (largely) on model organisms in controlled laboratory settings.The relationship between gene expression (transcript abundance) and sequence conservation was examined for protein-coding genes in coupled metagenome and metatranscriptome datasets generated by shotgun pyrosequencing of microbial community DNA and RNA, respectively. These datasets represent varied environments, including the oligotrophic water column from two subtropical open ocean sites in the Bermuda Atlantic Time Series (BATS) and Hawaii Ocean Time Series (HOT) projects, the oxygen minimum zone (OMZ) formed in the nutrient-rich coastal upwelling zone off northern Chile, and the surface soil layer from a North American temperate forest Tables  and 2. PP < 0.001, t-test; Table n = 53) detected among the RNA reads falling into this category  compared to DNA-derived sequences (metagenomes), with an average difference of 8.9% between paired datasets , we examined amino acid identities in expressed and non-expressed genes derived from the DNA dataset only. Hereafter, we operationally define 'non-expressed' genes as those detected only in the DNA datasets, whereas 'expressed' genes are those detected in both the DNA and RNA datasets , an expressed gene sampled from the environment will have a higher likelihood of finding a close match in the database, relative to a non-expressed gene. We therefore examined the discrepancy between expressed and non-expressed gene sets only for DNA reads whose top hits match the same reference genome. Under a null hypothesis of uniform evolutionary rates across a genome, all genes in a sample whose closest relative is the same reference genome should exhibit uniform divergence from the reference.Bradyrhizobium japonicum from the soil sample, the mean amino acid identity of expressed genes was significantly greater than that of non-expressed genes . These taxon-specific patterns argue against an overall bias due to varying levels of gene representation in the database. Rather, assuming that the sequences that match the expressed and non-expressed gene fractions of a given reference genome are indeed present in the same genome in the sampled environment , these results suggest that differential conservation levels, and not sampling artifacts, are driving the overall discrepancy between expressed and non-expressed genes.The link between expression level and sequence conservation was observed at the level of individual genomes. Figure Our data confirm an inverse relationship between expression level and evolutionary rate in natural microbial communities. However, it remains unclear to what extent gene expression level depends on a gene's functional importance to organism fitness  versus other potential explanations, such as 'translational accuracy or robustness' . It has P < 0.0009, chi-square). In some taxa, such as Prochlorococcus marinus str. NATL2A, core genome representation was over 30% greater among expressed genes relative to non-expressed genes. In contrast, for the HOT 500 m dataset, expressed genes were not enriched in core genes, which we speculate may be due to the activity of the microbial community at this depth (see Conclusions section below). Overall, however, the data support the broad trend that highly expressed genes are more likely to belong to an orthologous set shared across multiple taxa.Expressed genes were significantly more likely to fall within a core gene set shared across taxa. Figure The differential representation of core genes within expressed and non-expressed genes may influence the relative sequence conservation levels of these two gene fractions. Gene acquisition from external sources ) is an important source of genetic variation in bacteria . A consen = 60, as above), 80% showed higher expression levels (expression ratio) of non-core genes relative to core genes . This pattern seemingly conflicts with studies based on cultured organisms. For example, a prior comparative survey of 17 bacterial proteomes showed a relative enrichment of peptides representing proteins encoded within the core genome [Escherichia coli [Pseudomonas aeruginosa [Prochlorococcus [Surprisingly, within the expressed gene fraction, non-core genes were more highly expressed than core genes. Among the datasets representing the five most abundant taxa per sample . Notably, across all five samples, the expressed gene set was significantly enriched in genes involved in energy and nucleotide metabolism, transcription, and protein folding, sorting, and degradation . At the finer resolution provided at the KEGG pathway level, genes involved in oxidative phosphorylation, chaperones and protein folding catalysis, translation factors, and photosynthesis were consistently and significantly  overrepresented among expressed genes in all samples, whereas genes of peptidoglycan biosynthesis, mismatch repair, and amino sugar and nucleotide sugar metabolism were proportionally more abundant in the non-expressed fraction . It is unclear to what extent this same trend may be detected within clusters of related sequences within our samples, independent of comparison to an external reference database. We therefore examined variability in amino acid divergence within clusters of expressed and non-expressed protein-coding sequences for five representative samples, including shallow and deep depths from the OMZ and HOT oceanic sites, and the surface soil sample Table .Mean identity per cluster was consistently higher for DNA sequences in non-expressed clusters compared to DNA sequences from expressed clusters  will be relatively enriched in homologs that occur across multiple divergent taxa. By definition, therefore, DNA+RNA clusters will be relatively enriched in sequences differing at both the population level and at higher taxonomic levels , while DNA-only clusters will be enriched in sequences differing only at the population level. Given this explanation, we would predict that DNA+RNA clusters (with RNA sequences excluded) are larger than DNA-only clusters and that the DNA-only cluster set as a whole is enriched in high identity clusters. Indeed, DNA+RNA clusters are, on average, approximately 20 to 33% larger than DNA-only clusters (RNA sequences not included in counts) and DNA-only cluster sets, notably those of the OMZ samples, are enriched in clusters with identities greater than 98% Figure . These dThe discrepancy in sequence conservation between expressed and non-expressed genes coincided with differences in nucleotide composition and amino acid usage between these two sequence pools. GC content was substantially higher in the soil compared to the ocean samples (approximately 20 to 25% enrichment) and consistent between the DNA and RNA pools Table . In contP < 0.0002, chi-square; Table Consistent with the GC pattern, amino acid usage of protein-coding sequences differed significantly between the DNA and RNA samples Table , and 14.Among the ocean datasets, DNA-RNA shifts in amino acid frequency were strongly related to amino acid GC content Figure . In geneProchlorococcus [Prior studies describe a relationship among gene expression level, sequence conservation, and amino acid usage -42. Specrococcus ,39. GC rrococcus , or selerococcus . Assuminrococcus ,42, seleMicrobial metagenomes and metatranscriptomes are amalgams of thousands of taxonomically and functionally diverse microorganisms, each of which experiences unique evolutionary pressures. Such complexity might be expected to preclude the detection of bulk evolutionary signals in meta-omic data. Here, we show broad trends in protein coding sequence conservation that transcend variation in both taxonomic composition and habitat type.Specifically, we confirm that the hypothesized positive relationship between gene expression level and sequence conservation, which has been well established for individual taxa under experimental conditions , is a unPseudomonas aeruginosa [The mechanism linking evolutionary rates and expression level is still debated. Sequence conservation in highly expressed proteins has been hypothesized to be driven by selection acting to minimize the costs of protein misfolding, which should increase in tandem with expression level (protein copy number per cell) , though ruginosa . Though We show that the expressed gene set, compared to the non-expressed set, is more likely to contain genes that belong to an orthologous core genome shared across closely related sister taxa. This pattern was broadly consistent across the marine samples and the soil sample. Interestingly, the overrepresentation of expressed genes within the core set was not observed in the HOT 500 m sample Figure , which wOur datasets highlight similarities in gene expression and sequence evolution across very different microbial habitats, but differ markedly in other attributes. Notably, the soil community was a clear outlier with respect to functional gene content Figures , and 9 aThe composition of metatranscriptomic and metagenomic sequence datasets depends not only on intrinsic biological factors  but also on the physical and chemical environment at the time of sampling. Furthermore, interpretation of the resulting data can vary based on the analytical method  and on the availability and biases of the reference sequences to which the data are compared. Here, we attempt to rule out potential database artifacts by analyses at both the community and genome level. In so doing, our results suggest that environmental meta-omic datasets, despite their inherent complexity, can inform theoretical evolutionary predictions and reveal universal trends across ecologically and phylogenetically diverse microbial communities.We examined protein-coding sequences in coupled microbial metagenomes and metatranscriptomes from multiple depths at three distinct oceanographic sites and from surface soil in a temperate forest Table . Ocean dmirVana™ miRNA Isolation kit  for RNA isolation [Soil was collected from within a transition hardwood-white pine/hemlock forest in the Prospect Hill Tract of Harvard Forest  on 27 September 2010. Two cores were taken from 1 to 10 cm below the leaf horizon using a 10 mm diameter soil corer. These cores were homogenized, placed into 50 ml Falcon tubes, flash frozen in liquid nitrogen, and transported to the lab on dry ice. Visible pieces of plant material were removed from soil subsamples with sterile forceps. During plant removal, the subsamples were placed on a sterile surface, laid within