To do this, we conducted a systematic search for post-mortem, pharmacological, functional magnetic resonance and molecular imaging studies of dopamine function in bipolar disorder. Converging findings from pharmacological and imaging studies support the hypothesis that a state of hyperdopaminergia, specifically elevations in D2/3 receptor availability and a hyperactive reward processing network, underlies mania. In bipolar depression imaging studies show increased dopamine transporter levels, but changes in other aspects of dopaminergic function are inconsistent. Puzzlingly, pharmacological evidence shows that both dopamine agonists and antidopaminergics can improve bipolar depressive symptoms and perhaps actions at other receptors may reconcile these findings. Tentatively, this evidence suggests a model where an elevation in striatal D2/3 receptor availability would lead to increased dopaminergic neurotransmission and mania, whilst increased striatal dopamine transporter (DAT) levels would lead to reduced dopaminergic function and depression. Thus, it can be speculated that a failure of dopamine receptor and transporter homoeostasis might underlie the pathophysiology of this disorder. The limitations of this model include its reliance on pharmacological evidence, as these studies could potentially affect other monoamines, and the scarcity of imaging evidence on dopaminergic function. This model, if confirmed, has implications for developing new treatment strategies such as reducing the dopamine synthesis and/or release in mania and DAT blockade in bipolar depression.Bipolar affective disorder is a common neuropsychiatric disorder. Although its neurobiological underpinnings are incompletely understood, the dopamine hypothesis has been a key theory of the pathophysiology of both manic and depressive phases of the illness for over four decades. The increased use of antidopaminergics in the treatment of this disorder and new  However their modes of action have not been well established. By contrast antidopaminergic drugs do have well-understood class action at D2/3 receptors and have long been used to treat acute manic episodes. However, as will be explained below, newer drugs (particularly olanzapine and quetiapine) have been shown to have antimanic and antidepressant actions in bipolar disorder together with long-term benefit in preventing relapse to either pole of the illness.11 Furthermore drugs may be associated with significant side-effect burden and the risk of toxicity and/or teratogenicity in some instances.18 Hence better understanding of pathophysiology and drug action appears necessary to improve the use of current treatments and develop better alternatives.Despite these advances in treatment, many patients continue to experience high levels of disability.21 Early incarnations focused on mania, and the parallels between the behavioural consequences of amphetamine ingestion and the antimanic actions of antidopaminergic drugs. If hyperdopaminergia underlies the development of manic symptoms, then hypodopaminergia might underlie the depressive phase of the illness. Thus, opposite changes in dopaminergic function were hypothesised to underlie the opposing affective poles of the disorder.21 This theory did not explain how hyper- or hypodopaminergia would arise and subsequent versions proposed an additional component to the hypothesis, where an intrinsic dysregulation in the homoeostatic regulation of dopaminergic function leads to cyclical changes in dopaminergic neurotransmission,23 which would further distinguish the dopamine hypothesis of bipolar from theories of schizophrenia.24 Thus, faulty homoeostatic mechanisms responding to hyperdopaminergia in the manic phase of the illness are proposed to result in an excessive reduction in dopaminergic function, rapidly leading to a hypodopaminergic state and depression. In turn a faulty regulatory response to hypodopaminergia leads, to a switch back to mania.23 Implicit in this model is that a normalisation of dopaminergic function leads to remission and euthymia. Clearly, some kind of dysregulation must be required to account for the onset of episodes, but BD is also characterised by inter-episode mood instability.25 Any satisfactory theory must account for the randomness of much bipolar experience as well as the cyclicity. As current antimanic antipsychotics are all dopamine D2 receptor blockers, their use for mania has long supported the involvement of dopamine in mania, but the increasing use of some antidopaminergic drugs to treat bipolar depression and as maintenance drugs makes it timely to review the dopamine hypothesis of BD. Furthermore, a number of new lines of evidence relevant to the role of dopamine, in particular from molecular and functional neuroimaging, have developed in recent years. Thus, we synthesize evidence from pharmacological, in vivo neuroimaging and post-mortem studies addressing the role of the dopamine system in BD and then consider the treatment and drug development implications.The dopamine hypothesis of BD dates back at least to the 1970s.27 Following this, a dopamine transporter (DAT) knockout rodent model was developed, and dopamine transporter blockers other than amphetamine (such as GBR12909) have been used. Both these approaches have been shown to induce manic-like behaviour, such as hyperlocomotion and increased exploration.29 Subsequently, dopamine receptor stimulation using quinipirole (a dopamine agonist) was shown to induce manic-like behaviour.30 Interestingly, mood stabilisers such as valproate and carbamazepine reversed these effects.30 Recently, Sidor and colleagues demonstrated the impact of alterations in the regulation of circardian rhythm on dopaminergic activity and behaviour.31 In this study, mice with a mutation in a circadian clock gene displayed a manic-like phenotype as measured by hyperlocomotion in the daytime. Moreover, hyperlocomotion was linked to an elevated daytime spike in ventral tegmental area (VTA) dopaminergic activity, as well as increased dopamine synthesis and tyrosine hydroxylase activity.31 This was further validated using sustained optogenetic stimulation of the VTA, which also induced hyperlocomotion behaviour.31 Taken together this evidence suggests that hyperdopaminergia, induced either by increased dopamine release, dopamine transporter blockade or dopamine receptor stimulation, results in a mania-like phenotype in rodents. In contrast, lesions in dopaminergic areas (such as the VTA and substantia nigra) induce depressive behaviour, such as learned helplessness.32 Moreover, optogenetic stimulation of VTA dopaminergic neurons reverses depressive behaviour .33 These models thus provide evidence that hypodopaminergia may induce depressive-like behaviours and hyperdopaminergia may induce manic-like behaviour. Further research is needed to determine the key components of the dopaminergic circuit underlying this, particularly in relation to depressive behaviour.Initial animal models of mania were based on amphetamine-induced hyperlocomotion, and it was shown that lithium reverses this behaviour.35 However, to date only four studies,36 have been conducted, and only two focus on the same region . Thus, this finding still requires replication in other brain regions to determine if there is regional specificity. The literature on other dopaminergic receptors is more limited, with only two studies on the D1 receptor37 and one study on the D5 receptor.38 Another issue for the post-mortem studies is that they are affected by several confounding factors such as medication status, post-mortem interval, substance abuse and cause of death. Antidopaminergic treatment has been shown to increase D2/3 receptor levels in animals,39 and there is evidence this may also occur in schizophrenia.40 This suggests that prior treatment could also have affected the D2/3 receptor findings in BD. Finally, data on phase-specific changes are difficult to characterise as clinical status at the time of death remains unknown. Despite these limitations, post-mortem data supports the notion that an abnormality within the dopaminergic pathways, in particular involving D2/3 receptors, might play a role in the pathogenesis of BD.in vivo imaging studies of mania in 11C]-methylspiperone, when compared with healthy controls (HC) and non-psychotic mania patients,42 although, as this tracer has significant affinity for 5HT2 receptors as well,43 this finding requires replication with more selective tracers. Moreover, no significant difference in the striatal D2/3 density was noted in non-psychotic mania patients compared to HC.44 These studies also explored the relationship between manic symptoms  and dopamine synthesis capacity and D2/3 density, finding no significant correlations between these variables in patients with mania.45 However, in one of these studies D2/3 density was directly correlated with psychosis scores on the present state examination.41 Taken together, these data suggest that psychotic symptoms in mania may be associated with dopaminergic abnormalities, although the same cannot be inferred in non-psychotic mania patients.We have summarised the et al reported an upregulation of the dopamine transporter in drug naive euthymic bipolar patients, while Anand et al. observed a downregulation in a group consisting of both euthymic bipolar and bipolar depression patients.47 Additionally, one study has assessed the vesicular monoamine transporter protein (VMAT-2), located in presynaptic neurons and involved in the storage of dopamine. There was an increase in VMAT-2 in the thalamus and dorsal brainstem, but no significant alteration in striatum, in currently euthymic patients with a history of psychotic mania compared with HC.48 A positron emission tomography (PET) study using [11C]-SCH23390 found decreased D1 density in the frontal cortex but not in the striatum of bipolar patients compared to HC, suggesting a regional difference in D1 receptor distribution  (including nucleus accumbens) and prefrontal cortex have an important role in reward processing56 Changes in BOLD signal occur in the human nucleus accumbens, striatum and frontal cortex over a time course of seconds in response to anticipated reward.57 Consistent with the animal literature, event-related functional magnetic resonance imaging (fMRI) reward paradigms have demonstrated robust activation of the ventral striatum in relation to both the anticipation and receipt of reward,61 presumably related to dopaminergic neurotransmission.69Investigations in animals have shown that individual neurons signal reward-related information in the midbrain (substantia nigra and VTA), striatum, orbitofrontal cortex, amygdala and associated structures. Most dopamine neurons in the substantia nigra and VTA show brief, phasic responses that reflect the difference in value between received reward and predicted reward. The precise relationship between cell firing and function is still debated, but it would be misleading to think of dopamine neurotransmission simply in terms of a pleasure signal.82 This provides another indirect line of evidence for a dopaminergic abnormality in this disorder. We discuss the evidence in relation to specific mood polarities below.In BD several fMRI studies employing reward tasks support the existence of abnormal reward-related neural activity in the VS and frontal cortex ,81 compared with controls. Reward feedback related activation in the frontal cortex has also variously been reported as elevated,76 decreased (in euthymic and mildly depressed patients)81 or no different77 in bipolar I patients compared with controls. In euthymic bipolar II patients Caseras et al. reported elevated VS and prefrontal cortex activity for cued reward anticipation compared with controls.72 This finding, however, was not replicated in a more recent study of medication-naive bipolar II patients, which instead reported reduced anticipation-related activity in the right dorsal striatum in patients.82 Both studies reported no difference in VS activity during reward feedback in patients vs controls. Increased responses in the anticipatory phase of the monetary incentive delay task have been described in euthymic unipolar patients.83 Careful comparison of unipolar with bipolar cases will be necessary to distinguish network dysfunction associated with (hypo)mania from that with depression. All such studies need to be adequately powered and preferably address pre-specified hypotheses, not always a standard observed in imaging studies.Euthymic patients are of particular interest because they are at risk of mania. In these patients abnormalities in dopaminergic