From other studies, antigen presentation by B cells, not dendritic cells, is likely responsible for original antigenic sin. Recognition of restricted epitopes on the large glycosylated thyroid autoantigens  facilitates exploring the amino acid locations in the immunodominant regions. Epitope spreading has also been revealed by autoantibodies in thyroid autoimmunity. In humans, and in mice that spontaneously develop autoimmunity to all three thyroid autoantigens, autoantibodies develop first to Tg and later to TPO and the TSHR A-subunit. The pattern of intermolecular epitope spreading is related in part to the thyroidal content of Tg, TPO and TSHR A-subunit and to the molecular sizes of these proteins. Importantly, the epitope spreading pattern provides a rationale for future antigen-specific manipulation to block the development of all thyroid autoantibodies by inducing tolerance to Tg, first in the autoantigen cascade. Because of its abundance, Tg may be the autoantigen of choice to explore antigen-specific treatment, preventing the development of pathogenic TSHR antibodies.Evidence for original antigenic sin in spontaneous thyroid autoimmunity is revealed by autoantibody interactions with immunodominant regions on thyroid autoantigens, thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyrotropin receptor (TSHR) A-subunit. In contrast, antibodies induced by immunization of rabbits or mice recognize diverse epitopes. Recognition of immunodominant regions persists despite fluctuations in autoantibody levels following treatment or over time. The enhancement of spontaneously arising pathogenic TSHR antibodies in transgenic human thyrotropin receptor/NOD. The concept of “original antigenic sin” arose from findings in humans and mice infected with influenza virus , 2 and iA phenomenon interlinked with original antigenic sin is epitope spreading, a well-recognized feature of some autoimmune conditions such as type 1 diabetes mellitus and multiple sclerosis, as well as for the animal models of these diseases, namely NOD mice and experimental autoimmune encephalomyelitis (EAE). Spreading can involve increasing the number of epitopes recognized on the same autoantigen (intramolecular) and subsequent recognition of additional autoantigens (intermolecular) over time. An example of intermolecular spreading, in EAE, SWX mice immunized with myelin proteolipid protein (PLP) develop the (as expected) T cell reactivity to determinants on PLP and to determinants on myelin basic protein and myelin oligodendrocyte glycoprotein . SimilarThyroid autoimmune disease, Hashimoto’s thyroiditis, and Graves’ disease are the most common organ-specific autoimmune diseases affecting humans, far more common than type 1 diabetes mellitus and multiple sclerosis. In particular, approximately 1% of the population will develop Graves’ disease in their lifetime and ~15% of adult females have autoimmune thyroiditis, although usually subclinical , 10. AniWhether autoantibodies to TPO and Tg play a role in thyroid cell destruction is unclear, but they are excellent markers of the immune response to the thyroid. Moreover, B cells are increasingly recognized as powerful antigen-presenting cells by means of their membrane-bound antibodies that capture small amount of antigen for processing and presentation to T cells . TPO auintramolecular original antigenic sin and intermolecular epitope spreading in autoimmune thyroid disease. Our findings have important implications for understanding disease pathogenesis and for developing novel antigen-specific therapeutic approaches to control the development of thyroid autoantibodies and thereby prevent thyroid autoimmunity rather than treating the clinical disease.Here, we examine evidence involving thyroid autoantibodies for both It has long been recognized that antibodies induced experimentally to Tg interact with multiple, widely diverse epitopes on the large (600 kDa) dimeric Tg molecule, whereas human Tg autoantibodies interact with a restricted number of epitopes Figure  20–23).–23.20–23It should be emphasized that binding by the majority of patients’ autoantibodies to TPO is decreased following protein denaturation , 48, demIn this review, because of their dominance in the patient repertoire, we will focus on autoantibodies to TPO and Tg that interact with conformational epitopes.without intramolecular epitope spreading, for example, in families in which the proband has juvenile Hashimoto’s thyroiditis , which are responsible for hyperthyroidism in Graves’ disease, and TSH-blocking antibodies (TBAbs), which cause hypothyroidism. In rare patients, TSHRAb switching from TSAb to TBAb (or vice versa) has been observed [reviewed in Ref. ]. The deIt is of interest that injecting mice with TPO expressed together with MHC class II on a fibroblast line induced TPOAb that resembled autoantibodies from Hashimoto or Graves’ patients in terms of a high affinity for TPO and recognition of an IDR . In the Autoantibodies interact with epitopes in an IDR in other autoantibody-mediated diseases [reviewed in Ref. ]. For exOverall, the stability of human thyroid autoantibody recognition of an IDR is suggestive of “original antigenic sin.” It is possible that this concept may also apply to other human autoantibodies directed to an IDR on their respective autoantigens.induced to develop Graves’ disease by immunization with an adenovirus encoding the TSHR A-subunit gene, pretreatment with a non-pathogenic (or “inactive”) form of TSHR A-subunit protein attenuated hyperthyroidism by diverting pathogenic TSHR antibodies to a non-functional variety /NOD.h4H2 mice with the human TSHR A-subunit transgene targeted to the thyroid  as well as pathogenic TSHR antibodies   Figure . By usin) Figure . SimilarThe mechanisms responsible for original antigenic sin are not fully understood. However, because of the problems caused for vaccination against novel viral strains, approaches have been used to overcome original antigenic sin. These studies provide insight into the basis for this phenomenon, at least from the perspective of T cell epitopes.Bordetella pertussis toxin) that activate dendritic cells  thyroid cells, rather than dendritic cells. It should be noted that a restricted antibody response focused on the TPO IDR (as in humans) occurs in AKR/J-mice injected with cells expressing human TPO but not in mice of the same strain immunized with human TPO protein and adjuvant . The res in Ref. ].(i)C-terminal end of the molecule TPO is a homodimer (each 110 kDa) inserted in the plasma membrane, and TPO autoantibodies are directed against the ectodomain. The TPO epitope of an human autoantibody (expressed as a recombinant Fab) was identified using footprinting technology . In addi(iii)The TSHR, like TPO, is also membrane bound but pathogenic TSHR autoantibodies (as in Graves’ disease) are induced spontaneously to the heavily glycosylated TSHR A-subunit (~60 kDa) shed after cleavage of the membrane-bound receptor [reviewed in Ref. ]. Amino In addition to suggesting the likely cells involved in autoantigen presentation, IDR recognition simplifies what would otherwise be a gargantuan task, namely exploring the regions recognized by autoantibodies on large, glycosylated thyroid autoantigens:either Tg or TPO but none had TgPOAb activity . Unlike multiple sclerosis or EAE, original antigenic sin appears to be characteristic of thyroid autoimmunity as reflected by the recognition of an autoantibody IDR in humans and in the response of transgenic hTSHR/NOD.Finally, the pattern observed for intermolecular epitope spreading provides the rationale for antigen-specific manipulation to block thyroid autoantibody development by inducing tolerance to the first autoantigen in the antigen cascade, namely Tg. Because of its abundance, Tg may be the autoantigen of choice to explore antigen-specific treatment to block the development of pathogenic TSHR antibodies.SM and BR contributed to the concept, writing, and preparation of figures for this review.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. The handling editor declared a past co-authorship with author SM.


During pregnancy, regular physical activity is associated with the course of a healthy pregnancy, as it can increase physical fitness, and may lower the risk of pre-eclampsia, gestational diabetes, chronic hypertension, excessive gestational weight gain, macrosomia, and stillbirth , 2. GuidFor example, African-American and Latina women have higher risk of developing several complications, such as gestational diabetes mellitus (GDM), chronic hypertension, pre-eclampsia and Caesarean delivery in comparison to non-Latina white and non-obese women , 9. GDM Despite evidence that exercise improves lipid profiles in non-pregnant populations, in pregnant women this evidence is scarce. Dyslipidaemia during pregnancy is associated with GDM, pre-eclampsia, preterm birth  and otheRecent results support an effect of supervised physical exercise on triglyceride (TG) levels during pregnancy , 16, howTherefore, the purpose of this prospective, randomized study was to assess the benefits of supervised physical exercise during pregnancy, using a recommended physical activity dose for maternal lipids in low-income pregnant Latina women. Secondary aims were to assess the effect of exercise on obstetrical and neonatal outcomes.The present study is a secondary analysis randomized controlled trial (RCT) published previously , 19. Theclinicaltrial.gov [NCT00741312].Measurements were taken at baseline for participant women in their first trimester of gestation  and at the end of the exercise intervention period when participant women were in their third trimester of gestation  , 19. All−1 of moderate-intensity activity or 75 min·wk.−1 of vigorous-intensity activity); iii) nulliparous; iv) were in their 16th to 20th week of gestation; and v) with a live foetus confirmed by a routine ultrasound scan , . The experimental group showed a higher percentage in of Caesarean sections in comparison to the control group without significant differences . The experimental group showed fewer complications during delivery (postpartum hemorrhage moderate) than did the control group . Finally, the experimental group showed fewer complications in newborns compared to the control group   between groups Table . In the Our study shows that physical exercise reduces the c-LDL and TG increases in normal ranges, favouring fewer delivery and neonatal complications. Beneficial relations between physical activity and the plasma lipid profile in non-pregnant women have been demonstrated. Findings reported that the sensitivity of the regression slopes for HDL and TG imply that women may be more resistant to exercise-induced changes than men . A meta-Data from the OMEGA study reported that active women (early in pregnancy) had significantly lower mean TC and TG in comparison to those of women performing no recreational physical activity . Other sOur results showed that physical exercise reduces the excessive LDL-c and TG gain. Therefore, findings seem to suggest that early pregnancy physical activity could have a lasting impact on TG levels. In contrast, another study showed, surprisingly, that the exercise group had significantly higher LDL-c levels in the third trimester in comparison to the control group . These fA meta-analysis from 11 RCTs in sedentary women provides evidence that moderate-intensity physical exercise during physical activity in pregnancy does seem to positively influence excessive gestational weight gain . AnotherRegarding obstetrical and neonatal outcomes, our study showed that exercise performed over the second and third trimesters of pregnancy does not negatively influence gestational age, Caesarean delivery or neonatal growth. Previous studies investigating the effect of exercise during pregnancy and birth weight report inconsistent findings . SeveralA few limitations in this study should be considered and caution should be taken in the interpretation of the findings. Firstly, due to withdrawals, this study was underpowered to detect differential effects of supervised exercise training on blood lipids. Secondly, there was no assessment of other biomarkers or dietary nutrition intake, which could have shown additional information on metabolic health status. Thirdly, therapists and women were not blinded. However, comparison of the final study population with the women who did not complete the study did not identify any differences between groups. The pregnant participants in our study were healthy nulliparous with a low-income level, and are therefore not representative for all eligible Latina women.The strengths of our study include the consideration of important confounding variables in our analysis, such as compliance and obstetrical and neonatal outcomes. First, physical activity recommendations during pregnancy were evaluated. Second, women underwent a peak exercise test to more accurately prescribe the exercise intensities.In summary, exercise during the second and third trimester of pregnancy in sedentary women reduced excessive LDL-c and TG gain and favoured fewer delivery and neonatal complications without any adverse acute foetal responses to current exercise recommendations. Hence, implementation of an exercise-training programme is feasible and safe in low-income Latina women. The potential public health benefits of exercise are too great for obstetricians to miss the opportunity to effectively counsel pregnant low-income Latina women about this important health behaviour.