a bed of dry ice. Total microbial DNA and RNA were then extracted from these subsamples using PowerSoil Total RNA and DNA isolation kits , according to the manufacturer's protocol. This extraction protocol differs from the method used to generate the marine datasets, which employed the solation ,19,23,35in vitro  to produce microgram quantities of single-stranded antisense RNA. The amplified products  were converted back to double-stranded cDNA using the SuperScript® III First-Strand Synthesis System  for first-strand synthesis (random hexamer priming), and the SuperScript™ Double-Stranded cDNA synthesis kit (Invitrogen) for second-strand synthesis. cDNA was then purified , digested with BpmI  to remove poly(A) tails, and used directly for pyrosequencing.Total RNA was amplified and prepared for pyrosequencing using established protocols. Briefly, the proportion of ribosomal RNA transcripts  in total soil RNA was reduced via a published subtractive hybridization protocol using sample-specific rRNA probes ,19. Brie® AMPure® kit  and used for the generation of single-stranded DNA libraries and emulsion PCR according to established protocols . Clonally amplified library fragments were sequenced with full plate runs on a Roche Genome Sequencer FLX instrument using Titanium chemistry. Pyrosequencing datasets generated during this study have been deposited in the NCBI Sequence Read Archive under accession numbers listed in Table Soil DNA and cDNA were purified for sequencing via the AgencourtPyrosequencing reads matching ribosomal RNA genes were identified in cDNA and DNA datasets by BLASTN searches against a custom database of prokaryotic and eukaryotic small and large subunit rRNA sequences  taken from microbial genomes and the ARB SILVA LSU and SSU databases . Reads mLocal amino acid identities were recovered from within the top HSP for each sequence having a significant match (bit score >50) to a reference gene in the nr database; gaps were not included in identity calculations. Identities of multiple reads matching the same reference gene were averaged to obtain per gene identities, and these values were then averaged across all reference genes per dataset. Relative taxon abundance per sample was determined as the number of sequence reads matching the same reference taxon as a top hit in BLASTX searches of NCBI-nr. The relative transcriptional activity (expression level) per expressed gene was normalized to account for variations in gene abundance in the DNA pool, as calculated by the expression ratio: /Prochlorococcus sp., which was common in our samples, was defined as those genes present across all 12 of the sequenced Prochlorococcus strains, based on the analysis of [The proportional representation of expressed and non-expressed genes in the orthologous gene set shared between taxa was determined separately for the top five most abundant organisms in each of the samples. The core genome of lysis of . For allAverage-linkage clustering was performed in Cluster 3.0 using Pearson coefficients (centered) derived from pairwise correlations of KEGG functional category abundances Figures  and 9, aab initio gene-finding program Metagene [Sequence divergence was determined for clusters of related sequences within pooled DNA + RNA datasets. To avoid spurious clustering of non-coding regions ), amino acid sequences present in the DNA and RNA reads (those with significant matches to the nr database) were identified using the Metagene .  were examined to assess amino acid usage and GC content, using two approaches. First, amino acids were counted across all HSPs in alignments between sequence reads and their closest match in the nr database. Second, amino acids were counted across open reading frames that were identified using Metagene . These tt-tests. Significance was determined at alpha <0.05 following Bonferroni corrections for multiple comparisons.Proportional changes in amino acid usage, KEGG gene category and pathway abundances, and core genome representation in expressed and non-expressed gene fractions were compared using chi-square tests. Mean amino acid identities and expression ratios were compared using standard two-tailed BATS: Bermuda Atlantic Time Series; GOS: Global Ocean Sampling; HGT: horizontal gene transfer; HOT: Hawaii Ocean Time Series; HSP: high-scoring segment pair; KEGG: Kyoto Encyclopedia of Genes and Genomes; NCBI-nr: National Center for Biotechnology Information non-redundant protein database; OMZ: oxygen minimum zone.The authors declare that they have no competing interests.EFD and FJS conceived of the study. FJS carried out the computational analyses of pyrosequencing data and drafted the manuscript. JME participated in the computational sequence and statistical analysis, provided important coding support, and helped revise the manuscript. JAB helped design the study, provided critical analytical and data acquisition support, and helped revise the manuscript. AKS participated in data acquisition and computational sequence analysis and helped revise the manuscript. EFD coordinated the overall goals of the study, directed the analytical work, and helped to draft the manuscript. All authors read and approved the final manuscript.


Classical theories of automaticity assume that automatic processes elicited byunconscious stimuli are autonomous and independent of higher-level cognitiveinfluences. In contrast to these classical conceptions, we argue that automaticprocessing depends on attentional amplification of task-congruent processingpathways and propose an attentional sensitization model of unconscious visualprocessing: According to this model, unconscious visual processing is automaticin the sense that it is initiated without deliberate intention. However,unconscious visual processing is susceptible to attentional top-down control andis only elicited if the cognitive system is configured accordingly. In thisarticle, we describe our attentional sensitization model and review recentevidence demonstrating attentional influences on subliminal priming, aprototypical example of an automatic process. We show that subliminal priming(a) depends on attentional resources, (b) is susceptible to stimulusexpectations, (c) is influenced by action intentions, and (d) is modulated bytask sets. These data suggest that attention enhances or attenuates unconsciousvisual processes in congruency with attentional task representations similar toconscious perception. We argue that seemingly paradoxical, hitherto unexplainedfindings regarding the automaticity of the underlying processes in manycognitive domains can be easily accommodated by our attentional sensitizationmodel. We conclude this review with a discussion of future research questionsregar-ding the nature of attentional control of unconscious visualprocessing. Unconscious automatic processes are traditionally thought to occur autonomously andindependently of top-down control . Accordiautomaticity, which are essentially influenced by the classicalview, can be found in many areas of psychology and neuroscience such as in object orface recognition . In line with the assumptionthat priming is mediated by different processes, different forms of primingactivate distinct brain regions: Visuo-motor response priming recruitsoccipito-parietal areas  effects elicited by masked visual stimuli that are not consciouslyperceived. Different forms of priming can be distinguished, depending on therelation between prime and target . Masked al areas  known toal areas .As a complement to behavioral priming effects, event-related potentials (ERPs)recorded from the scalp capture task-specific priming processes on-line duringtask performance. Semantic priming modulates the N400 ERP component, a negativedeflection peaking at about 400 ms with a centro-parietal topography . The N40Before we review the latest findings demonstrating attentional influences onsubliminal priming as an example of an automatic process, we introduce ourtheoretical view of attentional control of unconscious visual processing in thenext section.Gain is a parameter in neural network modeling, whichinfluences the probability that a neuron fires at a given activation level  should depend on available attentional resources,and (b) are susceptible to top-down control by currently active taskrepresentations. Attentional sensitization of automatic processing by taskrepresentations is achieved by enhancing the sensitivity of task-relevant pathwaysand by attenuating the sensitivity of task-irrelevant pathways.preemptiveand reactive control  depends on attentional resources, (b) is susceptible to stimulusexpectations, and (c) is influenced by action intentions.Unconscious priming has been shown to depend on attentional top-downamplification and attentional resources similar to conscious visual perception:In a masked semantic priming study , an atteSubliminal processing of stimuli strongly depends on stimulus expectations thatinclude what kind of stimulus is likely to occur within a given situation.Expected subliminal stimuli receive attentional amplification and are furtherprocessed whereas processing of unexpected stimuli is attenuated . Of courMasked response priming has been shown to depend on action intentions: Ansorgeand colleagues  found thIn a continuation of this line of research, the capture of visuo-spatialattention by unconscious stimuli likewise was shown to depend on the matchbetween stimulus features and a top-down search template directed towards thetask-relevant visual features of the targets . Top-dowabove) or a lowered location  produced priming effects only during a spatialtarget task, but not during a target task with numbers of high and low numericalmagnitude , affective  or animacy (living vs. non-living) congruency of precedingsubliminally presented prime words elicited only priming effects on the targetdecision when the corresponding meaning dimension was also task-relevant in thetarget task . Similaragnitude . These fagnitude  receivesagnitude . Interesagnitude .Unconscious priming does not only depend on action intentions, but also on task sets,which are active during the presentation of the masked prime . Similartask sets as an adaptive configurationof the cognitive system which is necessary to efficiently perform a given task  designed to induce a specific task set . The different induction tasks were presented in separateblocks in order to avoid task switching effects between trials with