function would then indicate how vulnerability might be mediated. In medicated euthymic bipolar I patients, VS and prefrontal cortex activity to cued reward anticipation has been variously reported as increased,post hoc choice of analysis contrasts between these studies may account for the inconsistencies and non-replication. Further studies are required to explore the suggested differences between euthymic bipolar I, bipolar II and unipolar disorder.In summary, the precise nature of the abnormality of reward-related activity in euthymic bipolar patients remains unclear. However, abnormalities of reward processing are probably not confined to the manic phase of the illness. Important differences in task structure and the per se was increased in left ventrolateral prefrontal cortex in patients with bipolar-1 disorder, compared with healthy controls or patients with unipolar depressive disorder; it may represent a more generalised ‘arousal' response in the bipolar depressed group.73 Ventral striatal and prefrontal activation during reward feedback in bipolar depressive patients has been reported both as similar to controls79 and decreased.78Three studies have compared monetary reward processing in medicated depressed bipolar I patients and HC. Only one study analysed activity during cued reward anticipation; it reported no difference in anticipation-related activity between patients and HC in VS or prefrontal cortex, although there was blunting in the anterior cingulate cortex in depressed patients (bipolar and unipolar). Neural activity related to outcome anticipation 86 as well as major depressive disorder and alcohol addiction.84 In contrast, the fMRI findings in bipolar disorder summarised above do not provide consistent evidence for blunting. It may be important to consider only studies in the manic state, because depressed or even euthymic (often dysthymic) bipolar patients may well show blunting.82 In mania there was increased frontal activation to reward anticipation in two out of three studies. If this finding is confirmed in further studies, including direct comparisons with patients with schizophrenia, it could point to a key difference in the reward network between mania and schizophrenia. To date a small number of studies have compared reward processing in bipolar disorder with schizophrenia70 or major depressive disorder.78 Of these, one study reported blunted VS activation in depressed patients diagnosed with bipolar disorder compared with major depressive disorder, at reward feedback.78 Only one study compared reward processing in schizophrenia and manic patients with controls.70 Patients with schizophrenia and healthy controls showed an activation in the VTA on expectation of monetary rewards and nucleus accumbens activation during receipt vs omission of rewards. Manic patients, however, showed reduced differential activation in the nucleus accumbens on receipt vs omission of rewards compared to the healthy control subjects. Taken together with the findings in the frontal cortex discussed above, this small study does suggest that in mania there is a deficit in prediction error processing not seen in schizophrenia. It may further imply bipolar disorder specific changes in dopaminergic function, although how much of the fMRI response is driven by disrupted dopaminergic function remains to be determined. Further studies investigating differences in reward processing between bipolar disorder and other psychotic and affective disorders, and across illness phases, will shed light on the reward processing abnormalities specific to bipolar disorder.Recent fMRI studies and meta-analyses have demonstrated blunted activation of the reward network in the ventral striatum and frontal cortex in schizophrenia,90 Further, there are several case series of manic and hypomanic episodes in Parkinson's disease patients treated with Levodopa.93 Bromocriptine, another dopamine agonist, has also been shown to induce manic symptoms.95 Similarly, an increased risk of hypomania/mania was observed in bipolar patients who received stimulant  augmentation for bipolar depression.96 Secondly, pharmacological strategies to deplete tyrosine, a dopamine precursor, are known to reduce manic symptoms, although it remains to be established if this is specifically due to reducing dopamine levels.98 Alpha-methyl-p-tyrosine, which induces dopamine depletion by inhibition of tyrosine hydroxylase, also attenuates mania-like symptom in bipolar patients.100The role of dopaminergic abnormalities in BD has been extensively investigated using a variety of pharmacological approaches . Firstly102 In addition, a high prevalence of depression is seen in patients with Parkinson's disease and this has been linked to loss of striatal dopaminergic innervation.104 Finally, depletion of tyrosine has been shown to precipitate depressive symptoms in remitted patients with a history of major depressive disorder.106Reserpine and tetrabenazine depletes synaptic dopamine by irreversibly inhibiting vesicular uptake of monoamines and has long been known to induce depression.110 Olanzapine, risperidone, quetiapine, aripiprazole, asenapine, ziprasidone and cariprazine have been approved by the Food and Drug Administration (FDA) as monotherapy for the treatment of acute mania.111 Pertinently, a network meta-analysis of 68 randomised controlled trials (RCT), involving 16 073 participants, addressed the efficacy of different drugs in the treatment of acute mania. This showed that dopamine antagonists have larger effect sizes  relative to placebo: −0.56 (95% CI −0.69 to −0.43)), risperidone (−0.50 (−0.63 to −0.38)), olanzapine (−0.43 (−0.54 to −0.32)) than mood stabilisers for the treatment of acute mania (lithium) (−0.37 (−0.63 to −0.11)), carbamazepine (–0.36 (–0.60 to −0.11)), valproate (–0.20 (–0.37 to −0.04)).14Dopamine antagonists and partial agonists are increasingly used in the treatment of acute mania, bipolar depression and also as maintenance treatment.24 it is likely that reduced dopamine neurotransmission at least contributes to the clinical efficacy of these drugs. This assumption is supported by the greater efficacy of the cis- but not the trans-isomer of clopenthixol in the treatment of manic symptoms,112 as the cis-isomer is a high affinity D2/3 receptor blocker while the trans-isomer has much lower affinity for D2/3 receptors.112As the blockade of the dopamine D2/3 receptors is their common mechanism of action,111 Although lithium and valproate should be preferred, recent guidelines highlight that dopaminergic drugs have a place in maintenance treatment.113The FDA have approved olanzapine as monotherapy, quetiapine and ziprasidone as adjunctive therapy, and aripiprazole and risperidone long acting injections as both adjunctive and monotherapy for the maintenance treatment of BD.45 In a study consisting of 13 manic patients, 2 weeks of sodium valproate was shown to decrease presynaptic dopamine synthesis capacity,45 although this was not correlated with clinical improvement. Interestingly D2/3 density remained essentially unchanged after valproate treatment,44 despite the reduction in dopamine synthesis capacity with valproate, which is anticipated to alter D2/3 availability. Taken together these two could either suggest valproate blocks the capacity of the D2 receptor to respond to reduced dopamine synthesis, or that the capacity of D2 receptors to respond is intrinsically impaired in bipolar disorder. Although both possibilities are speculative at this stage, the latter is particularly interesting as it suggests that a failure of D2 adaptation could contribute to the pathophysiology of bipolar disorder, making the dopamine system more vulnerable to dopamine transporter driven changes in presynaptic function.Interestingly, it is possible that sodium valproate might exhibit antimanic effect through an action on the dopaminergic system.114 Furthermore, lithium acts on the adenyl cyclase and phospho-inositide, as well as protein kinase C pathways, which are part of the intracellular signalling pathway downstream of dopamine receptors.115 In addition, micro-dialysis studies show a reduction in extracellular dopamine levels in lithium treated animals.118 In summary, there is evidence to suggest that some non-D2/3 blocking mood stabilisers also act to reduce dopamine transmission, either through reducing presynaptic dopamine synthesis capacity in the case of sodium valproate, or post-synaptic dopaminergic signal transduction in the case of lithium. The degree to which these effects explain the therapeutic efficacy of these compounds remains to be determined.Although the exact mechanism by which lithium acts remains unclear, it is known to modulate signalling pathways downstream of dopamine receptors. Preclinical studies have shown that lithium reverses dopamine dependent behaviour by acting through the protein kinase B (AKT)/glycogen synthase kinase 3 signalling cascade.127 Although there have not been any RCTs investigating the efficacy of methylphenidate or amphetamines in bipolar depression, the available open-label and naturalistic studies point towards a benefit of stimulants in a selected group of patients with drowsiness and fatigue.131 Randomised controlled studies and open-label reports with other stimulant like agents such as modafinil and its R-enantiomer, armodafinil also indicate efficacy in bipolar depression although the development programme for armodafinil failed.138 There is evidence to support the potential use of monoamine oxidase inhibitors such as tranylcypromine in bipolar depression.140 It is speculated that tranylcypromine acts as dopamine releaser with about 1/10th the potency of amphetamine.140 There are thus several lines of evidence suggesting that dopaminergic augmentation is beneficial in bipolar depression.Three double-blind placebo-controlled studies have shown that short-term use of pramipexole, a D2/D3 receptor agonist, is efficacious as an augmentation strategy for the treatment of bipolar depression and positive results have also been seen in other open-label studies.15 The FDA has approved the combination of olanzapine and fluoxetine, as well as monotherapy with quetiapine or lurasidone, for the treatment of acute bipolar depression. A recent meta-analysis of 24 placebo-controlled trails (n=7307) revealed the following order of efficacy of drugs for the treatment of bipolar depression: olanzapine+fluoxetine⩾valproate>quetiapine>lurasidone>olanzapine, aripiprazole and carbamazepine.141 The analysis is interesting in showing that antidopaminergics on their own have efficacy in bipolar depression.However, there is also evidence that dopamine antagonists are effective in the treatment of bipolar depression.2A antagonists,142 whilst both lurasidone and aripiprazole have high affinity for 5HT1A receptors.143 Potentially supporting this explanation, there are no clinical trials reporting efficacy of pure D2/3 blockers in bipolar depression. Nevertheless, the doses used in the clinical trials correspond to the dose ranges associated with substantial dopamine D2/3 receptor occupancy,24 so dopamine receptor blockade is certainly compatible with antidepressant efficacy. The final consideration is that there has been a relatively small number of good quality RCTs for antidopaminergic treatment in bipolar depression, certainly in comparison to studies in mania. Studies that can directly test the mechanism underlying the mode of action of these drugs and pramipexole would be of great interest.The evidence thus suggests that both dopaminergic agonists and dopaminergic blockers, are effective in treating bipolar depression. This presents a paradox for understanding the role of dopamine in bipolar depression. However, actions at receptors other than dopamine ones may underlie the efficacy of the dopamine blockers. For example, as well as all being D2/3 receptor blockers, olanzapine and quetiapine are both relatively high affinity 5HTOur main findings for bipolar mania are that (i) there is consistent pharmacological evidence, especially from treatment studies, to support the hypothesis that a state of hyperdopaminergia can lead to mania; (ii) imaging studies support this hypothesis, with several studies reporting elevations in D2/3 receptor availability in psychotic mania and fMRI imaging evidence that identifies hyperactivity of the reward circuit in mania. Dopamine synthesis and receptor density appear to remain unchanged, at least in non-psychotic mania patients compared with HC.For bipolar depression (i) pharmacological evidence shows that dopamine agonists are potentially beneficial for bipolar depression, but