Selenium (Se) is an important micronutrient for many organisms, which is required for the biosynthesis of selenocysteine, selenouridine and Se-containing cofactor. Several key genes involved in different Se utilization traits have been characterized; however, systematic studies on the evolution and ecological niches of Se utilization are very limited. Here, we analyzed more than 5200 sequenced organisms to examine the occurrence patterns of all Se traits in bacteria. A global species map of all Se utilization pathways has been generated, which demonstrates the most detailed understanding of Se utilization in bacteria so far. In addition, the selenophosphate synthetase gene, which is used to define the overall Se utilization, was also detected in some organisms that do not have any of the known Se traits, implying the presence of a novel Se form in this domain. Phylogenetic analyses of components of different Se utilization traits revealed new horizontal gene transfer events for each of them. Moreover, by characterizing the selenoproteomes of all organisms, we found a new selenoprotein-rich phylum and additional selenoprotein-rich species. Finally, the relationship between ecological environments and Se utilization was investigated and further verified by metagenomic analysis of environmental samples, which indicates new macroevolutionary trends of each Se utilization trait in bacteria. Our data provide insights into the general features of Se utilization in bacteria and should be useful for a further understanding of the evolutionary dynamics of Se utilization in nature. This element is known primarily for its functions in redox homeostasis, and is recognized as a promising cancer chemo-preventive agent. In addition, Se is required for mammalian development, male reproduction and immune function ; (ii) 5-ficiency ; (iii) atrans-acting factors including Sec synthase (SelA), Sec-specific elongation factor (SelB), Sec-specific tRNA[Ser]Sec (a unique tRNA whose anticodon matches the UGA codon) and selenophosphate synthetase (SelD). In eukaryotes and archaea, the biosynthesis of Sec uses a similar mechanism as in bacteria. However, additional genes such as genes encoding the eukaryotic/archaeal Sec synthase, O-phosphoseryl-tRNA[Ser]Sec kinase and SECIS binding protein SBP2 are needed for incorporation of Sec into protein  is needed during SeU utilization  of all Sec-utilizing organisms showed the presence of a new selenoprotein-rich phylum and more selenoprotein-rich organisms in bacteria. Finally, analysis of environmental factors of all organisms revealed that different Se traits may favor specific ecological conditions such as habitat, oxygen concentration and temperature. As a whole, these data provide a better understanding of the general trends of Se utilization and evolution in bacteria.In this study, we applied comparative genomics approaches to more than 5200 sequenced bacterial genomes to investigate Se utilization in this domain. First, we analyzed the distribution of key components involved in different Se utilization traits and generated a large phylogeny map of Se utilization in bacteria. We also identified a subset of organisms that had ftp://ftp.ncbi.nlm.nih.gov/genomes/). A total of 5207 genomes were retrieved (as of January 2014). Information about environmental and other factors  associated with these organisms was acquired from either NCBI or Genomes OnLine Database (GOLD)  . The redundant selenoprotein sequences were removed using a custom script program. The presence of a putative Sec-encoding UGA codon and a downstream SECIS element was also analyzed using the bSECISearch tool  gene, which is a highly conserved housekeeping gene and has been widely used in many comparative metagenomic studies  were retrieved from the Joint Genome Institute Metagenome/Integrated Microbial Genomes (JGI-M/IMG) database , SeU (SelD/YbbB) and Se-cofactor (SelD/YqeB/YqeC) traits, in more than 2000 bacterial genomes .It has been proposed that SelD protein was required for all Se utilization traits, which defined the overall Se utilization. In this study, except for phyla containing one or two sequenced genomes , SelD was detected in almost all bacterial lineages with the exception of a small number of phyla or clades such as Chlamydiae, Mollicutes, Deinococcus-Thermus and Thermotogae. This implies that Se utilization is an ancient trait that once was common to almost all species in bacteria. However, only 1754 organisms  were detected to contain SelD, suggesting that most species have lost the ability to use this micronutrient over the long process of evolution. This is consistent with previous observations . All these results were consistent with our previous assumption that the presence of one Se trait may be beneficial to acquisition of others, probably partially due to the presence of SelD , 980 SeU-utilizing (18.8%) and 312 Se-cofactor-utilizing (6.0%) organisms. SelB has been suggested to be the signature of the Sec and YbbB of the SeU traits . For exaHelicobacter pylori that does not use Se , but lack a complete Se utilization trait (details are shown in Actinopolyspora iraqiensis IQ-H1 (Actinobacteria), Brevundimonas sp. BAL3 and Methylobacterium nodulans (Alphaproteobacteria), Xenorhabdus bovienii and X. nematophila  and Prochlorococcus sp. W7~W9 (Cyanobacteria). It is most likely that a novel and unknown SelD-based Se utilization trait is present in at least some of these organisms. On the other hand, the possibility that some of these selD genes are either pseudogenes or involved in non-Se-related processes could not be fully excluded.Previously, we have noticed the presence of very few organisms that had orphan SelD (containing SelD but lacking any other known components of Se utilization traits and selenoprotein genes) . These oselD in these organisms. Three genes were chosen as SelD-related candidates due to their genomic locations close to selD in at least three organisms belonging to different phyla, including genes encoding an isochorismatase-like protein (pfam00857), ABC transporter-related ATPase (pfam00005) and a cysteine desulfurase-like (pfam00266) protein  and Photobacterium profundum (Gammaproteobacteria), as well as between Pseudomonadale species (Gammaproteobacteria) and Betaproteobacteria  events for both Sec and SeU traits, such as HGTs of the entire Sec utilization pathway observed between Marinobacter species , M. daepoensis DSM 16072 was the only organism that contains the Sec trait, which might have acquired from Pseudomonas species . Moreover, in the majority of both donor and recipient organisms, the key genes (including selD) involved in the corresponding Se utilization traits as well as the only selenoprotein gene  in those Sec-utilizing organisms are either very close or even organized in single operons.Fusobacterium sp. 11_3_2 (Fusobacteria) sequences were within the Firmicutes/Clostridia node, and not within the Fusobacteria node as expected by vertical descent (Treponema phagedenis F0421 (Spirochaetes), which also acquired the Se-cofactor trait from Firmicutes/Clostridia  and used representative sequences for each of them to search for Sec-containing homologs in all Sec-utilizing organisms . The number of examined organisms was eight times more than previous studies .Syntrophobacter fumaroxidans (Deltaproteobacteria), which contains at least 39 selenoprotein genes . It was suggested that the Sec utilization trait might favor anaerobic and/or hyperthermic conditions, whereas the SeU trait might favor aerobic and mesophilic conditions . ConsideIn this study, we collected the information about ecological environments and some other factors for all organisms, and analyzed the contribution of each of these factors for organisms containing Sec, SeU and/or Se-cofactor traits. First, we found that both Sec and Se-cofactor traits favored a host-associated condition, whereas organisms possessing the SeU trait were aquatic species that were mostly isolated from sea or freshwater . Second,To further verify the relationship between Se utilization patterns and the habitat/environmental preferences of bacteria, we performed a comparative metagenomic analysis of Se utilization in 60 bacterial communities from different habitats or environments: aquatic (marine and freshwater), terrestrial and host-associated. Sample details are shown in selD gene revealed that it might not be essential for the majority of organisms in almost all environmental samples examined here, which is generally consistent with the above observation  have been regarded as the best alternatives to cereals in food and feed  and aquatic  environments could provide sufficient supply of Se. However, Se utilization in terrestrial bacteria may be heavily dependent on diverse Se levels in different areas. In addition, the substantial microbial taxonomic diversity in different ecological environments might also explain differences in Se utilization. Surprisingly, we could not identify the strong relationship previously reported between oxygen level and Sec utilization , as a siIn conclusion, we have performed a comprehensive comparative analysis of Se utilization in bacteria, which generated the largest map of Se utilization in this domain. Our data revealed a complex and dynamic evolutionary history for each Se utilization trait. Identification of organisms that only have SelD implies the presence of a new SelD-dependent Se utilization pathway in bacteria. Phylogenetic analyses of key components of Se utilization traits suggested that additional HGT events could be detected for each of them; however, co-transfer of two or more Se traits might be extremely difficult and has not been observed yet. Identification of new selenoprotein-rich phylum and selenoprotein-rich organisms as well as their selenoproteomes may provide important perspectives on the diversity and evolution of different selenoprotein families. Finally, comparative analyses of environmental conditions for all sequenced bacteria and metagenomic samples revealed new and important relationships between ecological environments and Se utilization.