differentinduction tasks. The induction tasks were followed immediately by a primed decisiontask . Across experiments, we systematically varied the type of induction task tospecify the attentional mechanisms which enhance or attenuate unconsciousprocessing. According to the proposed attentional sensitization model, taskrepresentations enhance and attenuate processing streams in order to facilitateprocessing in congruency with higher-level goals: Automatic processes that matchtask representations are assumed to be amplified, while other automatic processesshould be attenuated.Using the induction task paradigm, we systematically investigated the influenceof previously performed tasks on subsequent masked semantic priming within alexical decision task (word/non-word decision) in three experiments . We askeAcross experiments, the difficulty of the semantic and perceptual induction tasksas well as their verbal or non-verbal nature was systematically varied. InExperiment 1, participants performed an easy semantic word classification task(living/non-living decision) and a difficult perceptual letter classificationtask with words (deciding whether first or last letter of a word has a closed oropen shape). In Experi-ment 2, the difficulty of the induction tasks wasreversed: difficult semantic word classification (deciding whether a word refersto something dry or wet) versus easy perceptual letter classification (decidingwhether first or last letter of a word is the letter t). In Experiment 3,non-verbal induction tasks had the same level of difficulty: semanticclassification (living/non-living decision) versus perceptual classification ofobject pictures (round vs. elongated shape decision). At the short RPI, semanticpriming effects on RT and N400 ERP component were obtained when a semantic taskset was induced immediately before subliminal prime presentation, whereas apreviously induced perceptual task set attenuated priming.Comparable results were obtained regardless of the difficulty level and theverbal or non-verbal nature of the induction tasks. In line with the attentionalsensitization model, unconscious semantic processing is enhanced by a semantictask set and attenuated by a perceptual task set. At the long RPI, significantpriming was found after the perceptual induction task, but not after thesemantic task. The priming effects at the long RPI suggest that after 800 ms,the task set of the induction task was abandoned and a reconfiguration of thecognitive system in preparation for the upcoming lexical task took place : SemantiIn the second study , we contIn two further studies, we determined the nature of task sets which boostsemantic or visuo-motor priming. In the first study , we wanted to better characterize the task set that enhancesunconscious semantic processing. Previously, we found enhanced subliminalsemantic priming following a semantic word classification task, but reducedpriming following a perceptual letter classification task . Unlike autumn, bottle,ocean) or a consonant as first and as last letter . Priming following thisphonological induction task was compared with a semantic induction task, inwhich words had to be classified according to whether they refer to living ornon-living objects . We found somewhat smaller subliminal primingeffects following the phonological than the semantic induction task althoughthis difference in priming was not significant. In the second experiment, wordreading was discouraged in the phonological induction task by presenting wordswith a capital letter at one position . Inthis induction task, attention was allocated to phonological aspects of singleletters: Participants had to decide whether the capital letter was either avowel  or a consonant. In the semanticinduction task, participants again performed a semantic classification. In thelatter experiment, semantic priming effects were only observed following thesemantic classification, but not following the phonological letterclassification induction task. The results of these two experiments show thatattention to single letters/phonemes of a word strongly disrupts subsequentsemantic processing of unconsciously presented primes  to test the boundaryconditions for unconscious semantic processing to occur and contrasted them to asemantic induction task. In one experiment, we used a phonological inductiontask that required attention to the entire word and thus could permit wordreading . We asked whether this non-semantictask set permits subsequent subliminal semantic priming. In the word inductiontask, participants had to decide whether words comprised a vowel as first orlast letter .In the previous sections, we have reviewed recent findings demonstrating attentionalinfluences on unconscious visual processing. Accumulating evidence demonstrates thatunconscious visual processing is susceptible to attentional control similar toconscious visual processing: Subliminal priming effects, prototypical examples ofautomatic processes, are modulated by attentional resources, stimulus expectations,action intentions, and task sets. Hence, in contrast to classical theories ofautomaticity ,automatIn the next section, we will show that seemingly paradoxical, hitherto unexplainedfindings in many cognitive domains regarding the automaticity of the underlyingprocesses can be easily accommodated by our attentional sensitization model.automatic in the sense that it is involuntarily initiatedeven under unconscious viewing conditions. However, unconscious automaticprocesses are susceptible to attentional modulation and are not invariantlytriggered by the appropriate stimulus in a purely bottom-up fashion.Within the research of semantic processing, it has been argued that semanticprocessing is not automatic, but requires controlled access to conceptualmeaning . This isSimilar paradoxical findings regarding the automaticity of pro-cesses have beenreported in many other areas of psychology such as sensory-motor preparation, emotionThese examples demonstrate that the proposed attentional sensitization modelapplies to many domains and has the explanatory power to account for seeminglyconflicting empirical phenomena. If attentional sensitization of automaticprocesses is a general computational principle, the fascinating question ariseswhether it is possible to specifically enhance or attenuate broad classes ofunconscious cognitive and emotional processes in the healthy or patientpopulation. For instance, future studies could assess whether implicit memorytraces can be differentially influenced by the nature of previously activatedtask representations. Similarly, it could be tested whether automatic fearresponses to phobia-relevant objects (such as spiders or snakes) can beattenuated in phobic patients, when the activated task representationdeemphasizes visual object recognition and/or includes a positive emotionalstate.Our general experimental approach that combines a first task for inducing tasksets with subsequent unconscious or conscious presentation of the criticalstimulus would be an ideal tool for addressing these and related questions. Thenotion of attentional sensitization of unconscious cognition could also help toexplain and to further empirically investigate the modulatory effects ofhypnotic inductions on automatic processes. A modulation of automatic processesby hypnosis has been reliably demonstrated for the Stroop interference effect that depThe implicit top-down control of unconscious processing by attentional sensitizationreviewed in this article evidences the adaptability of the cognitive system inoptimizing ongoing processing toward the pursuit of an intended goal: Task-relevantinformation is prioritized and task-irrelevant, possibly interfering influences areattenuated, both at a conscious and an unconscious level. The unconscious processingstreams are thus under the control of higher-level attention to some extent. Theproposed attentional sensitization mechanism operates in such a fashion as toconsiderably reduce the risk that unintended and not goal-related unconsciousprocesses determine cognition and eventually influence behavior .Although much progress has been made to elucidate the attentional control mechanismsof unconscious visual perception, several open issues crucially deserve furtherinvestigations:1. If the proposed attentional sensitization model is a general theory, it shouldalso apply to other forms of unconscious pro-cesses such as visuo-spatial andemotional processing. In particular, conscious control of unconscious emotionalprocessing is clinically highly important because findings in this area might helpto design more efficient therapeutic treatment techniques for mood and anxietydisorders.2. In order to gain more insight in the specificity of the attentional sensitizationmechanism, it is necessary to compare top-down control of subliminal priming with avariety of stimuli  within one and the samepriming paradigm. The question arises whether or not processing of subliminallypresented pictures and words is boosted by the same task sets.3. Attentional control of unconscious visual perception was mainly investigated usingthe induction task paradigm or by manipulating action intentions. However, as theattentional sensitization model should also be valid for control settingsestablished with other techniques, it would be highly interesting to investigatetop-down control of unconscious cognition induced, for instance, by hypnoticsuggestion  or bysu4. The assumption of the attentional sensitization model that control of unconsciousprocesses is exerted by a prefrontal top-down signal, which in turn influences thesensitivity of processing pathways in posterior brain areas, should be tested inmore detail by means of fMRI and electrophysiological recording techniques.5. Finally, formal computational modeling of the proposed attentional sensitizationmechanism (see e.g., In conclusion, the present review described striking evidence for implicit top-downcontrol of unconscious processing by attentional sensitization. We demonstrated thatpreemptive top-down control of unconscious processes coordinates the processingstreams in congruency with higher-level task representations in various domains ofcognition and emotion. Hence, attentional sensitization of automatic processingoptimizes ongoing processing toward the pursuit of an intended goal and ensures ahigh degree of flexibility and adaptability of the cognitive system in unconsciousvisual processing.