the same seems true for dopamine blocking drugs; (ii) the imaging studies show replicated increases in dopamine transporter levels, but there is inconsistency and it is not clear if there are other alterations as well. Finally, post-mortem evidence suggests an upregulation of the D2/3 receptors in bipolar patients, but interpretation is limited by lack of information on phase of illness and medication status at the time of death.144 Tentatively, these studies suggest elevated D2/3 receptor availability and a hyper-responsive reward system in ventral striatum in mania, and an increase in striatal dopamine transporter availability in bipolar depression. Dopamine neurotransmission in the striatum is primarily terminated by reuptake of dopamine into the presynaptic dopamine nerve terminal by dopamine transporters. Thus, an elevation in striatal D2/3 receptor availability in mania would lead to increased dopaminergic neurotransmission whilst increased striatal dopamine transporter levels in depression would lead to reduced dopaminergic function. This suggests a model in which elevated D2/3 receptor levels lead to altered reward processing and the development of mania, which is followed by a compensatory increase in dopamine transporter levels to reduce dopaminergic neurotransmission. However, if, over time, D2/3 receptor levels reduce but dopamine transporter levels do not normalise, this would then lead to reduced dopaminergic transmission, leading to depression and, in turn, a compensatory upregulation of D2/3 receptor levels, precipitating a further phase switch. One can see how a failure of homoeostatic regulation of the dopaminergic system could lead to cyclical periods of elevated and blunted dopaminergic neurotransmission. This model might apply most precisely to rapid cycling bipolar disorder. However it could be a component of all manic episodes, with other systems acting to disrupt the regular cyclicity that is so obvious in a substantial number of patients.More than four decades on from the early conceptualisations of the dopamine hypothesis of BD, it has stood the test of time, and the evidence for elements have strengthened. This is particularly the case for mania where the strength of the clinical trial evidence for the benefit of dopamine antagonists and partial agonists, supported by meta-analysis, would require an improbably large number of negative studies for reversal. Added to this there is now molecular and fMRI imaging evidence in bipolar disorder. Further, preclinical studies using optogenetic methods and knockout mice have shown that dopaminergic neuron activation leads to manic-like behaviour and it is linked to circadian gene expression.per se may drive dopaminergic changes.145 It is worth noting that, none of the studies have investigated dopamine function in mixed states. As approximately two thirds of depressed patients have concomitant manic symptoms,146 any theory needs to explain mixed states as well as mania and depression. It would be hard to account for mixed states by dysfunction in dopaminergic function alone. Interestingly, asenapine seems to be more effective than olanzapine for mixed episodes.147 As asenapine has higher affinity for 5HT2A than D2/3 receptors, which could suggest a role of serotonergic system in mixed episode states. Finally elevations in D2/3 receptor availability would be predicted to increase dopaminergic neurotransmission via the indirect pathway, and reduce response to cues,148 while the fMRI findings in mania do not seem to fit with this. However, the majority of the patients in these studies were treated with dopamine antagonists and mood stabilisers .The key limitation of the dopamine hypothesis remains that its strongest supporting evidence comes from pharmacological studies, which offer an indirect and sometimes imprecise approach to studying dopaminergic function. Reserpine, alpha-methyl-para-tyrosine, amphetamine and 50 this is not consistent with the finding of elevated DAT in a euthymic state.47 It also remains unclear whether the dopamine hypothesis can completely explain bipolar depression and how dopaminergic dysregulation will be linked to the involvement of other neurotransmitter systems.Although our model suggests euthymia is a state of normalised dopamine, based on a study demonstrating no significant alteration in the dopamine release paradigm in euthymic patients compared to controls,115 it is not established if this is their mechanism of action. Determining whether dopaminergic mechanisms are common across classes of medication would be an important advance in understanding. Finally, it remains unclear if hyperdopaminergic activity is specific to mania or psychosis because many of the manic patients in the studies had psychotic symptoms as well. It is interesting to note that in schizophrenia molecular imaging studies indicate there is an elevation in dopamine synthesis and release capacity, but unaltered dopamine transporter and D2/3 receptor availability in striatum.40 PET studies have also demonstrated elevated striatal dopamine turnover, but blunted cortical and midbrain dopamine release in schizophrenia.150 In addition, blunting of the fMRI signal during reward tasks is observed in schizophrenia.85 In contrast, studies in bipolar disorder suggest dopamine synthesis capacity is unaltered, at least in non-psychotic mania, but there is elevated D2/3 receptor availability in psychotic mania. However, direct comparisons of dopaminergic function in psychotic mania and schizophrenia as well as longitudinal studies of dopaminergic activity across various phases of illness are needed to determine if there are differences between mania and schizophrenia, and between mania and bipolar depression (A critical limitation is the lack of longitudinal studies that investigate changes across phases of illness, including mixed states. Ideally future studies should focus on elucidating phase-related dopamine dysfunction by studying patients longitudinally in euthymic, manic and depressive episodes to determine the direction of causality. Clearly this will be difficult, although a focus on rapid cycling patients might make it feasible. Cross-sectional studies are more feasible and could test key elements of the dopamine hypothesis if well designed. Another key issue is why some antidopaminergics are effective for bipolar depression. Molecular imaging studies are needed to determine whether dopaminergic, serotonergic or other systems are involved in their mode of action. Finally, although studies have found an effect of valproate and lithium on dopaminergic function,pression .The dopamine hypothesis of bipolar disorder proposes that faulty homoeostasis between dopamine transporter and receptors underlies depressive and manic phases of the illness. The available evidence suggests elevated D2/3 receptor availability and a hyper-responsive reward system in mania, and possibly increased dopamine transporter availability in bipolar depression. Future longitudinal studies are needed to elucidate the precise phase-related changes in dopaminergic function and the specificity of alterations to mania over psychosis.


In addition, high DANCR and low lncRNA-LET were significantly correlated with lymph node metastasis and late clinical stage. DANCR associated with EZH2 and HDAC3 to epigenetically silence lncRNA-LET and then regulated GC migration and invasion. Taken together, these findings indicate an important role for DANCR–lncRNA-LET axis in GC cell migration and invasion, and reveal a novel epigenetic mechanism for lncRNA-LET silencing.Gastric cancer (GC) is one of the most prevalent gastrointestinal malignancies. Long noncoding RNA (lncRNA) DANCR is a newly identified oncogenic lncRNA. However, the functional role and underlying molecular mechanisms of DANCR involved in GC progress remain unclear. In the present study, we investigated the biological function and underlying mechanisms of DANCR in GC cell migration and invasion. The results showed that knockdown of DANCR inhibited migration and invasion of GC cells, whereas overexpression of DANCR showed the opposite effect. Further investigation demonstrated that lncRNA-LET was a  Gastric cancer (GC) is one of the most prevalent gastrointestinal malignancies and ranks the second leading cause of cancer-related deaths around the world . Even thThe human transcriptome contains not only a set of protein-coding mRNAs, but also a large number of noncoding transcripts that exert housekeeping or regulatory function, such as microRNAs, long noncoding RNA (lncRNA), tRNA, rRNA, siRNA, and piRNA ,7. LncRNIn the present study, we investigated the biological function and molecular mechanisms of DANCR in GC cell migration and invasion. We demonstrated DANCR as an important regulator of GC migration and invasion by suppression of lncRNA-LET. Notably, there is a remarkable up-regulation of DANCR and down-regulation of lncRNA-LET in GC tissue samples, indicating that DANCR–lncRNA-LET axis may be potential therapeutic target for GC metastasis.All patients provided written informed consent, and ethical consent was granted from the Committee for Ethical Review of Research Involving Human Subjects of the Xinxiang Central Hospital. Human GC samples and corresponding nontumorous gastric tissues were obtained from Department of Surgical Oncology, Xinxiang Central Hospital.2 in DMEM supplemented with 10% fetal bovine serum .The GES1, SGC7901, BGC-823, MGC803, AGS, MKN45, and MKN28 cell lines were obtained from the cell bank of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. Cells were maintained at 37°C in 5% COTotal RNA was isolated by using TRIZOL reagent (Invitrogen) according to its manufacturer’s protocol. For quantitative real-time PCR (qPCR) detection, RNA was reverse-transcribed to cDNA using a Reverse Transcription Kit (Transgene). qPCR detection was performed using SYBR Green Mixture (Roche). The specific primers are listed as follows: DANCR-F: CTGCATTCCTGAACCGTTATCT, DANCR-R: GGGTGTAATCCACGTTTCTCAT; LncRNA-LET-F: GGCTCTGTGGGATCAGTTATG, lncRNA-LET-R: GGCTGAGGAAGGTGGTATTG. GAPDH was used as internal reference.Cell lysates were separated by SDS/10% PAGE and transferred into PVDF membranes (Millipore), and incubated with primary antibodies overnight, anti-EZH2 , anti-HDAC3 , and anti-GAPDH . Blots were detected by ECL system (Millipore) and analyzed on an imager . GAPDH was used as internal reference.Cell migration and invasion were detected using Transwell and Matrigel Transwell (Corning Costar) membrane filter inserts in 24-well tissue culture plates respectively. Cells suspended in serum-free DMEM were seeded on the upper chamber of transwell filters. DMEM containing 20% FBS was added to the lower chamber and then incubated for 24 h. Cells in the upper side of the filter were removed, and the cells across the membrane were fixed with 4% formaldehyde, stained with Crystal Violet.siRNAs were purchased from Riobio Company . The target sequences of siRNAs for target gene were as follows: siDANCR-1: AGCCAACTATCCCTTCAGT, siDANCR-2: GAGCTAGAGCAGTGACAAT, siEZH2: TGCACATCCTGACTTCTGT, siHDAC3: CCTGCATTACGGTCTCTAT, silncRNA-LET: GTCTGATGTATCCACCCAT. The full-length DANCR and lncRNA-LET were synthesized by BGI company and cloned into pcDNA3.1 vector (Invitrogen). GC cells were transfected with siRNAs or plasmid vectors using Lipofectamine 3000 (Invitrogen) as the manufacturer’s protocol.BGC-823 cells were lysed with an immunoprecipitation (IP) lysis buffer (Beyotime). Total proteins were incubated with 3 μg of anti-EZH2 (Abcam) antibodies overnight on a rocking platform. And Protein G-agarose (GE) was then added to the immunoprecipitation mixture, and incubated for 6 h. After wash, the 2× loading buffer was added, the agarose was boiled for 10 min, and then subjected to Western blot analysis.The RNA pull-down was performed as previous described . BrieflyRNA immunoprecipitation (RIP) assays were performed using the EZ-Magna RIP kit (Millipore) following the manufacturer’s instruction. Five micrograms of anti-EZH2 (Millipore) and anti-HDAC3 (Abcam) antibodies were used.Chromatin immunoprecipitation (ChIP) assays were performed using the Hisense EZ-Magna ChIP kit (Millipore) following the manufacturer’s instruction. Five micrograms of anti-EZH2 (Millipore), anti-HDAC3 (Abcam), anti-H3K27me3 (Millipore), anti-H3Ac (Millipore), and anti-H4Ac (Millipore) antibodies were used.P value less than 0.05 was considered statistically significant.All statistical analyses were performed by using SPSS 17.0 software. The significance of differences was estimated by an ANOVA and a multiple comparisons test (LSD-t). To understand