Temporal discrimination is the ability to determine that two sequential sensory stimuli are separated in time. For any individual, the temporal discrimination threshold (TDT) is the minimum interval at which paired sequential stimuli are perceived as being asynchronous; this can be assessed, with high test–retest and inter-rater reliability, using a simple psychophysical test. Temporal discrimination is disordered in a number of basal ganglia diseases including adult-onset dystonia, of which the two most common phenotypes are cervical dystonia and blepharospasm. The causes of adult-onset focal dystonia are unknown; genetic, epigenetic, and environmental factors are relevant. Abnormal TDTs in adult-onset dystonia are associated with structural and neurophysiological changes considered to reflect defective inhibitory interneuronal processing within a network which includes the superior colliculus, basal ganglia, and primary somatosensory cortex. It is hypothesized that abnormal temporal discrimination is a mediational endophenotype and, when present in unaffected relatives of patients with adult-onset dystonia, indicates non-manifesting gene carriage. Using the mediational endophenotype concept, etiological factors in adult-onset dystonia may be examined including (i) the role of environmental exposures in disease penetrance and expression; (ii) sexual dimorphism in sex ratios at age of onset; (iii) the pathogenesis of non-motor symptoms of adult-onset dystonia; and (iv) subcortical mechanisms in disease pathogenesis. The purpose of this review is to present and analyze, from clinical, neurophysiological, and neuroimaging studies, the evidence as to the biological basis of temporal discrimination and the role of abnormal temporal discrimination in understanding the pathogenesis of adult-onset focal dystonia.The TDT  is defined as the shortest interval at which two sequential sensory stimuli are perceived as being asynchronous . The seqThe TDT is relatively easy to assess in the laboratory or even, by using a portable headset, in the participant’s home . The stiZ-scores.An early study of 80 healthy volunteers aged from 18 to 82 years found that TDT increased only in subjects older than 65 years . Age-relAn intriguing interaction of the relationship between age and sex, so far unreported by others, has been described , 9. YounThere are numerous comorbid disorders, which need to be screened for prior to study, which may affect TDT testing. For visual TDTs, corrected visual acuity needs to be assessed and patients with any condition resulting in loss of visual acuity, need to be excluded. For tactile TDT testing, impaired superficial sensation needs to be enquired about and examined. Also excluded are participants with a history of a neurological disorder or medication known to affect the basal ganglia. All participants should be screened for cognitive impairment using the Montreal Cognitive Assessment; cognitive impairment will affect the ability to understand and participate in the study.Although intuitively plausible, there had been no evidence, until recently, that intensive training from an early age has an effect on the efficiency of temporal discrimination. Musicians perform better than non-musicians in visual timing tasks including rhythm perception and duration discrimination ; howeverSub-second temporal processing of sensory information has been studied by several methodological approaches including temporal order judgment (TOJ), frequency discrimination task, time estimation tasks, and interval discrimination tasks –16. ThesFrequency discrimination and interval discrimination usually involve time interval comparison. Intervals elapsing between the two stimuli vary in the hundred milliseconds range, a procedure implying that the structures for encoding differ from those for temporal discrimination, which encompasses tens of milliseconds . Unlike Evidence, from lesional and neurophysiological studies, suggests that TDT involves the time-locked activation of both subcortical and cortical neural networks. Which neural structures primarily determine temporal discrimination is still open to debate; does temporal discrimination require cortical activation or is it purely dependent on basal ganglia–brainstem–cerebellar integrity?The role of the basal ganglia in temporal processing has been well known for decades. In an fMRI study in healthy subjects, Pastor and colleagues initially showed that, as well as primary somatosensory cortex and cerebellum, other areas, specifically active during temporal processing, included the pre-supplementary motor area (pre-SMA) and putamen . In a laIt is postulated that temporal discrimination is a measure of the acuity of an alerting circuit that signals the detection of biologically salient events (event or emergency) in order to modify on-going behavior (freeze or escape). Midbrain dopaminergic neurons produce short-latency responses to biologically salient events –30. A saThe pre-SMA probably plays a role in focusing attention on the discriminative task but is not determinant in encoding TDT values . TemporaAccumulating evidence indicates that the TDT task relates to the detection of novel, salient environmental change involving a brainstem–cerebellar–basal ganglia neuronal network with the superior colliculus as a principal cross-modal sensory input node. A comprehensive hypothetical model for the neural circuits involved in normal temporal discrimination requires basal ganglia functional integrity in a network that integrates incoming sensory information from the superior colliculus, thalamic nuclei and cerebellum and selects salient events for on-going behavior through the dopamine-mediated alerting system Figure . TemporaAdult-onset idiopathic isolated focal dystonia  is an uncommon movement disorder of unknown cause and pathogenesis . It presCurrent concepts concerning AOIFD are: (1) AOIFD is an autosomal dominant disorder with markedly low penetrance (which explains why most cases are sporadic). (2) The different phenotypes are not caused by different genetic mutations; discordant phenotypes are seen in 50% of affected proband-relative pairs  and are also seen in multiplex families . (3) EnvAbnormal temporal discrimination in AOIFD was initially described in 2001  and confWe consider that an abnormal TDT is a mediational endophenotype of AOIFD. This implies that the endophenotype and the disease are both caused by a genetic disorder and that the pathway from gene to disease passes through the endophenotype; one cannot acquire the disease without first having the endophenotype . A mediaAlthough many publications report cortical abnormalities in AOIFD, these abnormalities may be secondary, adaptive changes in response to the motor manifestation of dystonia. One structural abnormality, coherent with the endophenotype, abnormal temporal discrimination, is alteration in the size of the putamen. Unaffected relatives  with abnormal TDTs, when compared to relatives with normal TDTs have: (1) larger putaminal volumes by voxel-based morphometry , (2) redPutaminal enlargement is found in musician’s dystonia patients; the degree of putaminal enlargement correlates with the degree of keystroke irregularity (as a marker of severity of MD) . PutaminDiffusion tensor imaging (DTI) is a robust method used to analyze structural and functional connectivity changes and characterize microstructural white matter changes in multiple regions of the brain in dystonia patients. Unfortunately, no studies have examined functional connectivity in relation to temporal discrimination in adult-onset focal dystonia. DTI research in patients and unaffected relatives with and without abnormal temporal discrimination is warranted.The observations that abnormal TDTs in dystonic patients do not correlate with disease severity  are indeDystonia has been considered a network disorder in which different neurophysiological mechanisms have been consistently reported including reduced inhibitory activity, altered sensorimotor integration, and abnormally increased plasticity mechanisms. Abnormal temporal discrimination in AOIFD possibly reflects abnormal inhibitory interneuronal activity at different levels of the CNS, including subcortical structures  and S1 cortex , 43, 68.Since cortical plasticity mechanisms rely on a dynamic balance between excitatory and inhibitory interneurons , it is cBy using TDT testing in patients and their unaffected relatives, we can, by hypothesis-based research protocols, answer a number of fundamental questions on the pathogenesis of adult-onset focal dystonia Table .The profoundly low penetrance of adult-onset dystonia (10–15%) indicates that there are a number of non-genetic factors, which affect both disease development and its expression as a particular phenotype. Overuse, as in musician’s dystonia and writer’s cramp, is well recognized . HoweverNeurophysiological or imaging studies comparing unaffected relatives with normal temporal discrimination and unaffected relatives with abnormal temporal discrimination are powerful tools to assess pathomechanisms in dystonia. They have the advantage that secondary changes in the brain due to disease expression (secondary endophenotypes), such as remodeling in the cortex, do not complicate matters.Although adult-onset dystonia presents as a motor disorder, non-motor symptoms (comprising the “non-motor syndrome”) include sensory, neuropsychiatric, and sleep disorders ; these aTemporal discrimination in unaffected relatives of cervical dystonia patients and sex ratios in adult-onset dystonia phenotypes show similar patterns of age-related sexual dimorphism. AOIFD with onset below the age of 40 years is male predominant . After 40–45 years of age and, importantly, with increasing age there is a steady linear increase in the proportion of women affected in the craniocervical phenotypes. The male:female sex ratio (proportion of men) in AOIFD decreases with increasing mean age at onset; this association is highly significant; mean age of onset accounts for almost 60% of the variance in the proportion of men . This agConsistent evidence shows that TDT testing in dystonia has shed light into the pathophysiological mechanisms of dystonia. Future studies using the TDT in carefully constructed clinical research protocols may address important questions in relation to both disease penetrance and disease expression in adult-onset dystonia.Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: AC, MH, EM, SN, RB, OK, SO, and RR. Drafting the work or revising it critically for important intellectual content: AC, EM, SN, RB, OK, SO, RR, and MH. Final approval of the version to be published: AC, EM, SN, RB, OK, SO, RR, and MH. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: AC and MH.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.