Echocardiographic and hemodynamic analysis indicated that acute expression of caFGFR1 rapidly and directly increased cardiac contractility, while chronic expression resulted in significant hypertrophy with preservation of systolic function. Subsequent histologic analysis showed increased cardiomyocyte cross-sectional area and regions of myocyte disarray and fibrosis, classic features of hypertrophic cardiomyopathy (HCM). Analysis of downstream pathways revealed a lack of clear activation of classical FGF-mediated signaling pathways, but did demonstrate a reduction in Serca2 expression and troponin I phosphorylation. Isolated ventricular myocytes showed enhanced contractility and reduced relaxation, an effect that was partially reversed by inhibition of actin-myosin interactions. We conclude that adult cardiomyocytes are competent to transduce FGF signaling and that FGF signaling is sufficient to promote increased cardiomyocyte contractility in vitro and in vivo through enhanced intrinsic actin-myosin interactions. Long-term, FGFR overexpression results in HCM with a dynamic outflow tract obstruction, and may serve as a unique model of HCM.Fibroblast growth factors (FGFs) and their receptors are highly conserved signaling molecules that have been implicated in postnatal cardiac remodeling. However, it is not known whether cardiomyocyte-expressed FGF receptors are necessary or sufficient for ventricular remodeling in the adult heart. To determine whether cardiomyocytes were competent to respond to an activated FGF receptor, and to determine if this signal would result in the development of hypertrophy, we engineered a doxycycline (DOX)-inducible, cardiomyocyte-specific, constitutively active FGF receptor mouse model ( Hypertrophy is an adaptive mechanism by which the heart can respond to stress or injury. In response to postnatal growth, pregnancy, or exercise, the heart undergoes physiological hypertrophy, characterized by an increase in myocyte length, absence of fibrosis, maintenance of chamber dimension, and normal cardiac function. In response to injury or stress, such as a myocardial infarction or increased afterload, functional heart muscle compensates with pathological hypertrophy, which initially maintains cardiac function. Pathological hypertrophy is characterized by increased myocyte width, fibrosis, decreased ventricular chamber volume, and eventual cardiac dysfunction and failure ,2. Addit-/- hearts have decreased cardiomyocyte hypertrophy and impaired cardiac function compared to WT mice following MI, while FGF2-overexpressing hearts showed enhanced cardiomyocyte hypertrophy and preserved function .,30.Fgfr1To determine the effect of activating FGF signaling in cardiomyocytes, 12- to 14-week-old DTG animals and controls were induced, and cardiac function was monitored over time with echocardiography. LV diastolic posterior wall thickness (LVPWd) was significantly elevated in DTG mice within one day of DOX induction , or tau (Glantz) values of relaxation  and continued to increase through six months of induction , while ßMHC, Col1a1, and Col3a1 were not significantly elevated until 42 days of transgene induction .To confirm that the hypertrophy seen in DTG mice was pathological, qRT-PCR was used to examine the expression of markers of pathological ventricular remodeling . Anp andDouble transgenic mice expressing caFGFR1 exhibited all of the pathophysiological characteristics of hypertrophic cardiomyopathy (HCM). Because one of the most common clinical manifestations of HCM is exercise intolerance , it was One of the advantages of the Tet-on system is its potential for reversibility—the removal of DOX inactivates the rtTA and halts transcription of the transgene. To assess the reversibility of the caFGFR1-induced phenotype, 12- to 14-week old DTG mice and littermate controls were induced with DOX chow for six weeks and then fed normal chow for six weeks. LV mass index and LV internal diastolic diameter returned to near control levels after DOX removal . LV diasFgfr1 gene expression were performed. caFGFR1, as measured by the c-myc tag, was still expressed at relatively high levels after 42 days of DOX removal , although expression was significantly reduced compared to expression following three weeks of DOX induction , down 82% from the 299-fold seen following transgene induction for 42 days , which inhibits actin-myosin interactions and slows cross bridge cycling ,35, was To determine whether the altered contractile properties in DTG myocytes were the result of increased intracellular calcium concentration , myocytes were loaded with BAPTA-AM, a membrane-permeant calcium chelator . In contTwo classes of drugs that have been proven to reverse pathological remodeling and improve cardiac function in heart failure are angiotensin receptor antagonists, such as losartan, and ß-adrenergic receptor antagonists, such as propranolol . While tTo examine the clinical usefulness of this new model of HCM, DTG mice were given losartan (80 mg/kg/d) or propranolol (67 mg/kg/d) in their drinking water to determine if these drugs could prevent or lessen phenotype development  can be attributed to dominant mutations in sarcomeric proteins (over 800 mutations in 11 different contractile genes) . Many HCse model , suggestWe then turned our attention to other possible mechanisms for the development of hypercontractility and HCM, and hypothesized that expression of caFGFR1 resulted in cardiomyocytes that were hypersensitive to sympathetic stimulation. ß-adrenergic receptor activation in cardiomyocytes leads to synthesis of cAMP and activation of protein kinase A (PKA), which phosphorylates downstream targets, such as myofilament proteins , ion channels, and calcium handling proteins (phospholamban). Phosphorylation of these targets leads to increased inotropy and lusitropy in cardiomyocytes . HoweverDecreased TnI phosphorylation has important implications for the mechanism of phenotype development in this model. TnI is a sarcomeric protein that regulates contraction and relaxation. TnI inhibits the interaction between actin and myosin during diastole, when cytosolic calcium concentration is low. During systole, the calcium concentration rises, calcium binds to troponin C (TnC) and leads to a conformational change in the troponin complex that removes TnI inhibition of cross bridge formation and promotes sarcomere contraction. Phosphorylation of TnI by protein kinase A or C represents an important regulatory mechanism to modulate the contractile properties of the myofilament, as it increases the amount of calcium necessary for activation of contraction  and increases the rate of calcium dissociation ,42. ThisFurther studies will be necessary to determine precisely how acute activation of FGF signaling affects myofilament physiology. Mechanisms involved in TnI dephosphorylation are much less clear than those governing phosphorylation, although it has been demonstrated that protein phosphatases 1 or 2A  are capable of dephosphorylating TnI ,43–46. IAnother proposed model for the development and progression of HCM is abnormal intracellular calcium handling ,4. MouseTo assess the clinical usefulness of this model, we treated DTG mice with drugs shown to be effective in the management of HCM. ß-adrenergic receptor antagonists, such as propranolol, are used for the symptomatic relief of obstructive HCM to decrease the outflow gradient, although the precise mechanisms by which they improve cardiac function and promote reverse remodeling are unknown ,37. AddiThe studies presented here have only begun to address mechanisms of adult cardiac FGF signaling, as many significant questions remain. These studies make no assumptions regarding FGF ligands that may be involved in the regulation of cardiomyocyte contractility and hypertrophy, nor do we propose that activation of FGF receptors 2-4 will result in the same phenotype. Additionally, regulation of FGF signaling and activation of intracellular signaling cascades is poorly understood and beyond the scope of this paper. While multiple studies have demonstrated FGF2’s involvement in the development of pathological hypertrophy following injury, limited research has examined the expression or role of other FGF ligands or FGF receptors in the adult heart. These studies address consequences of FGF signaling on cardiovascular physiology and remodeling. While many classical mouse models of human HCM exist, our doxycycline-inducible cardiomyocyte-specific caFGFR1 mouse is unique in that it is inducible, reversible, and mediated by a non-sarcomeric protein. This model can be utilized to examine the common pathways that lead to phenotype development, as well as the extent of phenotype prevention and/or reversibility. This model also suggests the possibility that activating mutations in FGFR1 may directly lead to HCM or may modify the phenotype when combined with mutations in other proteins. Finally, perhaps the most striking finding from these studies is that low levels of activated FGFR1 have rapid and potent effects on cardiac contractility without any pathological effects, suggesting that targeted activation of the FGF signaling pathway may be beneficial to patients with heart failure and poor systolic function. Further investigation is necessary to more precisely elucidate the mechanisms responsible for the increased contractility following expression of caFGFR1, as well as pathways involved in the rapid progression from hypercontractility to HCM. File S1(DOCX)Click here for additional data file.Video S1(MOV)Click here for additional data file.