the biological function of DANCR in GC progress, we detected the DANCR expression level in several GC cell lines and a normal human gastric epithelial cell line (GES-1) A. The qPMigration and invasion are critical steps for cancer metastasis. We evaluated the migratory ability mediated by DANCR through a transwell assay. The results showed that knockdown of DANCR suppressed the cell migration in BGC-823 cells C. In conTo determine the underlying mechanism of DANCR in GC migration and invasion, we analyzes the gene expression profiles mediated by DANCR from a previous study . A potenTo detect whether DANCR promotes cell migration and invasion in an lncRNA-LET-dependent manner, rescue experiments were performed. We transiently overexpressed lncRNA-LET in DANCR-upregulated AGS cells C. The rer2 = 0.3019, P<0.0001; P=0.020, P<0.001) and late clinical stage . These data demonstrated that lncRNA-LET is regulated by DANCR in GC and DANCR–LET axis mediated the effect on GC metastasis.To confirm the pathological correlation between DANCR and lncRNA-LET in GC tissue samples, we analyzed the expression pattern of both DANCR and lncRNA-LET in 60 pairs of GC and corresponding nontumorous gastric tissues by qPCR. DANCR expression was significantly increased in GC tissues compared with nontumorous gastric tissues A. And GCFinally, we investigated the underlying mechanisms by which DANCR suppressed lncRNA-LET expression. It has been reported that lncRNA-LET expression is silenced by EZH2 and HDAC3 ,18. In aPrevious studies have demonstrated that lncRNA interacts with epigenetic modifier to epigenetically regulate target genes expression. To confirm whether lncRNA-LET was regulated by DANCR in this manner, we performed a RIP assay. The RIP assay demonstrated that DANCR could be significantly enriched by both anti-EZH2 and anti-HDAC3 antibodies compared with negative control IgG C. For fulncRNA-LET promoter region. The effect of DANCR silence on the binding level of EZH2 and HDAC3 or trimethylation levels of H3K27 (H3K27me3) or acetylation of histone H3 and H4 (H3Ac and H4Ac) in the lncRNA-LET promoter was determined by using a ChIP-qPCR assay. The results showed that DANCR silence decreased the binding level of EZH2 and HDAC3 in lncRNA-LET promoter, suppressed the occupancy level of H3K27me3, and increased H3Ac and H4Ac levels across the lncRNA-LET promoter regions (lncRNA-LET promoter regions were decreased (Given that EZH2 and HDAC3 regulate transcription of target genes by binding to promoter regions and modifying histone, we examined whether DANCR influenced the EZH2 and HDAC3 occupancy of the  regions E. On theecreased F. Moreovecreased G. These In the present work, we report that DANCR promotes cell migration and invasion through suppression of lncRNA-LET. And there exists a negative correlation between DANCR and lncRNA-LET expression in GC tissues. Mechanistic investigation demonstrates that DANCR associates with epigenetic modifier, EZH2 and HDAC3, which is crucial for lncRNA-LET silence.Emerging evidence have revealed the important roles of DANCR in cell differentiation and tumor progression. DANCR functions as a downstream regulator of SOX4-induced chondrogenic differentiation . DANCR dlncRNA-LET is critical for lncRNA-LET silence [lncRNA-LET. Subsequently, DANCR alters the level of H3K27me3, H3Ac, and H4Ac across the lncRNA-LET promoter regions to suppress lncRNA-LET transcription. DANCR also influenced the interaction between EZH2 and HDAC3. Above all, DANCR functions as a molecular scaffold of EZH2 and HDAC3 to suppress lncRNA-LET expression.LncRNA-LET is a well-known tumor suppressor gene in cancers. Deregulation of lncRNA-LET has been found in several cancers, such as gastric cancer, cervical cancer, nasopharyngeal cancer, and lung adenocarcinoma ,22–24. D silence ,18. In oIn summary, our present study is the first time to reveal that DANCR is up-regulated in GC tissues. DANCR enhances cell migration and invasion in GC cells through association with EZH2 and HDAC4 and epigenetically silencing another lncRNA, lncRNA-LET. Collectively, our findings provide a new perspective that regulatory network of different lncRNAs may act as a critical role in GC progress.


The electrical properties of the films were examined in a metal/insulator/metal configuration, with a highly-doped silicon bottom contact and a eutectic gallium-indium liquid metal (EGaIn) top contact. The junctions exhibit high yields (>90%), a remarkable resistance to bias stress, and current rectification ratios (R) between 20 and 200 depending on the structure, degree of order, and internal dipole of each molecule. We found that the rectification ratio correlates positively with the strength of the molecular dipole moment and it is reduced with increasing molecular length.We report on the synthesis and electrical properties of nine new alkylated silane self-assembled monolayers (SAMs) – (EtO) The idea of molecular electronics was proposed as a means to surpass the challenges present in downscaling existing technologies1245671011121415171819In this work, we developed nine new fluorinated benzalkylsilane molecules of varying internal molecular dipole due to the imposed length and termination, and we studied their electrical properties in relation to their chemical structure. We incorporated the self-assembled monolayers (SAMs) composed of these molecules into molecular diodes and obtained high yields and reproducible rectification ratios as high as 200, with good bias stress stability. We calculated the molecular dipole using density functional theory (DFT) and we found that the rectifying behaviour is stronger in the case of short molecules and is enhanced by the internal dipole. Our results provide evidence that the molecular structure impacts the electrical properties of molecular diodes through the internal dipole characteristic to each molecule, and may promote a rational design of compounds that can function as high-performance molecular rectifiers.2 surface, an alkyl chain with either 3 or 11 CH2 groups, a phenyl group to facilitate the formation of an “intermolecular top-link” to enhance ordering, and a fluorine-containing head-group providing different amounts and orientations of fluorine atomsE)-1-(4-(trifluoromethyl)phenyl)-N-(11-(triethoxysilyl)undecyl)methanimine (molecule 1), (E)-1-phenyl)-N-(11-(triethoxysilyl)undecyl)methanimine (molecule 2), (E)-1-(4-fluorophenyl)-N-(11-(triethoxysilyl)undecyl) methanimine (molecule 3), (E)- 1-(3-fluoro-4-trifluoromethyl)phenyl)-N-(11-(triethoxysilyl)undecyl)methanimine (molecule 4), (E)-1-(4-(trifluoromethyl)phenyl)-N-(3-(triethoxysilyl)propyl)methanimine (molecule 5), (E)-1-phenyl)-N-(3-(triethoxysilyl)propyl) methanimine (molecule 6), (E)-1-(4-fluorophenyl)-N-(3-(triethoxysilyl)propyl) methanimine (molecule 7), (E)- 1-(3-fluoro-4-trifluoromethyl)phenyl)-N-(3-(triethoxysilyl)propyl)methanimine (molecule 8) and (E)-1-(perfluorophenyl)-N-(3-(triethoxysilyl)propyl) methanimine (molecule 9). (E)-1-(perfluorophenyl)-N-(11-(triethoxysilyl)undecyl) methanimine  was also investigated; its properties, however, varied significantly from sample to sample, as well as for the same film, and therefore we decided not to include it in our analysis. The molecules were synthesized from amino trialkoxysilanes by using the condensation of commercially available amino alkyl triethoxysilanes with substituted benzaldehydes to prepare the precursors, following established procedures  wafers using solution or vapor-based deposition, as described in the Methods section.22 is <10°.In order to evaluate the quality of the SAM and the best method for assembly, water contact angle measurements were conducted using a Ramé-Hart Model 200 Contact Angle Goniometer; the results are displayed in We employed atomic force microscopy (AFM) measurements to characterize the surface roughness prior to SAM deposition. We measured a sample of 1 cm × 1 cm and imaged a surface area of approximately 2 × 2 μm in several spots using a Nanoscope IIIA AFM (Veeco Instruments), see 22The work function measurements on highly doped silicon were performed using a Trek model 325 electrostatic voltmeter configured for Kelvin probe measurements and calibrated using highly-ordered pyrolytic graphite (HOPG) as a standard. The calculations followed from e is the elementary charge, 2), where The electrical measurements were taken on metal/SAM/metal structures using a eutectic gallium-indium (EGaIn) conical-tip top contact in which the silicon substrate was held at ground and the bias was applied to the EGaIn contact in ambient conditions. In this configuration, the forward bias condition corresponds to a positive potential applied at the fluorinated end of the molecules. The eutectic liquid metal EGaIn was utilized as a soft top-contact because its use is non-damaging, reproducible, and well-studied in this context222331323334353637ab initio calculations at the density functional theory level, using VASP3941−4 eV. Calculations were performed in orthorhombic unit cells with a vacuum of at least 2 nm between periodic images. All structures were relaxed until the maximum force on any atom was less than 10 meV/angstrom. Dipole moments were calculated using VASP’s built-in functionality to calculate and correct for the dipole moment in all directions (i.e. the IDIPOL and LDIPOL tags).We performed J) on the applied voltage (V) for one film of molecule 3, in 25 different spots. The relative standard deviation for these measurements is <1.4. Measurements on different spots on the films are consistent, suggesting that our SAMs are uniform. In addition, for a particular SAM to be considered viable as a rectifier, it must be able to withstand many repetitive measurements with minimum changes in the electrical characteristics. This could involve a variability and instability in the measured output characteristics or, in the worst-case scenario, a “breaking” of the monolayer, indicating that there is now an irreversible conductive path between the two electrodes. The good operational stability, as clearly seen in In J-V) characteristics for each of the 9 molecules, taken during the forward and reverse bias conditions. We measured at least 10 devices for each type of SAM, in at least 25 spots each, and obtained similar results. As expected, the magnitude of the measured current depends on the molecular structure, with the long molecules generally exhibiting lower conductivities and less pronounced asymetries between the forward and reverse bias conditions.J value during the forward bias measurement, (V = +2 V) and the J value during the reverse bias measurement, (V = −2 V):We define the rectification ratio, R, as the ratio between the 5 = 183 ± 41 and R8 = 192 ± 42. These values are on par with some of the highest reported in the literature, with a thiophene-1,1-dioxide oligomer exhibiting an R of ~200, a ferrocene-alkanethiolate with an R = 150, and ferrocene placed on a monolayer of β-cyclodextrin demonstrating an R of 17015334446482O3 layer formed at the surface of the EGaIn electrodeIn In conclusion, we have designed, synthesized, and measured nine new rectifying self-assembled monolayers of different lengths and polar terminations. We found that these fluorinated benzalkylsilane molecules exhibit high yield when incorporated in molecular diodes, coupled with robust rectification ratios ranging from 20–200, depending on their structure. In addition, they show good uniformity over large surface areas and excellent bias stress stability. We found an increase in the rectification strength with enhancing the internal molecular dipole of each molecule. Our results suggest that the electrical properties of molecular diodes can be controlled by tailoring the molecular structures of the constituent molecules. This is a significant step toward controlling the performance of molecular rectifiers through manipulating specific structure-property relationships for use in macroscopic electronics as diodes, half-wave rectifiers, AC/DC converters, or other charge-restricting elements.1H and 13C nuclear magnetic resonance (NMR) and elemental analysis or high-resolution mass spectrometry , followed by a 10 minute exposure to UV-ozone and subsequent rinse in DI water before being dried in a stream of nitrogen. The UV-ozone step serves to both remove organic contaminants and to increase the density of hydroxyl groups on the surface. The monolayers were formed in a nitrogen  glovebox. We found that for the long SAMs  self-assembly from a 4 mMol solution in chloroform at 30 °C provided the films of highest quality. The short SAMs (molecules 5 through 9) were amenable to deposition