Molecular fluorescence spectrometry offers several important analytical advantages, including (a) achievement of very low limits of detection for intensely fluorescent analytes, (b) applicability to determine individual constituents of multicomponent samples without prior separation, (c) applicability to remote detection of analytes via fiber optic and/or laser probes, and (d) the high information content of luminescence measurements .A principal shortcoming of fluorescence spectrometry is the fact that most organic and inorganic molecules exhibit low luminescence quantum yields and thus cannot be detected by fluorometry without first being converted to fluorescent derivatives. Accordingly, many procedures for derivatizing nonfluorescent analytes to form luminescent products have been devised.Our work takes a somewhat different approach to fluorometric detection of nonfluorescent molecules. We exploit the fact that many small molecular fragments are intensely luminescent, including such common species as NH, OH, CN, CH, PO, and SH, as well as many atoms. Fluorescent fragments can be produced from virtually any nonfluorescent molecule. For analytical purposes, the key is to perform the fragmentation in a sufficiently reproducible manner that the fluorescence intensity of a fragment species can be related to the concentration of parent molecule present in the initial sample.We presently perform molecular fragmentations in the gas phase, to avoid “cage effects” which often decrease the efficiencies of fragmentation processes in liquid or solid media to unacceptably low levels. From among the large number of possible techniques for molecular fragmentation, we have chosen laser photolysis (LP) and electron impact (EI) as the most promising methods.In LP experiments, gaseous samples are fragmented by either the beam from an excimer laser (193 nm) or tunable radiation from an excimer-pumped dye laser. In EI methods, molecules are fragmented by 100-eV electrons from a conventional heated-filament electron gun. A certain fraction of fragment species are produced in electronically excited states and emit directly. If needed, a “probe” laser can be used to excite luminescence from fragments formed in their ground electronic states by LP or EI. Vacuum systems comparable in design and performance to those found in conventional mass spectrometers are usually used, though LP fragmentations can also in principle be applied directly to remote atmospheric sensing. Conventional fluorescence measurement and signal-processing techniques are used.We will describe studies of the analytical characteristics  for LP and EI fragmentation-fluorometric detection of a variety of nonfluorescent organic and organometallic compounds. The possibility of using low-temperature techniques (matrix isolation or supersonic expansion) to increase the selectivity of such measurements will be described. The complementarity of these measurements to mass spectrometry will be emphasized .


Motivational deficit is a common feature of negative symptoms in patients with schizophrenia. Patients with schizophrenia are impaired in goal-directed behaviour and effort allocation decision-making to pursue a potential reward. On the other hand, limited work has suggested that schizophrenia patients who experienced long-term social deprivation showed more severe negative symptoms. However, it is not yet fully clear the long-lasting impact of long-term social deprivation on motivation in these patients. The current study aimed to investigate the effect of long-term social deprivation on effort allocation pattern in patients with schizophrenia.We recruited 21 patients with schizophrenia institutionalized for more than 15 years and 20 patients with schizophrenia dwelling in the community and 24 healthy controls for this study. We administered the Effort-Expenditure for Rewards Task (EEfRT) to capture reward-based motivational salience, which requires participants make decision to choose a hard or easy task based on reward probability and magnitude. Moreover, a set of self-reported checklists including the Chapman Psychosis Proneness Scales, the Temporal Experience of Pleasure Scale, the Anticipatory and Consummatory Interpersonal Pleasure Scale and the Emotional Expressivity Scale were also administered to all the participants. For patients with schizophrenia, they also received rating score on the Positive and Negative Syndrome Scale (PANSS) and the Scale for the Assessment of Negative Symptoms (SANS).Institutionalized patients had exhibited significantly more prominent negative symptoms, especially in alogia subscale, attention subscale, and a trend of statistical significance in anhedonia subscale of SANSS. The two clinical groups did not differ in positive symptoms subscale and general psychopathology symptoms subscale of PANSS. Findings from one-way ANOVA analysis showed that both institutionalized patients and community-dwelling patients with schizophrenia did not differ from healthy controls in experiential pleasure and emotion expression. For performance in the EEFRT, amotivation was only observed in institutionalized patients with schizophrenia, they were significantly less likely to expend effort to pursue a potential reward than healthy controls in both medium (50%) probability and high (80%) probability level. Hence, as the reward probability increased, unlike healthy controls, institutionalized patients could not increase their hard task choices.Institutionalized patients with schizophrenia exhibited significantly more motivational deficits than healthy controls, and such impairment was not observed in community-dwelling patients. However, both institutionalized patients and community-dwelling patients with schizophrenia showed no deficits in self-reported scales measuring pleasure experience and expression. These findings further revealed that long-term social deprivation may be a vital contributor to severe motivation deficits of patients with schizophrenia.


Computational models of congenital heart disease (CHD) have become increasingly sophisticated over the last 20 years. They can provide an insight into complex flow phenomena, allow for testing devices into patient-specific anatomies (pre-CHD or post-CHD repair) and generate predictive data. This has been applied to different CHD scenarios, including patients with single ventricle, tetralogy of Fallot, aortic coarctation and transposition of the great arteries. Patient-specific simulations have been shown to be informative for preprocedural planning in complex cases, allowing for virtual stent deployment. Novel techniques such as statistical shape modelling can further aid in the morphological assessment of CHD, risk stratification of patients and possible identification of new ‘shape biomarkers’. Cardiovascular statistical shape models can provide valuable insights into phenomena such as ventricular growth in tetralogy of Fallot, or morphological aortic arch differences in repaired coarctation. In a constant move towards more realistic simulations, models can also account for multiscale phenomena  and importantly include measures of uncertainty . While their potential to aid understanding of CHD, surgical/procedural decision-making and personalisation of treatments is undeniable, important elements are still lacking prior to clinical translation of computational models in the field of CHD, that is, large validation studies, cost-effectiveness evaluation and establishing possible improvements in patient outcomes. Twenty years ago, pioneering research in the field of modelling congenital heart disease (CHD) showed how numerical simulations based on finite elements method (FEM) and computational fluid dynamics (CFD) could provide insight into local haemodynamics in the total cavopulmonary connection of Fontan patients.predictive capabilities rather than on haemodynamic insight, that now can be gathered directly in patients.The uniqueness and complexity of CHD anatomical arrangements (prerepair and postrepair) warrants a patient-specific approach, which can be facilitated by using computational models. The detailed insight into flow and structural phenomena that models can provide can add to our knowledge of CHD. Recent advances in the imaging realm, for example, 4D cardiovascular magnetic resonance (CMR) imaging flow quantification producing exquisite blood flow visualisation,This review will present engineering tools that can have a relevant role in decision-making, surgical planning and overall pathophysiological appreciation of CHD.Computer modelling can advance personalised predictive medicine, whereby an individual's unique anatomy and physiology are used to define the model, predicting outcomes of different treatments and helping to identify optimal strategies.4As mentioned, Fontan surgery has been one of the first modelling applications to CHD. Early work investigated the influence of the Fontan connection on caval haemodynamics as a determinant factor for surgical success.Quantification of magnitudes such as blood flow velocity, wall shear stresses (WSSs) and pressure can be critical in planning the treatment of other CHDs where a change in geometry can lead to haemodynamic variations. CFD simulations have been applied to the study of aortic coarctation (CoA), revealing how long-term morbidity can be explained by altered biomechanical indices such as time-averaged WSSs and oscillatory shear index.13Minimally invasive interventional procedures can benefit from patient-specific predictions for preprocedural assessment, when the operator lacks direct access to the implantation site, choosing the type and size of device based mainly on imaging data. Structural FEM simulations can provide information on the feasibility of device implantation, and have supported minimally invasive procedures such as percutaneous pulmonary valve implantation (PPVI) or CoA stenting.prior to the actual procedure. Postprocedural data were then acquired to assess the success of the implantation and were in agreement with computational predictions. This case demonstrates how the patient-specific approach may increase safety of preprocedural planning. Similarly, a modelling paradigm was employed for optimal device selection for a complex PPVI case  and, more recently, the development of a roadmap to introduce in silico clinical trials (http://avicenna-isct.org). In 2013, the US Food and Drug Administration (FDA) advocated the use of such systems as an additional innovative research tool,in silico trials as these ‘may potentially protect public health, advance personalized treatment, and be executed quickly and for a fraction of the cost of a full scale live trial’.21Patient-specific computational modelling has attracted considerable funding around the world,Third party companies, not directly involved in the Device Industry/Regulatory Agency interaction, but providing services to both by developing computational tools to design cardiovascular devices, are advancing in this direction, for example, leading engineering software company Dassault Systemes Simulia . They recently invested in the Living Heart Project initiative to build a computational platform of the human heart including multiphysics capabilities, creating a ‘complete 3D view of electrical impulses and muscle-fiber contractions able to replicate the true motion of the human heart’.24shape and size of the heart and its components are crucial for early diagnosis and management of patients with CHD. While non-invasive imaging techniques provide detailed 3D anatomical data, analysis of shape and structure in clinical practice is still often carried out via simple morphometry on 2D image slices, neglecting the abundance of available 3D information. Advances in medical imaging have led to growing image databases and population-based studies of cardiac anatomy.Considering CHD as ‘a gross structural abnormality […] of functional significance’ at birth,mean shape (or ‘template’) and shape variations around this mean. The combination of mean shape and its variations constitutes the so-called shape atlas, which integrates all shape information for a population of interest.descriptive and predictive  shape between healthy and diabetic subjects, finding regional LV shape features such as significantly increased septal bulging; analysing ventricular shape in women with pre-eclampsia for risk assessment; and investigating a characteristic LV shape of preterm born subjects.36In CHD, analysis of right ventricular morphology in patients with tetralogy of Fallot established correlations between distinct ventricular shape features and tricuspid and transpulmonary regurgitation fractions.Predictive models are very appealing. A computer-aided diagnosis system based on healthy participants and patients with connective aortic tissue disorder used shape modes as classifier for distinguishing between subjects, showing promising classification correctness.37in silico trials, shape atlases could be used to develop devices that fit the majority or different classes of CHD populations.The manifold anatomical variety in CHD calls for novel methods to investigate how shape affects function and ultimately patient outcome, so both descriptive and predictive SSMs could be clinically useful in a quest for shape biomarkers. Shape outliers could be automatically detected and followed-up more closely. Clustering techniques could uncover previously unknown shape subgroups, and subsequent classification techniques could explore if any of those subgroups is at a higher risk of following a pathological pathway or growth model. Regression and correlation of distinct shape features with clinical or functional parameters could identify shape biomarkers for adverse cardiac events and even inspire novel surgical approaches for repairing specific morphologies. Growth models could assist in treatment planning. Finally, in relation to above-mentioned As for every statistical model, increasing numbers will strengthen the models, building up on existing small sample studies that often focus on novel algorithms and moving toward larger-scale studies driven by clinical questions. This needs close collaboration between clinical and engineering centres, multicentre studies and pooling data from various imaging modalities or existing models.Having considered structural, haemodynamic and morphological variables, it is important to remember that a strong interaction exists between mechanical phenomena and biological processes. Computational models can, for example, output measures of WSS, that can be interpreted in the light of thrombus formation (low WSS) or potential vessel rupture (high WSS). A relevant CHD example can be represented by a validated computational model of repaired transposition of the great arteries showing regional increased WSS compared with an age-matched healthy control subject.9While technically challenging and not required for all applications, inclusion of multiscale phenomena can considerably strengthen the power of numerical models, particularly growth models, which in turn could be very relevant for CHD evolution predictions. Some of these associations are known, not just at a histological level .Recent advances in technologies such as 3D echocardiography can also enrich models, for example, including detailed valve morphologies.Another important development is the inclusion of uncertainty analysis and optimisation algorithms in the modelling process. An application of this uncertainty quantification in virtual surgery for patients with single ventricle showed statistical variability in the predictions, importantly allowing including a CI in the simulation results.Biomechanical modelling is a powerful tool, not just for CHD, and also in other areas of cardiovascular medicine, for example, simulating stent behaviour and stent implantation in coronary arteries in adult patients.