Comprehensive elucidation of genomic, transcriptomic, proteomic, and metabolomic networks that define normal and pathological states, in combination with reprogrammed patient cells are thus poised to become high value resources in modern diagnosis and prognosis of patient disease.Development of innovative high throughput technologies has enabled a variety of molecular landscapes to be interrogated with an unprecedented degree of detail. Emergence of next generation nucleotide sequencing methods, advanced proteomic techniques, and metabolic profiling approaches continue to produce a wealth of biological data that captures molecular frameworks underlying phenotype. The advent of these novel technologies has significant translational applications, as investigators can now explore molecular underpinnings of developmental states with a high degree of resolution. Application of these leading-edge techniques to patient samples has been successfully used to unmask nuanced molecular details of disease vs healthy tissue, which may provide novel targets for palliative intervention. To enhance such approaches, concomitant development of algorithms to reprogram differentiated cells in order to recapitulate pluripotent capacity offers a distinct advantage to advancing diagnostic methodology. Bioinformatic deconvolution of several “-omic” layers extracted from reprogrammed patient cells, could, in principle, provide a means by which the evolution of individual pathology can be developmentally monitored. Significant logistic challenges face current implementation of this novel paradigm of patient treatment and care, however, several of these limitations have been successfully addressed through continuous development of cutting edge  Disease anticipation prior to symptomatic presentation provides multiple pre-emptive opportunities for clinical management. Palliative or curative effectiveness is enhanced by early detection of disease-promoting factors, as early stage treatment can preclude catastrophic pathological progression. Translationally relevant risk factors can be identified through techniques that interrogate and assess biological marker status , where mDistinctions among high throughput methodologies confer specific advantages and disadvantages that determine efficiency and application of genomic, proteomic, or metabolomic approaches. For example, genes validated from tissue biopsies and measurement of glucose uptake with the glucose analogue tracer 18F-fluoro-2-deoxyglucose are among the tools that oncologists rely upon to diagnose and manage tumors . ExperimDifferential gene expression provides direct indicators of genetic background and transcriptome reprogramming as a consequence of evolving disease, but cannot provide information regarding dynamic functional changes. Peptide screening, a hallmark of proteomic analyses, can be employed to obtain this type of information ,8. For eAdvantages of genomic/transcriptomic and proteomic screens provide relevant data for clinical management of disease, though the techniques themselves require several days to weeks for quality-controlled processing and data analyses. Metabolic screening offers a distinct advantage in that samples can be assayed to provide feedback on disease states with greater celerity than the “-omic” approaches described previously. Though the prototype for metabolite analysis is glucose monitoring used in diabetes management , other eThese techniques allow deep interrogation of the molecular complement that comprises the functional background of a cell or tissue sample. Clinical application of these approaches gains significant leverage when combined with a pluripotent cell platform that can provide readouts of the molecular origin of disease phenotype.in silico methods. Indeed, bioinformatic approaches that employ systems biology principles to parse multidimensional biodata extracts multiple levels of integrated information, and application of such techniques to targeted stem cell populations may offer novel modalities of advanced pathology diagnosis.Current generation high throughput technology, originally developed to address rapid high volume sequencing needs , has galStem cells possess a unique potential to anticipate disease phenotypes as they harbor the fundamental molecular baseline that gives rise to genomic content and its derived transcriptomic, proteomic, and metabolomic strata. Interrogation of these “-omic” layers provides rich data to deconvolute systems biology of developmental programs, elucidation of which is critical to understanding clinical pathology etiology. Molecular cartography of pluripotent disease-prone backgrounds would facilitate pre-emptive diagnosis through comparison of healthy wild type templates with diseased states ,15,16, aEmbryonic stem (ES) cells can be harvested and profiled to establish a molecular baseline for comparison to diseased genomes. Recent data indicate that in addition to patient-donated material, patient-specific ES cells can be successfully cloned for potential therapeutic applications . Partly Full equivalency of induced compared to developmentally derived ES cells must be demonstrated to employ reprogrammed cells as proxies for a naturally occurring primordial baseline . Indeed,Systems-wide biodiagnostics, in which entire molecular complements can be assayed, provides an opportunity to comprehensively quantify molecular elements that underlie normal as well as clinically dysfunctional development. Current advances in cellular reprogramming methodology, along with the advent of high throughput technology and advanced bioinformatic computational approaches, presents a unique confluence of molecular tools and techniques by which diseased cells can be reprogrammed to a primordial state , then scThe genomic, transcriptomic, proteomic, and metabolomic elements of systems biology approaches provide biological information unique to each layer of the molecular network, and a variety of specialized techniques exist to interrogate each. From a molecular network ontogeny perspective, the genome is the origin of subsequent transcriptomes, proteomes, and metabolomes, and is a useful starting point for a discussion regarding roles the genome has played in translational diagnosis.The genome is the molecular blueprint composed of genic and non-genic sequences that ultimately determine organismal phenotype , and theImplementation of genome-wide analyses to stem cell (re)programming in the context of therapeutic application has yielded a rich body of novel data that provides details on changes in genomic regulatory elements, epigenomic landscape transitions, and three-dimensional chromatin shifts critical to the process of phenotype (re)acquisition ,33. ThesThe transcriptome is the full complement of RNA produced in response to signaling cues processed by, and transcribed from, the underlying genome, and technology and methods employed for genomic deconstruction are applicable to transcriptome resolution. Comprehensive transcript analysis is an attractive option for biomarker identification, as panels of differentially expressed genes (DEGs) are used to establish indices of disease progression . Prioritin silico approaches to properly resolve transcriptome dynamics of fate commitment/reversion  and function, including their physical and functional interactions . Modern Proteomic studies have increased our understanding of protein complexes and dynamics contributing to cell fate determination, of cell state transitions during development and reprogramming cells to pluripotency, and of the extensive molecular impact mediated by disease-targeted stem cell-based therapy. An expanded pluripotency network described by proteomic assessment of protein-protein interactions between known transcription regulatory factors required for maintenance of ES cells identified combinatorial effects of transcriptional activator complexes required for pluripotency, together with repressor complexes necessary to prevent expression of proteins associated with differentiated cells -49.Stoichiometric correlation between proteins and transcripts can vary , and proProteomic studies are also being used to define functional consequences of stem cell-based therapy. For example, extensive proteomic remodeling underlying structural and functional changes associated with onset of dilated cardiomyopathy was reversed by ES cell therapy, with derived protein networks exhibiting a pro-cardiogenic developmental response with concomitant demotion of dysfunctional disease-associated categories . UltimatThe metabolome consists of small molecular weight compounds that undergo chemical transformation within the cell. Metabolomics captures the complexity of global metabolism in the context of (patho)physiology, and a multitude of analytical tools have been developed to detect metabolite levels, such as enzymatic analysis, flame ionization, and Raman/Fourier transformed infrared and UV-VIS spectroscopy. Nuclear magnetic resonance (NMR) and mass spectrometry have become methods of choice due to their ability to resolve a wide range of chemical moieties in a high throughput manner . The majTo bridge the genotype-phenotype continuum, metabolite screening offers a minimally invasive diagnostic approach associated with high patient value and can provide a wealth of information as metabolite profiles serve as functional signatures of enzymatic activity . MetabolA growing number of studies have utilized metabolomic techniques to examine stem cell biology, critical for defining the baseline stem cell metabolic landscape and its perturbation in the diseased state, as well as identifying principal roles of energy metabolism in controlling stem cell fate -67. ThisGenomic, transcriptomic, proteomic, and metabolomic networks possess unique traits that endow these strata with qualities suitable for use as clinical tools, yet an added level of diagnostic sophistication may be accomplished by leveraging the integrated molecular architecture of these networks against complex disease phenotypes. Network biology, as part of a clinical management strategy, can potentially be employed to identify molecular candidates for pharmacological intervention ,72. DissBiological networks possess