by vapor treatment, where the silicon wafer was placed into a sealed jar along with 11 μL of the pure SAM compound at 30 °C. Both methods typically took 18 to 24 hours. To remove the excess adsorbed molecules on the monolayer surface, the samples were thoroughly rinsed in chloroform followed by IPA and then immediately dried in a stream of nitrogen.The SAMs were deposited on highly-doped silicon wafers with native SiOThe conical-tip EGaIn contact, which was used as a top soft contact for our molecular rectifiers, was created using a Micromanipulator probe holder, and a 0.5 mm diameter Micromanipulator probe tip. EGaIn was placed onto a sacrifical copper strip into which the probe tip was slowly lowered and then raised with the aid of the contact angle goniometer.How to cite this article: Lamport, Z. A. et al. Fluorinated benzalkylsilane molecular rectifiers. Sci. Rep.6, 38092; doi: 10.1038/srep38092 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The increasing number and global distribution of pathogens resistant to antimicrobial drugs is potentially one of the greatest threats to global health, leading to health crises arising from infections that were once easy to treat. Infections resistant to antimicrobial treatment frequently result in longer hospital stays, higher medical costs, and increased mortality. Despite the long-standing recognition of antimicrobial resistance (AMR) across many settings, there is surprisingly poor information about its geographical distribution over time and trends in its population prevalence and incidence. This makes reliable assessments of the health burden attributable to AMR difficult, weakening the evidence base to drive forward research and policy agendas to combat AMR. The inclusion of mortality and morbidity data related to drug-resistant infections into the annual Global Burden of Disease Study should help fill this policy void. Antimicrobial resistance (AMR) occurs when bacteria, viruses, fungi, and parasites adapt to antimicrobial drugs, resulting in drug inefficiency and persistent infections, with a subsequent increase in the risks of severe disease and transmission. AMR is a major global threat to the health of populations, endangering the ability to prevent and cure a wide range of infectious diseases –3. WhileThere are many challenges to estimating the burden of AMR. Primarily, there is limited and unreliable current and historical information on the geographical distribution, prevalence, and incidence of AMR and its health burden, making the burden of AMR difficult to measure and limiting our ability to devise geographically explicit strategies for its control , 11. DisAs the threat of AMR continues to grow, more work is needed to supplement current surveillance and methodologically inconsistent research regarding the global epidemiology and impact of AMR. Some studies have addressed the challenges to measuring the burden of AMR and also provide estimates of prevalence of resistance for particular pathogen-antimicrobial drug combinations in various locations , 3, 12. Here, we present a new project (“AMR project”) to provide rigorous quantitative evidence of the burden of AMR, to increase awareness of AMR, to support better surveillance of AMR, and to foster the rational use of antimicrobials around the world. An important aspect of this AMR project is the integration of AMR burden with the larger Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study at the Institute for Health Metrics and Evaluation.The GBD is an ongoing comprehensive global research program that provides comparable estimates of mortality and disability resulting from 328 disease and injury causes, as well as from 84 risk factors, across age and sex groups, over time and space –17. ThisThe strategy we will pursue in estimating the burden of AMR has several dimensions. First, because of the important role of sepsis involving a drug-resistant organism as an intermediate pathway to death from AMR, we will analyze the burden of all forms of sepsis in the GBD. Second, we will collate and analyze the data gathered by the public and private sectors on resistant bacteria present in various human samples . Third, we will systematically review published and unpublished sources on the relative case-fatality rate for drug-resistant versus drug-sensitive infections for different clinical syndromes. These components will allow estimation of the burden of resistance for different drug-organism combinations. Over time, we will systematically expand the number of combinations included in the GBD assessment. The initial objective of the burden of disease assessment for AMR will include a comprehensive synthesis of existing global AMR data on selected bacterial pathogens (listed in Table Results from this effort will contribute to the assessment of the burden of AMR over time, allowing an evaluation of current and past magnitude and geographical dispersion of the hazard, thus providing essential health intelligence to guide interventions and policies, as well as a benchmark for measuring the impact of interventions on future burden. By incorporating this work into the GBD, it will enable comparison of the loss of healthy life due to AMR for populations around the world relative to other causes of disability and mortality by age, sex, and location over time. In addition, updates to data and results will enable ongoing annual assessment of the incidence of AMR to track progress towards reducing its burden. This aim will benefit from and be supported by similar streams of work within this AMR project that will map the prevalence and incidence of resistance of selected bacteria-antibacterial drug combinations at the highest geographic resolution possible Table . Analyse


The excess mortality in craniopharyngiomas is attributable to their size, site and the traditional surgical approach; aggressive resection predisposes to hypothalamic complications such as obesity, somnolence, thirst disorders and neurocognitive dysfunction. Recently, treatment has been modified to partial resection and radiotherapy. The role of the endocrinologist has expanded from identification and replacement of hormone deficits to include management of hypothalamic disease. Future treatment of craniopharyngioma with neo-adjuvant chemotherapy to minimise surgical resection may improve the outcomes for these patients. There is now a better understanding of the factors that contribute to both the morbidity and mortality associated with craniopharyngioma, which has led to a change in clinical practice and an expansion of the role of the endocrinologist.Craniopharyngiomas are large tumours that extend beyond the sella and suprasellar region into the hypothalamus.2 Surgical resection of these lesions is difficult because of their adherence to surrounding hypothalamic structures. The traditional surgical approach was to attempt gross total resection (GTR), often via a frontal or pterional craniotomy. Aggressive surgery was associated with high rates of panhypopituitarism and diabetes insipidus, morbid obesity, disorders of appetite and thirst, somnolence, poikilothermia and a range of neurocognitive deficits.8 Despite attempts at surgical clearance, rates of tumour recurrence were as high as 40 %.3Although the histology is described as benign, craniopharyngiomas form micropapillary extensions into surrounding hypothalamic tissue, which promote a gliotic reaction.10 Routine management has changed to address some of the morbidities associated with craniopharyngioma.Traditional methods of management of craniopharyngioma are also associated with high standardised mortality, despite adequate replacement of pituitary hormone deficiencies.12 Hypothalamus-sparing surgery in children with craniopharyngioma results in lower rates of obesity than in children who have undergone a traditional surgical approach.11In the last 10 years there has been a change in surgical approach to a modified transsphenoidal resection in parallel with radiotherapy and radiosurgery. It is not possible to conduct large-scale trials for this rare tumour, but published data indicate that less-aggressive surgery is not complicated by an increase in tumour recurrence rates.13 There may be less benefit from growth hormone (GH) replacement therapy in reduction of visceral adiposity and improved lipid profile.14 Although GH replacement does not appear to reduce the mortality associated with craniopharyngioma, GH therapy is safe for these patients and does not lead to tumour recurrence.15 Craniopharyngioma is also associated with higher rates of diabetes insipidus that may be complicated by adipsia or polydipsia.16 It is unclear whether modification of hormone replacement therapy will reduce the morbidity and mortality associated with craniopharyngioma; however, the recognition of the unique abnormalities seen in craniopharyngioma patients does prompt the need for further research into accurate hormone replacement in this cohort.Analysis of large pituitary disease registers has revealed that patients with craniopharyngioma differ from other hypopituitary patient groups in the rates of hormone deficiency and response to replacement therapy. Data show that they have higher rates of pre-receptor activation of cortisone to cortisol by the enzyme 11βHSD1.17 The concerns about bariatric surgery must be carefully weighed against the complications of obesity, on an individual basis. Obesity in craniopharyngioma has also been treated successfully with exenatide (12.8 % weight loss),18 and by the combination of diazoxide and metformin (stabilisation of weight gain).19 It is likely that early intervention to prevent obesity will be most effective; once hypothalamic obesity is established both somnolence and immobility render it difficult to treat. There is a spectrum of sleep disorders in craniopharyngioma including obstructive sleep apnoea (OSA), somnolence and narcolepsy and all exert a negative impact on quality of life. Diagnosis of the underlying sleep disorder is essential for appropriate treatment; OSA may respond to continuous positive air pressure (CPAP) therapy4 and somnolence may respond to melatonin or modafinil.20 Management of thirst disorders is complex; monitoring for dysnatraemia identifies those with hypernatraemic dehydration who require intravenous rehydration and anticoagulation to prevent thrombosis.16 Active management of hypothalamic disease has led to an improvement in quality of life for many craniopharyngioma patients; we routinely screen for sleep and thirst disorders, and adopt a preventative approach to obesity.Better recognition of hypothalamic complications of craniopharyngioma has led to more active management of a number of conditions. Obesity is multifactorial and treatment should encompass a personalised approach. Bariatric surgery may be useful in some patients; a metaanalysis of a study and a number of case reports have shown that such an approach can be effective and safe in halting or reversing weight gain in craniopharyngioma patients with obesity.21 BRAF inhibitors are already in clinical use for melanoma, and a case of response to a BRAF inhibitor, vemurafenib, in a craniopharyngioma patient has been reported.22 A formal clinical trial of this drug class in craniopharyngioma is in development.The ideal therapeutic tool for the team managing craniopharyngioma patients in the future would be a chemotherapeutic agent that could shrink craniopharyngioma tissue away from the hypothalamus and allow a surgical resection without damage to hypothalamic nuclei. The identification of a BRAF mutation in the papillary subtype of craniopharyngioma has revealed an exciting potential target for a chemotherapeutic approach.19It is unlikely that neoadjuvant chemotherapy for craniopharyngioma will entirely abolish hypothalamic complications of this disease, therefore individualisation of the approach to hypothalamic complications in craniopharyngioma patients will be a priority in the future. Identification of those at highest risk for hypothalamic obesity and intervention with either bariatric procedures or pharmacological therapy before the development of morbid obesity has the potential to reduce morbidity.Management of patients with craniopharyngioma has always been a challenge. The role of the endocrinologist has now expanded to meet the needs of these patients beyond the area of hormone replacement in a multidisciplinary and patient-orientated approach. The pooling of data between centres and the development of clinical care recommendations, both within Europe and beyond, will be important for the future well-being of craniopharyngioma patients.