A meta-analysis of 295 patients found higher odds of achieving clinical remission and remaining in clinical remission among patients on combination therapy with specialised enteral nutrition and Infliximab (IFX) compared with IFX monotherapy . In conclusion, evidence-based knowledge on impact of diet on anti-TNF treatment response for clinical use is scarce. Here we propose a mechanism by which Western style diet high in meat and low in fibre may promote colonic inflammation and potentially impact treatment response to anti-TNF drugs. Further studies using hypothesis-driven and data-driven strategies in prospective observational, animal and interventional studies are warranted.We wanted to investigate the current knowledge on the impact of diet on anti-TNF response in inflammatory bowel diseases (IBD), to identify dietary factors that warrant further investigations in relation to anti-TNF treatment response, and, finally, to discuss potential strategies for such investigations. PubMed was searched using specified search terms. One small prospective study on diet and anti-TNF treatment in 56 patients with CD found similar remission rates after 56 weeks among 32 patients with good compliance that received concomitant enteral nutrition and 24 with poor compliance that had no dietary restrictions (78% versus 67%,  Inflammatory bowel diseases (IBD), including Crohn’s Disease (CD) and ulcerative colitis (UC), are chronic lifelong illnesses of early onset that substantially affect the life quality of the patients and their families ,2,3. Up The management of IBD has changed significantly during the last two decades ,7. The iMany patients ask their health care professionals about recommendations for dietary intake, and for now we are not able to provide evidence-based answers. Dietary recommendations that can help to improve the outcome of anti-TNF drugs are thus highly warranted by patients. Furthermore, knowledge on diet that supports good treatment response is important for the health care system in order to improve the utilization of the health care resources, as the resources spent for the care of IBD patients are expected to increase exponentially in the coming years .Given the important role of environment on the development of IBD, we hypothesised that lifestyle may impact the effect of anti-TNF drugs. We therefore set out to investigate the current knowledge on the impact of diet on anti-TNF response. Furthermore, we wanted to identify dietary factors that warrant further investigations in relation to anti-TNF treatment response, based on knowledge of their effects on gut inflammation. Finally, we discuss potential strategies for such investigations. For review of diet and risk of IBD please refer to recent papers by Lee et al. and Andersen et al. ,15. PubMed was searched using the search terms shown in n-3 and n-6 polyunsaturated fatty acids (PUFA), and protein, particularly animal protein, in IBD development ..61].Ruminococcus gnavus, Ruminococcus torques, Akkermansia muciniphila, and bacteria in the Bifidobacterium genus) are defined as mucus-degrading bacteria that are able to grow on mucin as its sole carbon substrate [Ruminococcus gnavus and Ruminococcus torques and a reduced abundance of Akkermansia muciniphila compared to controls have been reported [Bilophila wadsworthia) for microbially produced hydrogen sulphide [Mucolytic bacteria  [High intake of food containing organic sulphur and sulphate additives, such as meat, processed meat, milk, and wine, may increase the amount of sulphate for microbially produced hydrogen sulphide ,73. In fworthia) ,74 have The resultant hydrogen sulphide from either low intake of fibre, high intake of meat and other sulphur, or both may reduce the disulphide bonds in the mucus network, rendering the mucus layer penetrable to bacteria ,75. ThenAdditionally, diet may affect the systemic immune response. Intake of low-glycemic index diet was found to lower secretion of TNF-α and IL-6 from stimulated peripheral blood mononuclear cells from obese humans .p = 0.51). A meta-analysis of 295 patients found higher odds of achieving clinical remission and remaining in clinical remission among patients on combination therapy with specialised enteral nutrition and Infliximab (IFX) compared with IFX monotherapy . The animal studies provide understanding related to effects of high animal fat and fish oil diets on rates of colitis and cytokine expression. However, caution has to be taken when translating results from therapeutic studies in animals to humans with IBD. Here we hypothesised that Western style diet, high in meat  and low in fibre, may seriously impact treatment response in IBD. Such a diet could affect gut microbiome metabolism in a way that promotes gut inflammation and negatively affects medical treatment. There are some previous findings that support such processes in IBD, in particular UC. In fact, high sulphide levels have been found in UC patients [Diet may impact the treatment response to anti-TNF drugs. We found that evidence-based knowledge on impact of diet on anti-TNF treatment response for clinical use is scarce. One small prospective study on diet and anti-TNF treatment in 56 patients with CD found similar remission rates after 56 weeks among 32 patients with good compliance that received concomitant enteral nutrition and 24 with poor compliance that had no dietary restrictions  is taking place. In colon, metabolites resulting from microbial degradation of the luminal content may drive the inflammation. Similarly, different mechanisms driving inflammation in CD versus UC may add to the complexity. In accordance, a recent study of genotype-phenotype associations in IBD found that disease location is an intrinsic aspect of disease. The authors demonstrated that IBD is better characterised as three groups  instead of CD and UC .It has been suggested that plant fibre may be important for restoring the mucus layer ,79. SuppDiet and other lifestyle factors may potentially impact the treatment response to anti-TNF drugs. However, evidence-based knowledge for clinical use is generally scarce, although vitamin D supplementation to vitamin D-deficient IBD patients seems rational. Our results suggest complex interactions between diet, microbiome, and genetics in defining immune response and treatment response in IBD. Here we propose a mechanism by which a Western style diet high in meat and low in fibre may promote colonic inflammation and potentially affect treatment response to anti-TNF drugs. Further studies of the biological mechanisms of various food items, such as red meat and fibre rich foods, using hypothesis-driven and data-driven explorative strategies in observational studies, animal studies, and interventional studies, may help in understanding how diet may affect treatment results.