a discriminant set of characteristics that can be quantified ,75. ThisSystems biology integrates multiple disciplines to create a novel area for translational application, yet faces a plethora of challenges that constrain full implementation. High throughput techniques are expensive and availability to clinical populations at present is financially prohibitive, but as technology improves and assay costs diminish, application of these techniques to the greater clinical population becomes feasible . For exaAnother parameter associated with high throughput approaches is generation of massive amounts of biodata . LogistiTechnology development in the post-genomic area has given rise to advanced high throughput methodologies and modern, integrative bioinformatic tools that permit an unprecedented level of molecular resolution. Dissection of these discrete, yet interrelated molecular strata, in combination with the ability to reprogram phenotypically committed cells to a pluripotent state that provides a unique and patient-specific embryonic pool of cells, enables feasible dynamic and individualized diagnostic strategies for translational application -93. For ad hoc diagnosis and real-time molecular tracking of patient pathology and disease progression.Resetting diseased cells to a pluripotent state provides opportunities to track patient-specific changes at primary (genomic), secondary (transcriptomic), tertiary (proteomic), and quaternary (metabolomics) molecular network strata. Construction of an accessible electronic archive, to house large volumes of biodata produced from high grade bioinformatics analyses, is critical for establishing a dynamic clinical resource essential to fully realize comprehensive


An institutional task force on upper gastrointestinal tumours is active at the European Institute of Oncology (EIO). Members decided to collate the institutional guidelines on management of liver tumours (primary and metastatic) into a document. This article is aimed at presenting the current treatment guidelines as well as ongoing research protocols and trials in this field at the EIO.A steering committee convened to assign tasks to individual members. Contributions from experts in each treatment area were collated in a single document, in order to produce a draft for subsequent review from the aforementioned committee. Six drafts have been discussed and the final version approved.Surgical, medical oncology, interventional radiology, nuclear medicine and radiation therapy approaches, their roles in management of liver tumours and ongoing research trials are presented and discussed in this article.At the EIO a multi-disciplinary integrated approach to liver tumours is standard and several ongoing research projects are currently active in this field. Liver transplantation may overcome the problem of the low resectability rate in highly selected patients suffering from hepatocellular carcinoma (HCC), but it has very limited applicability, and non-resectional therapies with palliative  intent currently provide the mainstay of treatment. Careful patient assessment is required to select those patients affected by colorectal metastases (CRMs), with good prospects of achieving substantial benefits by means of integrated treatments, including surgery, interventional radiology and chemotherapy . A wide variety of treatment modalities are now available, each having its limitations, a number of which are reviewed here since they are routinely applied in our Institution. Such a wide range of treatment modalities requires a constant multi-disciplinary approach, where the management of each patient can be adapted to their particular needs. In the near future, the combination of these new technologies with advances in resectional surgery will offer significant improvements in the treatment of liver malignancies.HCC is a global disease killing more than a million people each year, to whom surgery offers the only real chance of cure, even if the majority have an irresectable disease because of tumour stage or advanced cirrhosis . MetastaHepatic resection is currently the only treatment able to offer a significant prognostic improvement in cirrhotic patients suffering from large HCCs, while liver transplant is still the best choice for very small lesions. Resection is also the best option for treatment of HCC developed in a normal liver ,4.In specialist centres, the surgical mortality rate of liver resection carried out in cirrhotic patients for HCC is under 3%; the five-year overall survival rate of these patients is more than 20%. In terms of crude survival rates, liver resection and transplant have the same results ,6. NeverA similar approach is currently proposed for surgical treatment of CRMs  initiallIn highly selected patients, harbouring large solitary lesions, we perform an embolization of the portal branch affected by the CRM  to obtain, after 4–6 weeks, a compensatory hypertrophy of the remnant liver, thus avoiding the risk of post-operative liver failure. Another approach that we use regularly in selected cases is the so-called ‘repeat-hepatectomy’; based on the regenerative properties of the liver, it is possible to perform metastasectomies in different operations, even when liver deposits are found in most segments and a single operation is not initially feasible.Between December 1995 and May 2005, 88 patients affected by colorectal liver metastases underwent hepatic resection with curative intent. Twenty-seven of these patients  were treated with neo-adjuvant chemotherapy. A seven-year survival analysis was performed. Chemotherapy included mainly 5-fluorouracil, leucovorin, and either oxaliplatin or irinotecan for a median of eight courses. Results: 16 patients (59%) were affected by synchronous, and 11 (41%) by metachronous metastases. During pre-operative chemotherapy tumour regression was obtained in ten cases (37%), stable disease (SD) in another ten cases (37%), and progressive disease (PD) in the remaining 7 (26%). The five-year overall survival for CT responders was 64%, which statistically differed from non-responders at CT  see . Among aA comparison between responders and non-responders to neoadjuvant chemotherapy is shown.Ultrasound examination of the liver, at open operation or with laparoscopic access to the peritoneal cavity, is the most accurate determinant of the extent and location of intra-hepatic tumour deposits . It is eLaparoscopic determination of incurability rather than determination at laparotomy results in less morbidity, lower costs and reduced hospitalization time . UltrasoFormal hepatic resection may be accompanied by considerable blood loss and the requirement for inflow occlusion, where ischaemia/reperfusion cycles and haemorrhage represent the major morbidity and mortality attributed to the surgery. Various techniques have been described to substantially reduce intra-operative blood loss during liver resection including the Cavitron ultrasonic aspirator (CUSA), microwave coagulation, water jet dissection, ultrasonically activated shears and the harmonic scalpel.Each of these techniques may or may not be coupled with inflow occlusion or vascular isolation, potentially further impairing perioperative hepatic functional reserve. New techniques, which result in sealing during parenchymal dissection, will potentially result in less bile leakage and haematoma formation at the hepatic resection margin as well as less post-operative hepatic dysfunction. The use of ultrasonic dissection of liver tissue has become a standard option in our institution: Using this technique, damage to vessels and biliary ducts can be avoided, while hepatic parenchyma is separated and cleared through a cavitational effect which occurs at the tip of the vibrating rod of the device , converting water to steam. The ultrasonic probe separates parenchymal cells because of their high water content by the cavitational effect without injuring structures having high content of fibrous tissue, like blood vessels and bile ducts.™) has been safely used in haemorrhoidectomy -octreotide or 90Y-DOTATOC), and the beta-gamma emitter 177Lu ([177Lu-DOTA0-Tyr3]-octreotate or 177Lu-DOTATATE). The total amount of radioactivity is fractionated in several cycles up to a cumulative activity sufficient to irradiate the tumour but below the kidney or the bone marrow dose thresholds .Yttrium-90 and Lutetium-177 are used as labels for their physical properties, which allow sufficient irradiation of the liver masses, due to a direct and a ‘cross-fire’ effect on surrounding receptor-negative cells . Our experience of receptor radionuclide therapy with 90Y-DOTATOC began in 1997, first with the dosimetric studies, then with two phase I studies , followed by the evaluation of the response to therapy and the evaluation of kidney protection. In 2004, we started a phase I–II, open-label, non-controlled, two-step sequential study aimed at defining the efficacy and the acute and late toxicity of 177Lu-DOTATATE in patients affected by neuroendocrine tumours. The study is ongoing and to date we have enrolled the pre-fixed number of 51 patients.With the pharmacokinetic and dosimetric studies, we established that both 90Y-DOTATOC and 177Lu-DOTATATE can be administered without causing serious toxicity . The tumDosimetric studies indicate that 90Y-DOTATOC and 177Lu-DOTATATE are suitable for efficient receptor radiotherapy. High absorbed doses to tumour lesions (>80–100 Gy) may result in a high percentage of cure . In our n=14), gastric (n=3), small intestine (n=34), rectum (n=3), functioning (n=16) and non-functioning (n=29) pancreatic, and unknown origin (n=14) endocrine carcinomas. Patients received cumulative activities of 90Y-DOTATOC ranging from 7.4 to 38.2 GBq, in two to 12 cycles. In a 5–81-month follow up, objective responses (CR+PR) were observed in 42 patients (38%), while 58 patients (50%) stabilized. Responses ranged from three to 76 months (median 20).We selected and treated with 90Y-DOTATOC a group of 114 patients affected by pure neuroendocrine tumours mainly of gastro-entero-pancreatic origin, the large majority of which (94%) with progressing liver metastases. Liver metastases developed from bronchial , which is a progressive, vascular-based