New biomaterials are sought for the development of bioengineered nanostructures. In the present study, electrospun nanofibers have been synthesized by using poly and poly  as building polymers for the first time. To further functionalize these materials, nanofibers of PMVEMA-Ac and PMVEMA-ES containing a conjugated polyelectrolyte  were achieved with both forms maintaining a high solid state fluorescence yield without altered morphology. Also, 5-aminolevulinic acid (5-ALA) was incorporated within these nanofibers, where it remained chemically stable. In all cases, nanofiber diameters were less than 150 nm as determined by scanning and transmission electron microscopy, and encapsulation efficiency of 5-ALA was 97 ± 1% as measured by high-performance liquid chromatography. Both polymeric matrices showed rapid release kinetics in vertical cells (Franz cells) and followed Higuchi kinetics. In addition, no toxicity of nanofibers, in the absence of light, was found in HaCaT and SW480 cell lines. Finally, it was shown that loaded 5-ALA was functional, as it was internalized by cells in nanofiber-treated cultures and served as a substrate for the generation of protoporphyrin IX, suggesting these pharmaceutical vehicles are suitable for photodynamic therapy applications. To a large degree, these advances are due to the development of novel materials that allow the design of nanostructures with physicochemical properties that could overcome the limitations that hinder further progress in biomedicine7. The multiple design possibilities in both (nano)size and (nano)structure presented by this technology make it a very versatile tool with potential for use in various applications and with the ability to be incorporated into almost any biomedical device8. In addition, this technology offers new advantageous uses because of the properties that emerge only when molecules are nanoscopically arranged; properties not present in their macroscopic arrangement9.Nanotechnology has gained new impetus recently thanks to the solutions it offers for both the development of new biomedical applications and the improvement of conventional ones10. From a clinical point of view, these contributions are of great interest because they might improve the efficiency of the therapeutic and diagnostic compounds by, for instance, increasing their circulating half-lives and specificity, and thus reducing the necessary dosage and side-effects6.Among the many applications that nanotechnology can contribute to, nanoencapsulation can add significantly to the protection of pharmaceutical agents against environmental degradation, to site-specific targeted delivery and/or to controlled release14. By using appropriate polymer/s, such assemblies can be designed, for instance, to contain molecules of very different nature, size and water solubility and to control their release and maintain concentrations at therapeutically relevant levels for prolonged periods15. Besides, they can be shaped and size-adjusted to match the requirements of the delivery route/device of choice. With regard to this latter feature, the most popular forms, while versatile, are mainly nanoparticulated ones, although polymer nanofibers are becoming increasingly important because of their unique suitability to medical applications such as tissue scaffolds and dressings17.As to the materials that can be used for this purpose, polymers have the advantage of comprising a large variety of biocompatible and biodegradable molecules, including many suitable for the construction of different types of drug-loadable nanostructures that meet clinical needs, through relatively facile and short protocols17. Different techniques including vapor deposition19, phase separation21, self-assembly24 and centrifugal spinning27 can be used to synthesize such nanofibers, but electrospinning is the most widely adopted one due to its advantages for implementation/commercialization purposes31.The size scale of the nanofibers, similar to that of cellular components, can mimic that of the extracellular matrix of biological tissues, and interactions at the molecular level could be exploited to enhance the therapeutic potential of functional agents loaded within nanofibers31. Electrospun polymer nanofibers are also highly tunable and it is possible to produce 3D structures through simple mechanical modifications in the set-up17. In addition, for drug release applications in particular12, nanofibers generated by electrospinning are highly amenable to loading with molecular cargo, up to 60% w/w in some cases30, and encapsulation yields close to full efficiency32.In general, and in contrast to other techniques, the fabrication of nanofibers by electrospinning is simple, cost-effective and scalable4. Therefore, selecting the most suitable polymeric materials is critical and characterizing a wide variety of compounds is of great benefit to the bionanotechnology community.Another factor that contributes to the versatility and flexibility of the technique is the possibility of using both natural and synthetic electrospinnable polymeric compounds, individually or by combining different polymers, in order to achieve nanofibers with desired physico-chemical and, consequently, functional properties38. These synthetic compounds have been used for medical purposes including encapsulation of pharmaceutical agents in nanoparticles36. Further, electrospun nanofibers made of blends of polymers including PMVE/MA with loading capacity have recently been reported40.Copolymers of alternating methyl vinyl ether and maleic anhydride  and its derived forms, which are sold commercially under the generic trademark Gantrez, are biodegradable molecules that have shown low toxicity and high biocompatibility and display useful bioadhesive propertiesHere, two forms of PMVE/MA (PMVE/MA-Ac and PMVE/MA-ES) polymeric material were assessed for the capacity to create drug-loadable electrospun nanofibers. Also, the distribution of a potential load along the electrospun film was analyzed by loading with either a red- or blue-emitting fluorene-based cationic polyelectrolyte (CPE), as a fluorescent marker. Further, the encapsulation capacity of these electrospun nanofibers was assessed using a clinically-relevant pharmaceutical agent, the photosensitizer 5-aminolevulinic acid (5-ALA), which is widely used in photodynamic therapy (PDT). Its release dynamics and compound stability are analyzed for all combinations of polymeric nanofibers mentioned above (PMVE/MA forms ± CPEs).Initial efforts were focused on the optimization of the conditions to obtain electrospun nanofibers of PMVE/MA derivatives. The concentration of each polymer form seemed to be critical for achieving nanofibers with uniform size and shape. Briefly, preliminary optimization studies performed in our lab led to the selection of the general electrospinning settings described in the materials and methods section, including a polymer concentration of 20% w/w for the initial electrospinning solutions. As a result, nanofibers were obtained with both PMVE/MA forms, but only those based on PMVE/MA-Ac met proposed quality standards (ie continuous nanofibers with uniform size and shape and no bifurcations or thickenings), whereas PMVE/MA-ES ones were irregular and with beads  of nanofibers synthesized under optimal conditions with each PMVE/MA form are shown in Fig. Subsequently, the morphology of nanofibers synthesized under the above conditions but using a polymer blend with the PMVE/MA derivatives as base compounds was described. The chosen companion polymers were fluorene-based CPEs to function as reporters of successful inclusion, detectable through their fluorescent properties. This feature helps to assess the ability of a base polymer to accumulate properties of other compounds in the final nanostructure as well as to check the homogeneity of the blend throughout the entire synthesis process.Optical micrographs of electrospun nanofibers made with all polymer combinations, w/ or w/o 16.7% w/w 5-ALA, are shown in Fig. Changes in the fluorescence spectra of a compound are indicators of its stability. Thus, they were recorded for all polymer blend solutions that generated homogeneous nanofibers. Fluorescence spectra for these blends Fig.  were simin vitro transdermal release methodology was used to analyze single 5-ALA-loaded nanofibers composed of PMVE/MA-Ac . Likewise, treatment with non-loaded (no 5-ALA) PMVE/MA-ES nanofibers was also included. Different concentrations of 5-ALA were also tested to perform a calibration curve for this compound. After 24 h, cell viability was determined by Thiazolyl Blue Tetrazolium Bromide (MTT) assay as described in the material and methods section . In none of the cases did the viability levels drop below 85% with 5-ALA treatment.The viability values obtained for nanofiber solutions revealed that PMVEMA-ES nanofibers do not show significant toxicity levels in the SW480 cell line. For these cells, the lowest percentage of cell viability was 88.3 ± 5.9% when treated with the solution of non-loaded single polymer nanofibers. Whereas SW480 was more sensitive than HaCaT to 5-ALA, the HaCaT cell line was more sensitive to treatment with nanofibers than SW480. In this case, cell viability levels in HaCaT ranged between 64.9 ± 6.5 and 74.2 ± 1.0%, with no significant difference between treatments.It might happen that the conditions during the process of synthesis of the nanofibers may degrade the compounds intended to be encapsulated. Even though no changes were found in the elution peaks of encapsulated 5-ALA in comparison to control 5-ALA samples (data no shown), thus indicating no major changes in the molecular structure, assays to ensure its activity are required. In this sense, the intracellular accumulation of PpIX in cells treated with 5-ALA from electrospun nanofibers would demonstrate the stability of the structure of the encapsulated molecule. Therefore, in an attempt to verify this issue, HaCaT and SW480 cells were treated for 24 h with a solution of 5-ALA-loaded and non-loaded PMVE/MA nanofibers in the same manner as described for determining cell viability in the previous section. CPE-blended nanofibers were not used, as their absorption and/or emission spectra overlap with PpIX  from expected ones (PpIX detection in cells treated with 100 μg/mL of non-encapsulated 5-ALA) in either of the cell lines used  and allows the determination of the relative amount of PpIX that is produced and accumulated in cells. Thus, increased concentrations of 5-ALA up to 100 μg/mL correlated with detected levels of PpIX, with intracellular PpIX accumulation plateauing at this concentration in both cell lines Fig. . As for sed Fig. . SpecifiThe present study also analyzed how the storage conditions inside the PMVE/MA-ES electrospun nanofibers affect the activity of the loaded compound over time. For this purpose, the activity of 5-ALA loaded in nanofibers from two different batches was examined in both cell lines for three months, from the moment of their production. Each time-point therefore corresponded to the mean (±sd) of two experiments performed in triplicate in both cell lines. It was observed that over the course of three months the activity of encapsulated 5-ALA remained stable and did not change significantly from when each batch was synthesized .40, and in none of them was it employed as a single polymer source, but rather as complementary material to confer other characteristics such as biocompatibility, biodegradability or rigidity to the general nanostructure. For instance, electrospun nanofibers were made of a blend of gelatin and PMVE/MA at a 4:1 ratio with a final concentration of 10% and