It has always been an ambitious goal in medicine to repair or replace morbid tissues for regaining the organ functionality. This challenge has recently gained momentum through considerable progress in understanding the biological concept of the regenerative potential of stem cells. Routine therapeutic procedures are about to shift towards the use of biological and molecular armamentarium. The potential use of embryonic stem cells and invention of induced pluripotent stem cells raised hope for clinical regenerative purposes; however, the use of these interventions for regenerative therapy showed its dark side, as many health concerns and ethical issues arose in terms of using these cells in clinical applications. In this regard, adult stem cells climbed up to the top list of regenerative tools and mesenchymal stem cells (MSC) showed promise for regenerative cell therapy with a rather limited level of risk. MSC have been successfully isolated from various human tissues and they have been shown to offer the possibility to establish novel therapeutic interventions for a variety of hard-to-noncurable diseases. There have been many elegant studies investigating the impact of MSC in regenerative medicine. This review provides compact information on the role of stem cells, in particular, MSC in regeneration. Being first isolated in 1966 from bone marrow, mesenchymal stem cells (MSC) are adult stromal nonhematopoietic cells, well known for their potential to differentiate into osteoblasts and osteocytes , 3. ThesBeside their potential to differentiate into osteoblasts in the process of osteogenesis, there have been several other regenerative roles attributed to MSC. These cells can serve as pericytes , 22 wrapThese findings were enough evidences for scientists to speculate a promising role for MSC in regenerative therapy. In the past years, MSC have been used in clinical trials aiming for regeneration of tissues such as bone  and cartSeveral clinical trials are running to identify different aspects of MSC application in terms of safety and efficacy. In the recent decade, somatic stem cells have become attractive tools for cell therapy and regenerative medicine due to their proliferation and differentiation potential as well as established isolation and propagation protocols that promote a high standard of purity and functionality of the cells when applied in vivo. Adult stem cells (ASC) and progenitors, in particular mesenchymal stem cells have been derived from a variety of tissues such as umbilical cord and umbilical cord blood, placenta, bone marrow, epithelium, and white adipose tissue. These cells have been characterized, expanded, and applied for transplantation procedures in which allogeneic adult stem cells give rise to committed cells such as osteocytes, adipocytes, and chondrocytes as well as functional vessels in the process of neovasculogenesis –49.The proliferation rate of adult stem cells and in particular MSC is a crucial parameter for stem cell therapeutic interventions like patient-specific tissue regeneration. There are, however, limitations with regard to the amount of tissue that can be taken from the patient, the limited propagation capacity of the cells that are isolated from the tissue, and restrictions in terms of passage number of the cells to be utilized for regenerative therapy. Therefore, it has been of great interest to enhance the proliferation rate of  stem cells, especially in terms of patient-specific regenerative therapy. In this regard, low-level laser therapy (LLLT) has been tested in vitro to stimulate and enhance proliferation capacity of the cells. According to systematic review conducted by Ginani et al., LLLT is increasingly used as a method to enhance proliferative potential of adipose tissue-, dental pulp-, periodontal tissues-, and bone marrow-derived stem cells to date . BalliniConsidering the tissue-specificity property of stem cells in determining their regenerative potential, it is of interest to test and compare the impact of LLLT on proliferative potential of stem cells that are derived from different tissues to ensure a more effective regenerative strategy approach.MSC are, however, not considered as the only cellular mediators for enhancement of regenerative therapy, as embryonic stem cells (ESC) and, later on, induced pluripotent stem cell (iPSC) technology through cellular reprogramming were introduced and aimed to push regenerative cell therapy beyond its existing limits.Pluripotent, inner blastocyst cell mass-derived cells are defined as embryonic stem cells (ESC) that can proliferate without limitation, possess the potential of self-renewal, and are able to differentiate into different cell types derived from all the three germ layers . These cDifferentiating hESC to numerous cell types including osteoblasts, cardiomyocytes (CM), hepatocytes, neurons, and endothelial cells (EC) to be used in cell replacement therapy (CRT) has been increasingly taken into consideration . HoweverDuring the past few years, several studies were carried out to investigate differentiation of ESC into dopamine-producing neural cells , 59 and ESC research offers great promise for understanding mechanisms of cell differentiation which ultimately leads to discovery of novel treatments for diseases such as myocardial infarction , 62. PluThe therapeutic potential and benefit of ESC, however, have been a matter of debate and raised ethical concerns due to the opinion that the process of deriving embryonic stem cells results in severe damage to the embryo. Moreover, the existing complications and some as-of-yet unclarities in differentiation potential and proliferation rate of ESC pose risk of undesired complications such as teratoma formation and cancer development. Therefore it is not an approved procedure in several countries. Although research has overcome many of these limitations to date, ESC are still not fully approved for being used in cell therapy procedures and regenerative application .Other groups of potential playmakers in regenerative therapy, induced pluripotent stem cells (iPSC), have come to the scene by Takahashi and Yamanaka who successfully produced induced pluripotent stem cells (iPSC) using mouse embryonic and adult fibroblast cells and introducing four transcription factors SOX2, OCT 3/4, KIF4, and c-myc to cells . Later, + cells and reducing proinflammatory cytokines in a lymphocyte population admix [Several studies on the implication and capacity of iPSC technology for therapeutic approaches have been carried out through which iPSC were generated from committed and somatic cells , 70. TheKang et al. also established a method for generating iPSC-MSC with morphological characteristics and surface marker expression profile similar to that of BM-MSC . The iPSThe iPSC technology has undoubtedly raised hope in regenerative biology; however its use in regenerative medicine did not appear as a facile, straight-forward procedure, as iPSC technology led to complications in cell therapy and regeneration . The genIn comparison with ESC and iPSC, adult somatic stem cells do not cause ethical and severe health issues and are therefore widely used in regenerative research. There have been, however, limitations concerning the in vitro expansion and pluripotency when using adult somatic stem cells for therapy . NeverthFurther investigations are still required for application of ESC and iPSC in regenerative medicine until these cells would be considered as effective tools for clinical regenerative therapy. For this reason, other options such as new sources of ASC, in particular, MSC as an important adult stem cell subfamily, have been considered for establishment of successful and progressive cell-based regenerative and therapeutic procedures. The need for novel cell sources is obvious because of increasing need of regenerative cell therapy for diseases that are, as of date, difficult, if not impossible to be cured.MSC are present in several adult tissues. Despite similar morphology and phenotypic properties amongst MSC that have been isolated from various tissue sources, their regenerative potential has been shown to differ. It has been previously described that activated aging mechanism in MSC has an impact on their regenerative potential, probably due to DNA damage accumulation  and/or iBone marrow (BM) was one of the first tissues that had been used for isolation and propagation of mesenchymal stem and progenitors. Bone marrow aspirate is rich in hematopoietic and nonhematopoietic stem cells, endothelial progenitor cells (EPC) derived from embryonic hemangioblasts, and mesenchymal stem cells (MSC). MSC have been shown to participate in hematopoiesis or bone marrow regeneration , 84. TheAdult adipose tissue is rich in fibroblast-like cells with multidifferentiation potential –92. In 2Several studies revealed that cells isolated from Wharton's Jelly (WJ), a component of umbilical cord extracellular matrix, express stemness characteristic and multipotency , 103. ThThe potential of UC-MSC to participate in neovasculogenesis , 109 andThere have been, however, contradictory reports in terms of surface markers that are expressed on UC-MSC surface . AccordiUC-MSC have been shown to maintain a high differentiation potential in vitro as these cells have shown the ability to differentiate into adipocytes, chondrocytes, osteoblasts, muscle cells, cardiomyocytes, beta cells, endothelial cells, neurons and dopaminergic neurons, and so forth , 118–121It has been shown that regenerative potential of UC-MSC can differ if the cells are obtained from an individual with metabolic disorders such as type 1 diabetes. Kim et al. indicated that UC-MSC derived from diabetic pregnant women show lower potential of osteogenic and adipogenic differentiation, whereas their surface marker expression profile is not significantly affected. The cell population doubling has also been shown to diminish in UC-MSC from diabetic mothers when compared to UC-MSC from healthy individuals . This fiUmbilical cord blood (UCB) has always been considered as a source of hematopoietic stem cells (HSC) , 123. NoDental tissues are specialized tissues and they do not undergo continuous remodeling as has been indicated in other bony tissues; therefore, stem cells that are obtained from dental tissue might show a restricted differentiation capacity compared to BM-MSC , 128.Dental pulp stem cells (DPSC) are amongst different human dental stem and progenitor cells that have been isolated and characterized to date . DPSC poαVβ3 during differentiation of these stem cells into osteoblasts [The osteogenic differentiation potential of the cells isolated from dental follicle (DF) has been investigated by Mori et al. . This steoblasts . The dateoblasts .The studies that have been carried out on dental stem cells and their regenerative potential have raised promise for using dental tissue-derived MSC in fracture healing as well as regenerative bone formation interventions due to disease or loss of the tissue .The amniotic membrane is a part of the placenta that protects the fetus during pregnancy and provides nutrient transport to fetus . The amnAmniotic membrane-derived mesenchymal stem cells (AMN-MSC) have been shown to have the potential to differentiate into all three mesodermal lineage cells as well as endodermal lineage cells . They haThe regenerative potential of MSC isolated from different tissues has been shown to undergo alteration according to the tissue of isolation , 149. ItAMN-MSC were also shown to successfully participate in neurogenesis, whereas such a regenerative potential has not been distinguished in UC-MSC , 153. AmDespite the fact that DPSC and BM-MSC are regulated by similar factors and they also possess a similar protein expression profile, these populations have been shown to alter significantly in their proliferative capacity in vitro and, more importantly, in their regenerative capacity in vivo . BM-MSC BM-, dental pulp- (DP-), and adipose tissue- (AT-) derived MSC have revealed a greater promise in regenerative therapy since these adult stem cells might promote patient-specific regenerative interventions.MSC are attractive alternatives for regeneration of the injured and/or deficient cells and tissues due to their multipotent differentiation capacity as well as their immunomodulatory and anti-inflammatory properties through cellular crosstalk and production of bioactive molecules . MSC havThe combination of multipotent endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) is an additional key tool for stem cell therapy. These cells are localized in bone marrow stroma as well as vascular inner and outer layer and perivascular niches and are capable of forming mature endothelial cells and mesenchymal cell lineages such as osteoblasts, chondrocytes, adipocytes, and myoblasts , 83, 160Mitogen activated protein kinases (MAP kinases) have been also shown to regulate MSC differentiation to osteo- or adipogenic lineage  with a sBone and cartilage injuries occur as a result of bone fracture, or joint diseases such as rheumatoid arthritis or osteoarthritis. These disorders have a costly economic and social impact on the quality of life amongst middle-aged patients. Despite the progress in orthopedic surgery, bone and cartilage repair have remained a major challenge because large injuries do not heal spontaneously –169. TheStudies on mesenchymal stem cells have opened a new horizon for bone and cartilage tissue engineering. Because of their multipotent capacity, MSC lineages have been successfully used in animal models to repair articular cartilage and regenerate bone , 173. ReThe potential of MSC and progenitor cells in prospective cell-based regenerative models has been investigated by Lohberger et al. . The stuHuman mesenchymal stromal cells isolated from bone marrow (BM) and alveolar bone have been compared according to their regenerative potential by Pekovits et al. . The stuComplete healing occurs when the regenerated tissue has been integrated into the neighboring host tissue and the differentiation process has been thoroughly performed , 179. HoAs indicated earlier, MSC can differentiate into different mesodermal cells and they also possess transdifferentiation ability to preserve phenotypes of neural ectodermal and epithelial cells . It has During development, surface ectoderm gives rise to the corneal epithelium . It has In a preclinical study using rabbits, BrdU labelled BM-MSC were seeded on fibrin scaffolds and were transplanted into the alkali damaged cornea. The BrdU positive cells were shown to participate in the process of cornea healing which clearly indicated the ability of BM-MSC to differentiate into corneal epithelial cells .The result of several in vitro experiments supported the idea that MSC are able to resemble cornea epithelial cell phenotype under certain conditions; however, up till now, the in vivo data has not shown supportive evidence that justifies the in vitro results.Recently, adipose tissue-derived MSC (AT-MSC) have shown the ability to differentiate into the corneal epithelium . AlthougA number of studies have revealed the potential of umbilical cord mesenchymal stem cells (UC-MSC) and bone marrow mesenchymal stem cells (BM-MSC) to differentiate into corneal endothelial cells , 192. HoImmune-mediated disorders like type 1 diabetes (T1D) severely affect quality of life in several millions of patients all over the world. T1D leads to a shorter life span of the patient, has various side effects including cardiovascular and ophthalmic disorders and neuropathy. The disease puts economic pressure both on the health system and the patient. Therefore, great effort has been made to develop innovative therapeutic strategies for cell-based therapy through stem cell immune modulation, autologous/allogeneic stem cell transplantation, and small molecule mediated beta cell regeneration for treatment of T1D.The use of MSC in cell-based therapy in T1D has been investigated by a number of scientific groups all over the world –203.The potential of bone marrow-derived MSC in immunomodulation of immune-mediated disease T1D and cell-based regenerative models has been investigated by Fiorina et al., 2009 . In thisHuman mesenchymal stromal cells isolated from BM and peripheral blood (PB) have been tested in a humanized mouse model by Lee et al., 2006 . In thisOther studies have also focused on MSC derived from adipose tissue (AT)  and placInvestigations on the mesenchymal stromal cells have opened a new horizon for diabetes research. Because of their multipotent capacity, MSC lineages have been used successfully in animal models to suppress immune reactions that cause beta cell death and the onset of T1D . Recent Stem cells derived from a variety of sources are promising tools for regenerative cell therapy. Although stem cell therapy has opened a new horizon in regenerative medicine, there are still several obstacles that need to be overcome before this novel treatment tool can be used in large scale in clinics. However, it is obvious that regenerative stem cell therapy has been transformed from scientific fiction to a feasible medical procedure. Regenerative stem cell therapy has created a lot of hope amongst scientists and physicians for finding more effective treatment strategies; nevertheless, it is essential for this new spectrum to develop further through high quality investigations and an effective contribution of researchers and physicians to perform advanced clinical trials aiming to facilitate MSC application for clinical therapy.