entity, potentially degenerating to cirrhosis and liver failure . Thus, tRadiobiologically, the liver is characterized by a physiological parallel architecture; meaning that a close relationship exists between radiation absorbed dose and irradiated volume in terms of risk of functional damage. Thus, it is possible to deliver high radiation doses to a small portion of liver parenchyma without excessive risk of hepatic failure.The development of highly selective radiotherapy techniques, like three-dimensional conformal therapy, recently allowed the irradiation of smaller portion of liver parenchyma with higher doses, with similar risk of developing RILD, but higher chances of obtaining a ‘curative’ effect. It is possible to deliver, in a liver irradiation course, doses higher than 70 Gy, allowing treatment enhancement and dose-escalation protocols. Published series have reported local control rates of 68% and median survival periods ranging from 16 to 20 months.Furthermore, interest lies in a specific partial liver irradiation modality: stereotactic hypofractionated radiotherapy . It may For this irradiation modality, it is mandatory to guarantee an accurate target definition and adequate treatment reproducibility. Thus, specific efforts are required to guarantee improved patient setup and target localization, as well as to minimize, through specific systems of breathing control, tumour respiration-related motion.At the EIO, since May 2003, the Radiotherapy Division has been exploring this kind of treatment for primitive and metastatic liver cancer localizations, with particular efforts in patient selection, and integration with other loco-regional treatments. Specifically, treatment procedures: dose and fractionation schedules have been investigated, as well as the implementation of breathing control modalities. Forty-eight stereotactic liver treatments have been performed in 40 patients , both for primary HCC (five) and for metastatic liver localizations (43). In 19 cases, treatments have been performed with a declared palliative intent; otherwise, in 29 cases both the limited number (no more than three synchronous lesions at the time of irradiation), and the small size of lesions , as well the absence (or control) of extra-hepatic disease, configured a ‘curative’ purpose see .Irradiation doses ranged from 15 Gy in a single fraction (two cases) to 36 Gy in three fractions; the majority of patients being treated with total doses exceeding 24 Gy. Compliance to treatment has been satisfactory and acute toxicity was confirmed to two cases of gastric bleeding, due to the close position of the treated lesion to the stomach or the small bowel. As far as ‘curative’ treatments are concerned (29 treatments), 22 cases are actually evaluable for response to treatment, having a minimal follow-up of three months; for them, the first site of eventual disease progression (PD) has been evaluated in order to define the possible efficacy of stereotactic irradiation. In ten cases, no PD occurred at all (45%); in the remaining five cases (23%) experienced a PD at the site of irradiation (in two cases as a unique progressive disease), while seven cases (32%) evidenced a PD in another part of liver parenchyma (four cases) or as a systemic diffuse disease (three cases) but not at the site of treatment see . InteresIt is important to remember that stereotactic radiotherapy, as well as other high-dose partial liver irradiations, should not be considered antithetic to other loco-regional approaches, but as a complementary treatment. Thus, clinical research should be dedicated to the optimization of the technical aspects, and at the same time to the evaluation of the specific selection criteria for the different therapy options and for their correct integration.The criteria for resectability of colorectal liver metastases have been significantly revised and expanded over the past decade, and factors such as the tumour number, tumour size and presence of extra-hepatic disease should no longer be used to exclude patients from consideration for surgical resection, especially when a combined treatment can be proposed. Combining hepatic resection with radiofrequency ablation can expand the number of patients who may be submitted to liver surgery, particularly as larger lesions that are less effectively treated with ablation can be resected and small lesions can be ablated. Radiofrequency ablation has applicability in patients who do not meet the criteria for resectability but are candidates for liver-directed therapy based upon the presence of liver-only disease. In those patients with extensive disease and limited hepatic reserve, portal vein embolization or a two-stage hepatectomy approach can sometimes permit surgical resection. Similarly, the use of pre-operative chemotherapy can be associated with conversion of some patients from unresectable to resectable, but perhaps more importantly pre-operative chemotherapy can help assess the underlying biology of the patient’s disease. Patients with extensive extra-hepatic disease, or those who progress on systemic chemotherapy, should probably not be considered for hepatic resection. Over the past decade, chemotherapeutic and biologic agents have been found having significantly greater activity against colorectal cancer. These newer agents have led to higher response rates, exceeding 50% with combination therapies that include oxaliplatin and irinotecan. This superior efficacy of chemotherapy agents has allowed the treatment of a subset of previously unresectable patients and has ‘converted’ them so that they can undergo liver surgery following tumour downsizing. About 15–20% of patients with initially unresectable disease have significant tumour downsizing to the point that the metastatic disease can ultimately be considered resectable. As such, patients with extensive disease who respond to chemotherapy should be re-assessed by a hepatic surgeon. By reconsidering the initial unresectability of patients, hepatic resection and long-term survival may be achieved in a sub-group of patients who otherwise would have a poor outcome. Ultimately, a therapeutic approach that includes all aspects of multi-disciplinary and multi-modality care is required to select and treat this complex group of patients.Patients with HCV-related HCC have generally a better liver function than alcoholic patients, and are less prone to develop liver failure after aggressive procedures. This fact might explain the better results of arterial chemo-embolization in patients with viral disease compared with patients with alcoholic cirrhosis. Conversely, it has also been suggested that hepatic recurrences after surgery or ablative therapy are more frequent in patients with HCV-related HCC, possibly due to a higher incidence of new tumours. HCC screening is now widely performed in industrialized countries, leading to a better knowledge of liver carcinogenesis and an increasing rate of small HCC at diagnosis. Important advances have occurred concerning curative treatment of small tumours; even if liver transplantation, which is the best curative option for the long term as it is able to remove the tumour and the underlying cirrhosis (preventing therefore the occurrence of new tumours), can be performed in only limited number of well-selected patients. Other curative options, resection and percutaneous ablation mostly by radiofrequency, are able to cure the tumour mostly when small and well circumscribed. Due to a lower mortality and morbidity and a less deleterious influence on liver function, radiofrequency is to be performed in an increasingly larger number of patients with cirrhosis as new techniques now allow treatment of tumours more than 3 cm in diameter. Nevertheless, tumour recurrence rate is high (10–20% per year) in cases of resection or radiofrequency due to either intra-hepatic metastasis or occurrence of a new HCC, a case that is particularly frequent in patients with HCV infection. This fact justifies post-therapeutic surveillance of patients and the search for preventive treatments.In metastatic liver disease, a multi-disciplinary approach, tailored to each patient, has shown an improvement in the therapeutic results. Receptor radionuclide therapy is preferentially performed in minimal residual disease. Thus, our multi-disciplinary approach includes the symptomatic control of hypersecretory syndromes, with somatostatin analogues and/or interferon alpha-2b, and cytoreduction with surgery or chemoembolization, in order to reduce the amount of tumour to be subsequently treated with radiopeptide therapy using 177Lu-DOTATATE.The development of highly selective radiotherapy techniques, like three-dimensional conformal therapy, recently allowed the irradiation of a smaller portion of liver parenchyma with higher doses, without excessive risk of hepatic failure. Stereotactic hypofractionated radiotherapy may represent an effective irradiation approach, delivering therapeutic doses in a single or a few treatment fractions, with practical advantages of patient’s compliance, better integration with other treatment modalities, and comparable outcome in terms of toxicity and local control, both for hepatocellular carcinoma and colorectal liver metastases. Preliminary data confirm the feasibility of the modality, although extensive follow-up data are still warranted to explore the potential value of stereotactic irradiation on overall clinical outcome.In conclusion, an institutional task force on upper gastrointestinal tumours is active at the EIO. Members decided to collate the institutional guidelines on management of liver tumours (primary and metastatic) into a document. Contributions from experts in each treatment area were collected in a single document, in order to produce a draft for subsequent review from the aforementioned committee. Six drafts have been discussed and the final version approved, representing the content of this article. Surgical, medical oncology, interventional radiology, nuclear medicine and radiation therapy approaches, their roles in management of liver tumours and ongoing research trials have been presented here and discussed, focusing on the value of the multi-disciplinary approach to liver neoplasms.