loaded with zinc oxide-containing nanoparticles, as part of a gelatin scaffold designed for wound healing39. In another study, nanofibers loaded with silver nanoparticles were produced by electrospinning a 1:1 w/w polystyrene-PMVE/MA blend and used to coat experimental membranes, providing antimicrobial properties40.This work is the first report of the synthesis and characterization of drug-loadable electrospun nanofibers using a PMVE/MA derivative (either PMVE/MA-Ac or PMVE/MA-ES) as the single polymer source. So far, there are only a few reports of PMVE/MA being used41.In our study, after optimizing general electrospinning settings, in order to synthesize nanofibers with desired morphological (regularity) and size  standards by using these materials, some difficulties were found with PMVE/MA-ES. However, the concentration of the polymers in the electrospun solution was found to be the critical point, which might be explained by changes in the viscosity of these solutions. The significant influence of the viscosity on the morphology and size during the electrospinning process has been reported elsewhere38. In this work, electrospun nanofibers were also generated by blending any of the PMVE/MA forms with fluorene-based CPEs (either HTMA-PFP or HTMA-PFNT), and therefore confirming that they can work as building polymers with assembly capacity for other compounds. The fluorescence properties of CPEs also facilitated their use as reporters to confirm the homogenous distribution, along the nanofibrous electrospun mat, of the molecular components involved and better characterize the morphology of the nanostructures.The mixture of polymeric materials for the creation of nanostructures has the advantage of combining the positive features from each compound involved. Usually, these blends comprise a (building) polymer with the ability to form nanofibers easily and thus serving as support for the integration of other material/s with complementing activities, but which may not be suitable for use as building polymers. As previously discussed, PMVE/MA and its derivatives display a number of interesting abilities suitable for this building role, ie low toxicity, biodegradability and adhesive capacity useful as a vehicle for controlled administration of drugs in epithelia42. This hypothesis, together with the fact that no shift was observed for PMVE/MA-Ac nanofibers when combined with HTMA-PFP, might explain why nanofibers of PMVE/MA-Ac blended with HTMA-PFNT gave no suitable nanofibers when electrospun.Regarding the blends used in this study, none of these fluorene-based CPEs are capable of building electrospun nanofibers by themselves; however, using another electrospinnable polymer species, which assumes the building role, they not only allow nanofiber formation but also provide their fluorescent properties to the main structure. This means that the electrospinning process under described conditions did not modify, significantly at least, their molecular structures. Solid state fluorescence of CPE-PMVE/MA blends showed interesting emission spectra of the corresponding CPEs. In both cases, blue-shift emission occurs with respect to their corresponding spectrum in water solution. This observation has previously been linked to the possibility that the conjugate chains in the polyelectrolytes are ordered or stretched and without forming aggregates17. The ease with which 5-ALA was encapsulated is probably due to its low molecular weight, hydrophilicity and low reactivity. Furthermore, since there were no significant differences in the encapsulation yields obtained for each type of synthesized nanofibers, the encapsulation capacity of PMVE/MA nanofibers seems not to be limited by either the PMVE/MA derivatives, or the complementary copolymers when used. However, assays to determine maximum content loads were not performed since they were focused on a fixed concentration (16.7% w/w) that approximately corresponds to the average concentration already used in approved biomedical products, Levulan® (20%) and Ameluz® (7.8%) in USA and EU, respectively.In terms of encapsulation capacity, in all synthesized nanofibers from this work high encapsulation yields of 5-ALA were obtained (97 ± 1%). In a recent study, poly nanofibers containing 10% w/w 5-ALA were also achieved and, although no data on encapsulation yields were given, activity values were similar between encapsulated and non-encapsulated 5-ALA46 were considered (zero-order (Mt/M∞ = K0 t), first-order (Ln(Mt/M∞) = −K1 t), Higuchi (Mt/M∞ = Kh t1/2) and Korsmeyer-Peppas (Mt/M∞ = K tn) models) in order to adjust transdermal release assay data. From these adjustments, it was found that all nanofibers diffused according to Fick’s law and loaded 5-ALA diffused in a manner more accurately described by a Higuchi model43, which is typified by solid flat matrices. In turn, solutions showed values of n = 0.25–0.23, ie values lower than 0.45, corresponding to a quasi Fickian diffusion, according to Dash et al.46. The stabilization before 3 h and high release rates in the first stages (30 min) observed for all release profiles make these polymers, when nanostructured as nanofibers, highly effective delivery systems for compounds such as 5-ALA, which has a half-life at physiological pH and 50 °C of about 3 h 47. Among them, PMVE-MA-ES nanofibers appeared to be the most efficient vehicles since they reached up to 100% release of 5-ALA after 3 h.From the pharmacological point of view, four classical kinetic release models48. These results are consistent with those obtained in vitro showing that encapsulated 5-ALA is able to penetrate cells and be used to produce the PpIX photosensitizer just as non-encapsulated 5-ALA is, in concordance with the results of Yoo et al.17. In addition, our study demonstrates that 5-ALA lability47 is prevented during long periods since its encapsulation into PVME/MA-ES nanofibers helps to maintain its stability and biological activity for at least 3 months under our storage conditions.In this sense, the quantification by HPLC of encapsulated 5-ALA, when analyzing either its release kinetics or encapsulated yields, also reveals information on the stability of the encapsulated compound. Possible modification or degradation events on the molecule may be associated to changes in the elution dynamics of the original compound. After examining and comparing the elution profiles, no differences were found between loaded and non-loaded 5-ALA, indicating that the encapsulation process and the compounds used for this purpose did not seem to affect this compound, congruent with the fact that electrospinning is, so far, the most popular methodology to create drug-loadable nanofibers50. Together with the fact that the biocompatibility of these polymers in nanofiber form37 was also partially confirmed in this work, since no significant effects on viability were observed when treating sensitive cell lines with them, we consider that electrospun nanofibers of PMVE/MA forms are effective vehicles for topical delivery of therapeutics. Nevertheless, further in vivo assays will be required in order to assess their actual potential.Consequently, we propose that encapsulation into these electrospun nanofibers might be applicable to other compounds already demonstrated to be suitable for encapsulation by PMVE/MA nanostructuresN,N,N-trimethylammonium)hexyl]-2,7-(fluorene)-alt-1,4-phenylene} bromide  and poly-{-2,7-(fluorene)-alt-1,4-} bromide  were synthesized and characterized in our laboratory as previously reported by Molina et al. (2009)51 and Kahveci et al. (2016)52, correspondingly. For the synthesis of nanofibers, both forms derived from PMVE/MA, PMVE/MA-Ac (Mw: ~216 kg.mol−1) and PMVE/MA-ES (Mw: ~130 kg.mol−1), were purchased from Sigma-Aldrich . The loadable pharmaceutical agent chosen, 5-ALA, was also purchased from Sigma-Aldrich. Chemical structure inputs of all these compounds, as main components of synthesized nanofibers, are shown in Fig. Both fluorene-based CPEs, poly{[9,9-bis with L-glutamine and sodium bicarbonate (DMEM), fetal calf serum (FCS), 5000 units penicillin – 5 mg/mL streptomycin solution, phosphate-buffered saline (PBS), polystyrene culture flasks and 96-well plates, were purchased from Sigma-Aldrich as well.MTT, ethanol, anhydrous isopropanol, hydrochloric acid and dimethyl sulfoxide (DMSO) were obtained from Merck. Milli-Q water was obtained from a Synthesis A10 system (Millipore). PpIX was purchased from Echelon Biosciences .2O with constant stirring during 1 h. For CPE-PMVE/MA blended nanofibers, 7.5 mg of either HTMA-PFP or HTMA-PFNT in DMSO were dissolved per gram of previously described PMVE/MA solutions. Those with HTMA-PFP were stirred for 20 h and HTMA-PFNT ones for 48 h in dark.Different solutions were prepared, all at room temperature (RT), depending on the materials used for each type of electrospun nanofiber synthesized. To obtain unloaded single-polymer nanofibers, 20% w/w PMVE/MA-Ac  and 25% w/w PMVE/MA-ES  solutions were prepared by dissolving in H2O) was added to polymer solutions (of PMVE/MA derivates with or without CPEs) to reach a concentration of either 33.2 mg/mL or 41.5 mg/mL for PMVE/MA-Ac or PMVE/MA-ES formulations, respectively. Thus, 5-ALA final concentration was 16.7% w/w respect to the amount of both PMVE/MA derivate. All of these solutions were prepared in darkness prior to use.For the synthesis of 5-ALA-loaded (single and blended) nanofibers, all solutions were prepared as previously described for each type of nanofibers. 5-ALA . For the assays to measure cell viability, a 5 mg/mL MTT stock solution in PBS was prepared and stored at −20 °C until used.The lysis buffer, used for lysing and solubilizing cellular components prior to PpIX determination assays, was composed of 10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Triton X-100, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol and 0.1% sodium dodecyl sulfate (SDS).PpIX stock solution was prepared at a concentration of 750 µM in 35% v/v HCl, from which there were prepared 7.5 µM working solutions in hydrochloric acid as well.The electrospinning setup consisted of a disposable 2 mL plastic syringe (Becton Dickinson), which was filled with the polymeric solutions, coupled to a blunt end stainless steel hypodermic needle 316 (20 Gauge) (Sigma-Aldrich) which is connected to the positive output of a high voltage power supply Series FC . The flow rate was controlled by means of a syringe infusion pump KDS 100 Series (KD Scientific). A surface covered with aluminum foil, and located 10 cm from the tip of the needle, usually worked as collector system. Common glass slides  for fluorescence microscopy and solid state fluorescence spectroscopy assays or copper TEM grids with 3 mm of diameter  for TEM analysis were coated instead for these particular applications. The polymeric solutions were electrospun at 15 kV using a flow rate of 0.5 mL/h. The thickness of the final mats was controlled by modulating the deposition time. The whole process was carried out in darkness and at RT. The relative humidity seemed not to be critical under these conditions since the process worked satisfactorily at levels ranging 35–60%. Finally, in order to evaporate solvent excess the membranes were kept in a fume hood overnight. Synthesized nanofibers were then stored protected from light at RT and dry atmosphere until used.The morphology and size of nanofibers were analyzed by TEM with a Jeol 1011  apparatus. The average diameter of the different types of nanofibers was obtained by using the image processing and analysis software Image J  on TEM images. SEM images were taken with a JEO 6100  apparatus.Likewise, in order to confirm