Users can upload their own reference databases to tailor the metagenomics annotation to focus on various taxonomic and functional gene markers of interest. MetaStorm offers two major analysis pipelines: an assembly-based annotation pipeline and the standard read annotation pipeline used by existing web servers. These pipelines can be selected individually or together. Overall, MetaStorm provides enhanced interactive visualization to allow researchers to explore and manipulate taxonomy and functional annotation at various levels of resolution.Metagenomics is a trending research area, calling for the need to analyze large quantities of data generated from next generation DNA sequencing technologies. The need to store, retrieve, analyze, share, and visualize such data challenges current online computational systems. Interpretation and annotation of specific information is especially a challenge for metagenomic data sets derived from environmental samples, because current annotation systems only offer broad classification of microbial diversity and function. Moreover, existing resources are not configured to readily address common questions relevant to environmental systems. Here we developed a new online user-friendly metagenomic analysis server called MetaStorm ( The field of metagenomics has arisen following the advent of next-generation DNA sequencing. Through new technologies, such as Illumina and pyrosequencing, it is now possible to directly shot-gun sequence DNA extracted from various environmental samples, without the need for cloning. Metagenomics is particularly promising for advancing the understanding of the structure and function of microbial communities residing in natural, human, and engineered environments. To date, metagenomic data sets have been obtained from different regions of the human body , 2, 3, sVarious computational resources have been developed for taxonomic and functional annotation of metagenomics data sets. These resources can be classified into two main categories: 1) Web services organized as a collection of different computational resources that facilitate the storage, analysis, and retrieval of metagenomic data . Users are first required to login into the MetaStorm website, select (or create) the project they wish to analyze, and select the desired method (Assembly/Read matching). Once in the project profile page, users need to insert sample information , select reference databases, upload raw FASTQ files, and finally run the annotation pipeline. To simplify the process of data submission, MetaStorm does not require external files such as Excel spreadsheets for sample description and provision of metadata . This interactive tool also allows users to remove samples and projects or re-run the samples with different pipelines, visualizing the results as needed.Submission of metagenomic data is made by an interactive web interface . Selecting the appropriate pipeline depends of several parameters including: the design of the experiment, the previous knowledge about the experiment, the research hypothesis and goals. For instance, if the objective is to characterize the most abundant taxonomy in the community, the assembly pipeline may suffice [Once stored in the MetaStorm server, raw reads are queued for taxonomic and functional annotations. MetaStorm incorporates two pipelines, the assembly-based pipeline and the read-matching pipeline : reads are trimmed and filtered out by TRIMMOMATIC [MMOMATIC  to removAssembly: IDBA-UD [ IDBA-UD  is a wid IDBA-UD , 38, 39.Gene prediction: Once a set of scaffolds are assembled, PRODIGAL [PRODIGAL  . CurrenFunctional annotation: Predicted genes (translated proteins from PRODIGAL) are matched to the user selected reference databases using the DIAMOND BLASTP aligner [ aligner . We use  aligner , and min aligner , whereas aligner .marker gene analysis [The read matching pipeline conducts taxonomic and functional annotation of metagenomic data comparing the raw sequence reads to a reference database. This approach is also called analysis . For taxanalysis  and thenanalysis , a fast analysis  using Boanalysis  that useanalysis  which usanalysis  aligner analysis  , all descendants from that node will be displayed as a pie chart. The overall abundance of a taxonomy level can also be displayed as a pie chart. Node colors represent relative abundance. All visualization formats are available for the taxonomic annotation methods.MetaStorm offers interactive visualization, allowing users to see in detail the main features of the sequence make-up of each sample. A taxonomic tree encodes relative abundance information of different lineages in the sample. For example, in Fig 4A) that relate functional categories with the genes involved in each category. Users can select the reference database to analyze and all the tables in text format can be downloaded. When analyzing individual samples, read/gene counts are normalized using a linear scale between 0 to 100.Functional relative abundance is described by a set of interactive pie charts and bar plots  are not available in other visualization tools, such as MG-RAST or EBI-Metagenomics.Visualization techniques employed by MetaStorm include: heat maps, stacked bars, and interactive trees (taxonomy annotation). As for single sample visualization, the response tree shows relative abundance for each node (taxa) and also for each taxonomic hierarchical level, allowing a high level of specificity. This type of interactive visualization features (1) the focus of knowledge based on user runs and 2) the projected run time for reporting MetaStorm results. Expectedly, small customized databases will report results faster than full reference databases. A novice user can use this database for analysis and jump to the specific biological problem, thus saving the computing time. Moreover, the search tool enables users to find similar existing metagenome samples in MetaStorm (public ones) and include them for more comprehensive comparison studies. Comparison across different samples is made feasible by the normalization criteria implemented in MetaStorm. Finally, all the raw and generated files for the metagenomic analysis can be downloaded in a variety of formats by clicking on the download button of each section in the visualization page.Similar to MG-RAST and EBI-Metagenomics, all the information on a project tagged public, such as raw read files, processed files, description files, and visualization tables, are freely available through MetaStorm. From the home page, the user can access descriptions of all the recently listed (public) projects and the reference databases that other users submitted. A search tool is available for users to identify potential sets of reference sequences that can match their analysis. MetaStorm’s reference sharing capability aims to support section customizable reference database). Those databases have been used as default by several current metagenomics resources. While the assembly pipeline implemented by MetaStorm is similar to that of the MetaHIT pipeline [Compared to other metagenomic resources, such as MG-RAST and EBI-metagenomics, MetaStorm extends the analysis and visualization of metagenomic samples by: 1) adding a fully developed assembly-based annotation pipeline, in addition to the read matching pipeline deployed by these Web servers; 2) offering a customized analysis where the user can select and upload reference databases, which enables focus on specific genes of interest as well as inter-project comparison; and 3) interactive visualization capabilities, including an interactive taxonomic tree, which permit users to interrogate and compare specific aspects of the sequence data. MetaStorm includes a wide variety of databases used for metagenomics analysis  and the assembly pipeline. MetaStorm, unlike any other web resources, incorporates user reference customization, which will help to streamline the annotation process when a research hypothesis requires specific and customized databases.