Complicated crown-root fracture of maxillary central and lateral incisors is common in case of severe trauma or sports-related injury. It happens because of their anterior positioning in oral cavity and protrusive eruptive pattern. On their first dental visit, these patients are in pain and need emergency care. Because of impaired function, esthetics, and phonetics, such patients are quite apprehensive during their emergency visit. Successful pain management with immediate restoration of function, esthetics and phonetics should be the prime objective while handling such cases. This paper describes immediate treatment of oblique crown root fracture of maxillary right lateral incisor with reattachment procedure using light transmitting fiber post. After two and half years, the reattached fragment still has satisfying esthetics and excellent function. The increased incidence of traumatic injuries to anterior teeth is a consequence of leisure activities, where the most common injuries are crown fractures. These manifestations can vary from simple enamel-dentin fracture to complicated crown-root fracture or root fracture . It has Various methods and techniques were advocated to restore fractured teeth. In the late 60's, temporary and permanent restoration of traumatized teeth in young individuals represented a severe challenge. Methods like resin crowns, stainless steel crowns with or without window preparation, pin-retained inlays, and complex ceramic restorations were in use . All theAcid-etch technique and advances in dentinal adhesives combined to drive a growing minimally invasive treatment philosophy among dentists, as evidenced by a renowned interest in the tooth reattachment procedure . This isThe advantages of tooth fragment reattachment over conventional composite restoration are conservatism, favorable wear mechanism, color match of the remaining crown portion, preservation of incisal translucency, maintenance of original tooth contours, preservation of identical occlusal contacts, color stability of the enamel, cost effectiveness, and convenient single visit treatment –15. The Nowadays a number of successful reattachment cases and research are reported in literature , 16–22. A 23-year-old patient reported to the dental hospital after 5 days of trauma and sustained a complicated crown-root fracture to the maxillary right lateral incisor . The fraSince the fractured fragment of the maxillary left central incisor was attached to the underlying root at just one point so it was removed and stored in distilled water to be used at a later stage Figures  and 3. IAfter two and half years, the treated tooth has satisfying excellent esthetics and function Figures  and 10. A patient with fractured anterior teeth usually reports with pain and is emotionally upset about his or her appearance . Quick r


Ischemia and metabolic crisis are frequent post-traumatic secondary brain insults that negatively influence outcome. Clinicians commonly mix up these two types of insults, mainly because high lactate/pyruvate ratio (LPR) is the common marker for both ischemia and metabolic crisis. However, LPR elevations during ischemia and metabolic crisis reflect two different energetic imbalances: ischemia (Type 1 LPR elevations with low oxygenation) is characterized by a drastic deprivation of energetic substrates, whereas metabolic crisis  is associated with profound mitochondrial dysfunction but normal supply of energetic substrates. The discrimination between ischemia and metabolic crisis is crucial because conventional recommendations against ischemia may be detrimental for patients with metabolic crisis. Multimodal monitoring, including microdialysis and brain tissue oxygen monitoring, allows such discrimination, but these techniques are not easily accessible to all head-injured patients. Thus, a new “gold standard” and adapted medical education are required to optimize the management of patients with metabolic crisis. Managing traumatic brain injury is like navigating the ocean. Dangers (secondary insults) are everywhere, but, usually, easy to prevent. In this ocean, just imagine cerebral energetic disturbances as an iceberg, where ischemia is the tip Figure . All cliIn Intensive Care Unit (ICU), cerebral post-traumatic metabolic disturbances have commonly been characterized by an increase of the lactate/pyruvate ratio (LPR) above 40, as measured by clinical intracerebral microdialysis. High LPR has originally been attributed to compromised cerebral perfusion and impaired oxygen delivery. However, high LPR may actually reflect several pathological events or compensatory mechanisms . After tIn the literature, confusion persists with articles dealing with metabolic crisis and that potentially refer to any type of metabolic disturbances . The discrimination between different types of metabolic disturbances is, however, crucial because each type requires an adapted management to avoid deterioration of the patients. In addition, there is currently no consensual terminology for the specific pattern of metabolic disturbance characterized by elevated extracellular LPR and normal oxygenation. Indeed, the original work of Vespa and colleagues referred to this kind of disturbance as “metabolic crisis without brain ischemia” , whereasTo understand the singularities of each type of post-traumatic metabolic disturbance , some physiological aspects of the cerebral metabolism need to be considered.Neurons and astrocytes can efficiently utilize lactate, pyruvate, glutamate, and glutamine as energetic substrates, in addition to glucose . AstrocyAn increase in glucose utilization is commonly observed in head-injured patients in the first days after injury. Several roles have been proposed for this post-traumatic hyperglycolysis, including restoration of the ionic balance. This compensatory hyperglycolysis can induce a transient accumulation of lactate in the extracellular compartment of the brain in head-injured patients , 7. UndeIt is commonly admitted that ischemia is one of the most deteriorating post-traumatic insults. Experimental studies of traumatic brain injury have shown that cerebral oxidative metabolism is reduced due to ischemia, because of oxygen and glucose deprivation , 16. In Vespa’s article published in 2005 may be considered as the princeps article introducing the concept of the metabolic crisis , 17. OccThere is a gap of knowledge regarding the exact mechanisms underlying metabolic crisis. Nevertheless, two main hypotheses have been proposed: mitochondrial dysfunction and excessive increase in metabolic demand.tiO2), but not with a decrease in extracellular pyruvate level , have unfortunately failed to improve the oxidative metabolism or normalized the biomarkers of metabolic crisis , 36–39. Protocol-driven management is currently used to treat most head injuries . This aptiO2) monitoring, has allowed the detection of metabolic crisis. However, these techniques do not meet the requirements for determination of a gold standard: sensitivity and specificity. Indeed, (1) the microdialysis markers of metabolic crisis are not sufficiently specific, and (2) PtiO2 primarily reflects a compromise between local CBF and oxygen delivery and therefore gives only very indirect information on cerebral oxygen metabolism (The most important factor to consider in future clinical trials is the segregation of patients with metabolic crisis. To date, in everyday clinical practice, only multimodal monitoring, i.e., microdialysis and brain tissue oxygen (Ptabolism . Moreovetabolism . So, bectabolism . Future Although metabolic crises are frequent and deleterious after traumatic brain injury, they have not been much studied so far. The challenge of the coming years will be to clearly define the specific mechanisms underlying metabolic crisis in order to improve its diagnosis and to optimize therapeutic treatment. Medical education about metabolic crisis will be a key factor for optimal management of head-injured patients with metabolic crisis.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.