Endochondral ossification (EO), by which long bones of the axial skeleton form, is a tightly regulated process involving chondrocyte maturation with successive stages of proliferation, maturation, and hypertrophy, accompanied by cartilage matrix synthesis, calcification, and angiogenesis, followed by osteoblast-mediated ossification. This developmental sequence reappears during fracture repair and in osteoarthritic etiopathology. These similarities suggest that EO, and the cells involved, are of great clinical importance for bone regeneration as it could provide novel targeted approaches to increase specific signaling to promote fracture healing, and if regulated appropriately in the treatment of osteoarthritis. The long-held accepted dogma states that hypertrophic chondrocytes are terminally differentiated and will eventually undergo apoptosis. In this mini review, we will explore recent evidence from experiments that revisit the idea that hypertrophic chondrocytes have pluripotent capacity and may instead transdifferentiate into a specific sub-population of osteoblast cells. There are multiple lines of evidence, including our own, showing that local, selective alterations in cartilage extracellular matrix (ECM) remodeling also indelibly alter bone quality. This would be consistent with the hypothesis that osteoblast behavior in long bones is regulated by a combination of their lineage origins and the epigenetic effects of chondrocyte-derived ECM which they encounter during their recruitment. Further exploration of these processes could help to unlock potential novel targets for bone repair and regeneration and in the treatment of osteoarthritis. The mobility provided by a robust locomotor skeletal system is a major determinant of human health and quality of life. Loss of mobility is a leading cause of ill-health and ultimately death. Two common skeletal problems include defective bone healing and osteoarthritis (OA); despite increasing incidence their treatment remains unmodified for many years  they secrete. The continuous transition of cartilage to bone in this endochondral ossification (EO) process is the source of all longitudinal bone growth. A distinct osteoblast cell type is responsible for shaping and maintaining bone formation upon this cartilage ECM template. Of course, some bones arise intramembranously, wherein embryonic mesenchymal cell condensations differentiate directly into osteoprogenitor cells and later into osteoblasts (OB), which secrete an unmineralized osteoid bone matrix around blood vessels. The compact layer of mesenchymal cells surrounding the skeletal element becomes the periosteum; OB on its inner surface deposit osteoid to form layers of bone Hall, .Endochondral ossification is initiated by migration of mesenchymal cells to form pre-cartilage condensations, which then undergo differentiation into chondrocytes that secrete ECM rich in aggrecan (Acan) and collagen type II. Calcification of this cartilaginous ECM in this primary ossification center is rapidly followed by its partial replacement and use as a template upon which bone is deposited by OB. This process involves the “propulsion” of the growth plate toward the epiphysis by a burst of proliferation, column formation followed by differentiation into pre-, early- and late, “terminally” differentiated, hypertrophic chondrocytes, associated with an increase in cell volume and deposition of their transient, calcified cartilage matrix  promoter/enhancer, and their descendants, contributed to osteoblast progenitors before Runx2 expression; using inducer Col2-creER, Sox9-creER, and Acan-creER approaches these authors reported that early postnatal cells progressively contribute to multiple mesenchymal lineages  reveals (i) transient long bone shortening that is restored before adulthood; (ii) diminished bone mass with defective architecture and lower fracture resistance; (iii) restriction of TIMP3 Tg impact to only endochondral bones; and (iv) deficiencies in osteoblast proliferation and differentiation that persists in vitro. Our data show that bone formation relies partly upon the cartilage ECM encountered during osteoblast recruitment (Javaheri et al., To test this hypothesis, we created mice with severely modified cartilage ECM remodeling by targeting TIMP3, a member of the metzincin family of MMP inhibitors that inhibits a wide spectrum of ECM-modifying enzymes. Our work in TIMP3-deficient mice and mice overexpressing TIMP3 specifically in chondrocytes, in vivo by the ECM they encounter. Since growth plate calcified cartilage serves as a template not only for bone formation but also osteoclast-mediated resorption, this implies that osteoclast behavior may also be modified in TIMP3 Tg mice to subsequently influence bone formation. However, our pilot studies suggest this is unlikely as we find no significant changes in osteoclast numbers in TIMP3 Tg mouse bone (Javaheri et al., in vitro isolation, supporting the likelihood that “imprinted” epigenetic modifications are contingent upon the ECM the OB encounter (Poulet et al., While our findings confirm the existence of a cellular chondro-osseous continuum, they also crucially pinpoint how behavior of cells with divergent origins is controlled Recent evidence challenges a long-held paradigm regarding EO, indicating that chondrogenesis and osteogenesis are more closely linked than appreciated and, that cells derived originally from the cartilage niche, as opposed to solely osteoprogenitors, transform into bone-forming OB and ultimately osteocytes. We offer a fundamental extension to this paradigm proposing that these OB are epigenetically influenced by ECM encountered during this transition. We need to ascertain how hypertrophic chondrocyte transdifferentiation fate is controlled and which specific ECM factors regulate this transition to impact bone quality and integrity. This new concept challenges us to re-evaluate cartilage and bone stages in EO, such that we treat them as a continuum rather than entirely independent. It also prompts us to revisit EO as an “evolutionary” shift in which cartilage might provide new cells that can instead form bone rather than simply as a means of lengthening skeletal elements. As impaired bone healing and OA both involve EO it is conceivable that tissue engineering approaches might exploit these developmental principles, embracing the newly established cellular chondro-osseous continuum to better control cell fate decisions.Conceived and designed the idea: BJ, GG, and AP. Wrote the mini review: BJ, SC-S, IK, GG, and AP. Revision and finalizing the mini review: BJ, GG, and AP.The authors declare that all financial, commercial or other relationships that might be perceived by the academic community as representing a potential conflict of interest are disclosed.


The specific role of serotonin and its interplay with dopamine (DA) in adaptive, reward guided behavior as well as drug dependance, still remains elusive. Recently, novel methods allowed cell type specific anatomical, functional and interventional analyses of serotonergic and dopaminergic circuits, promising significant advancement in understanding their functional roles. Furthermore, it is increasingly recognized that co-release of neurotransmitters is functionally relevant, understanding of which is required in order to interpret results of pharmacological studies and their relationship to neural recordings. Here, we review recent animal studies employing such techniques with the aim to connect their results to effects observed in human pharmacological studies and subjective effects of drugs. It appears that the additive effect of serotonin and DA conveys significant reward related information and is subjectively highly euphorizing. Neither DA nor serotonin alone have such an effect. This coincides with optogenetically targeted recordings in mice, where the dopaminergic system codes reward prediction errors (PE), and the serotonergic system mainly unsigned PE. Overall, this pattern of results indicates that joint activity between both systems carries essential reward information and invites parallel investigation of both neurotransmitter systems. Among the brain’s neuromodulators, serotonin  is likely the most ambivalent one with regard to its supposed importance for behavior and level of understanding. Serotonergic drugs are widely used in psychiatric disorders, abused as recreational drugs and liabilities in the serotonergic system have been identified as important etiological factors in many prevalent disorders. On the other hand, 5-HT is commonly described as “mysterious” in scientific contexts  and punishments (noise) in marmosets, Rygula et al.  found thThe DRN has since long been identified as one of the primary brain areas that promote self stimulation that strongly reinforces behavior via the dopaminergic system  DRN 5-HT neurons displayed strong reward- but not aversion-related activity modulations in freely behaving mice , which has been ascribed as a central function of the DA system. Recently, it was found that different subpopulations of dopaminergic neurons with specific projection regions respond differentially to reward, novelty and aversiveness. Projection neurons to the ventral striatum reflect reward, but appear insensitive to aversive events and novelty , and their most commonly abused variants such as cocaine, amphetamine and Methylenedioxymethamphetamine (MDMA or ecstasy) are, after cannabis, the most widely abused illegal drugsAnother way to assess behavioral control over reward behavior in laboratory settings is to combine intracranial self stimulation (ICSS) of rewarding areas (often the medium forebrain bundle) with administration of drugs. DA releasing agents have been shown in this setting to increase self stimulation, which is interpreted as a context dependent facilitation of addictive behavior. Interestingly, selective 5-HT releasers inhibit ICSS (Olds and Yuwiler, Human studies that combine pharmacological manipulations of the 5-HT system and imaging techniques, have not yielded an unambiguous picture (Macoveanu, These findings translate to human abuse behavior and also appear to dissect a drug’s rewarding properties, mapping wanting to DA and speculatively liking to 5-HT, in an additive manner. Epidemiological data suggest that MDMA has a significantly lower abuse rate compared to monoamine releasers with less serotonergic action, like amphetamines (Degenhardt et al., In short, consistent with subjective drug effects, positive reward may be reflected by a combination of DA and 5-HT, whereas punishment could be reflected by 5-HT and absence, or reduction, of DA (Cohen et al., + or TpH2+ neurons, on the other hand, does not reproduce rewarding effects (Miyazaki et al., We put effort in this review to include especially findings from studies employing recent state-of-the art methods, such as optogenetics. These promised to resolve many contradictions regarding the 5-HT system by unequivocally identifying and interfering with specific neural populations, for which thus far identification was relatively unspecific using mostly either firing patterns or receptor dependent changes in activity. However, even these specific methods yield contradictory results. For example, doubtlessly direct DRN stimulation is rewarding, as known from self stimulation studies (Miliaressis et al., + DRN projection neurons to the bed nucleus of the stria terminalis induces fear and anxiety (Marcinkiewcz et al., + neurons in the DRN itself, does not display this effect (Correia et al., On the other hand, it is well established that the group of raphe nuclei is involved in different physiological functions depending on their topography (Hale and Lowry, Furthermore, it is crucial to incorporate the possibility of co-release of other neurotransmitters from monoaminergic neurons, which can lead to very different effects when comparing drug studies, e.g., employing agonists or releasers, which circumvent co-release, with stimulation studies, which induce co-release depending on the exact method used to target specific neurons Hu, . ConsideAfferent and efferent projections, hodological properties, the time course of individual neural activity bridging short latency and longer lasting activity modulations, all position the serotonergic system ideally to extract motivationally salient information and induce focused attention to guide goal oriented actions towards rewards. Human and animal studies that manipulated 5-HT levels strongly support serotonin’s role in affective processing, but the direction of association and specificity for aversive or rewarding events remains controversial. Optogenetic studies in rodents suggest that DA and 5-HT jointly guide learning by positive coding of reward PE signals, whereas absence of DA and presence of 5-HT is associated with reward omission, and possibly punishment PE signals. Furthermore, the DRN controls release of 5-HT to many projection regions and can recruit dopaminergic reward circuits via glutamate. Activation of purely serotonergic cells was found to be mostly non-rewarding or purely inhibitory. Taken together, this could be interpreted such that manipulations increasing 5-HT signals without a consecutive DA increase, mimics aversive events. On the other hand, direct DRN stimulation, specific stimulation of glutamate-releasing and co-releasing DRN cells, induces rewarding effects comparable to direct VTA stimulation (McDevitt et al., Highly specific methods to stimulate neuromodulatory systems reveal the complexity and entanglement between neuromodulatory systems on multiple levels, but do not provide simple answers. As more data is collected, it appears that judging the effect of one neuromodulatory system alone is unphysiological. However, converging between pharmacological effects in humans and optogenetic stimulation in animals, DA and 5-HT combined provide signals that interdependently are sufficient to guide reward related behavioral adaptations, and induce subjective reward. This complexity has direct consequences for the interpretation of pharmacological, genetic, and correlational studies in humans and indicates that DA and 5-HT do not have opposing functions (Daw et al., AGF wrote the manuscript, AGF and MU discussed, corrected and conceived the manuscript.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.