and characterize the integration of CPEs to the nanofiber structure, nanofibers were observed and photographed by using a Leica DMI 3000B inverted microscope with a 63× objective and equipped with a Leica EL6000 compact light source and a Leica digital camera DFC3000G.The presence of CPEs in synthesized nanofibers as well as their fluorescence properties associated to imaging abilities were analyzed by solid state fluorescence spectroscopy with a QuantaMaster steady-state spectrofluorimeter . For this purpose, its solid state sample holder was set at 340° and 3.25 mm and the emission fluorescence spectra were obtained by exciting the nanofibers on a glass slide at 370 and 510 nm wavelengths. Each spectra was registered from the average of three different positions of the material.in vitro using Franz diffusion cells (PermeGear®). Cellulose acetate membranes Cuprophan® (Medicell) were mounted on the Franz cells receptor chambers, which were connected by a 9 mm diameter  cavity to their corresponding donor compartments. The cell receptor was filled with a 5 mL volume of 1 mM phosphate buffer (pH 7) that was in contact with the upper membrane, which was allowed to equilibrate for 30 min. Then, nanofiber mats were cut into pieces of 0.64 cm2, weighted, arranged facing upward into the donor and wetted with 150 µL of 1 mM phosphate buffer (pH 7). The corresponding viscous polymeric solution with 5-ALA used to synthesize each type of nanofiber was used as reference. Continuous magnetic stirring of the volume in the receptor chamber was kept at 600 rpm during the whole process. The system, comprising six Franz cells, was connected to a thermostatic bath (Selecta Digiterm-100) which maintained temperature at 32.0 ± 0.5 °C like the temperature at the skin surface53. An aliquot of 400 μL in the receptor compartment was removed at each settled time-point after drug application. Withdrawn volumes were immediately replaced with fresh receptor buffer. This assay was performed twice for all nanofiber types using a different production set each time. Samples were analyzed by HPLC immediately after their collection to avoid 5-ALA degradation in polar solutions.The cutaneous absorption dynamics of 5-ALA loaded into different experimental electrospun PMVE/MA types of nanofibers were analyzed 2O with 0.1% v/v trifluoroacetic acid and 2% acetonitrile with 0.1% v/v trifluoroacetic acid, a flow rate of 1 mL/min and an injection volume of 10 μL, similarly as it is described in previous studies54.In order to assess the amount of 5-ALA loaded into the nanofibers and diffused in the skin permeability assays as well as any degradation sign of the molecule, the detection of 5-ALA was optimized using HPLC with a Merck/Hitachi HPLC system and an Alltech 3300 Evaporative Light Dispersion (ELSD) detector (Grace Davison Discovery Sciences) coupled to it (ELSD-HPLC). The isocratic mobile phase used was 98% H2) of greater than 0.999  previously (n = 3). The drug concentration was measured as µg of detected 5-ALA per µg of total theoretically loaded 5-ALA and represented as percentage.By using these settings, a standard curve of detected 5-ALA, previously dissolved in 1 mM phosphate buffer (pH 7) with final concentrations ranging 20–250 μg/mL, was performed by ELSD-HPLC before running the experimental samples. A quadratic correlation was found between concentration and detected peak area in the range of selected concentrations with regression coefficient values  and the human adenocarcinoma cell line SW480 (ATCC® Ref.: CCL-228™) (gently provided by Dr. Miguel Saceda) from colon tissues, were used for the The potential toxicity on tested cell lines of all combinations of nanofibers as well as of their components individually was analyzed by measuring the changes in cell viability by MTT assay. Briefly, cells at 90–100% confluence, which were seeded in 96-well plates 24 h before, were treated with different concentrations of each type of nanofiber and compound (previously dissolved in 1 mM phosphate buffer (pH 7)) in cell culture media for 24 h (100 µL/well). Then, treatments were replaced with fresh cell culture media (100 µL) with 0.5 mg/mL of MTT (from 10 × concentrated stocks). Cells were then incubated for 2 additional hours, media carefully removed and the colored formazan product was dissolved in 100 µL of DMSO and measured at 570 nm (reference at 620 nm) with a SPECTROstar® Omega absorbance microplate reader . The optical density was directly correlated with quantity of cells and expressed in percentages relative to control group (untreated cells). All experiments were performed in triplicate and results are shown as means with standard deviation (sd) from three different experiments.55. In order to check the functionality of the 5-ALA loaded into the different types of nanofibers, the amount of PpIX converted from that 5-ALA internalized into cells was measured as previously described by Jin Ju Yoo et al.17. To this end, cells at 90–100% confluence, which were seeded in 24-well plates 24 h before, were treated with different concentrations of each type of nanofiber and 5-ALA (previously dissolved in 1 mM phosphate buffer (pH 7)) in cell culture media for 24 h. Then, cells were lysed with 100 μL/well of lysis buffer, dispensed in black 96-well plates and the accumulated PpIX in cells was measured with a Cytation 3 cell imaging multi-mode plate reader  at 412 nm (excitation) and 665 nm (emission) wavelengths  higher than 0.95 for both cell lines in all experiments  and singlet oxygen in PDTnts Fig. . All expt-test or one-way ANOVA (depending of the variables of the experiment). GraphPad Prism v5 software was used for performing the statistical analysis. Both GraphPad Prism v5 and Microsoft Excel were used for creating the graphs.Data were statistically analyzed by either unpaired All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Molecular spins have become key enablers for exploring magnetic interactions, quantum information processes and many-body effects in metals. Metal-organic molecules, in particular, let the spin state of the core metal ion to be modified according to its organic environment, allowing localized magnetic moments to emerge as functional entities with radically different properties from its simple atomic counterparts. Here, using and preserving the integrity of transition metal phthalocyanine high-spin complexes, we demonstrate the magnetic doping of gold thin films, effectively creating a new ground state. We demonstrate it by electrical transport measurements that are sensitive to the scattering of itinerant electrons with magnetic impurities, such as Kondo effect and weak antilocalization. Our work expands in a simple and powerful way the classes of materials that can be used as magnetic dopants, opening a new channel to couple the wide range of molecular properties with spin phenomena at a functional scale. Molecular magnets are molecules with an inherent non-zero spin that can exhibit magnetic ordering. Here, the authors show that such molecules can change the many-body ground state of nonmagnetic metals at a functional scale with magnetic phthalocyanines. Accordingly, when MPc molecules are placed on a metallic substrate, the resulting magnetic moment is short-ranged and typically requires localized techniques, such as scanning tunneling microscopy, to study its inherent local spin physics11Metal-organic molecules have been explored for their applications in (opto)electronics134567910Magnetic doping has witnessed a surge of interest in the past decade4S=1/2 arising from the oxidation state of the core metal ion, Cu2+  phthalocyanine (CuPc), a paramagnetic metal-organic molecule with spin angular momentum in situ large area fabrication of metal/molecule/metal stacks in ultra-high vacuum conditions (see Methods). Our samples consist of two Hall bars, one of Au (100 Å)/MPc (t)/Au (100 Å), where t is the nominal thickness of the MPc, and the other is a bare-gold control reference with nominal thickness of 200 Å  is the resistivity at T=150 K of the gold reference sample, and ρ(150 K) the resistivity at T=150 K of the Au/CuPc/Au stack. Rs) and residual resistivity ratio (RRR) for each CuPc sample is included in the T2 dependence at high temperatures, confirming the good quality of the deposited goldTmin the resistivity of the CuPc samples increases monotonically down to 2 K for all thicknesses. Tmin shifts towards higher temperatures whereas the resistivity upturn increases in magnitude roughly proportionally with the molecular thickness, although with some deviations that can be explained by the dispersion of the deposition conditions. The observed dependence of Tmin and of the resistivity with temperature and molecular thickness points out to the presence of Kondo effectT=0 K, ρ0, as well as Tmin  for each CoPc sample is included in the We have extended our experiments with another related molecule, cobalt (II) phthalocyanine (CoPc) , for obt1/2 ref. . In the 1/2 ref.  and a ch1/2 ref. , clearlyodel see  yield TK.6±0.2 K . This reIn conclusion, we have demonstrated the doping of a gold metal thin film with CuPc and CoPc molecular magnetic impurities. Using a simple device architecture and varying the thickness of the deposited MPc, we modulate the magnetic interaction and extend it from a molecular level to a functional scale. Measuring the metal we are able to sense the molecular magnetism. Metal-organic molecules, being versatile for the incorporation of transition metals (magnetic and nonmagnetic), suggest wider limits for the range of materials that can be used as magnetic dopants. As long as a molecular spin state can be stabilized in the metal host, transition metal free organic radicals, such as trismethyl, can also be sought for molecular spin doping, as well as molecules with higher spin states. Moreover, by extending the spin state, metal organic molecules might be enhancing the interaction of the magnetic impurities and the host material, giving rise to a stronger coupling. Because of the intrinsic properties of organic molecules, such as photosensitivity, molecular spin doping can be used as a new platform to combine magnetic and optical effects. This can be extended to the wide range of properties that organic molecules exhibit. Device applications that envision the coupling of the molecular properties to the spin state of the molecule can lead to new approaches to explore quantum information at a functional scale.2 Si/SiO2 substrates, where before gold evaporation an adhesion layer of 30 Å of aluminum was deposited by effusion cell evaporation and plasma oxidized for 6 min. A layer of 100 Å of gold was then deposited by e-beam evaporation using shadow masking techniques for the simultaneous patterning of two equal Hall bars on the chip carrier at a rate of 0.1 Å s−1. Without breaking vacuum, 10 to 50 Å of high purity (>99%) copper (II) phthalocyanine acquired from a commercial supplier (Sigma-Aldrich) were evaporated at a rate of 0.1 Å s−1 on one of the Hall bars using shadow masking. Again, in situ, 100 Å of gold were deposited on top of the two Hall bars at the low rate of 0.1 Å s−1. All the evaporations were performed in situ and in ultrahigh vacuum conditions (10−8–10−9 mbar), with the reported thickness values calibrated with depositions onto Si/SiO2 substrates.The devices were fabricated on 10 × 10 mmW of 1 mm and a channel length L of 2.25 mm were considered for the purpose of normalized resistivity and magnetoconductance calculations.Transport characterization was carried out with standard four-probe measurements, using Keithley 6221 nanovoltmeter and Keithley 2182 DC current source, with an excitation current of 10 μA, in a Quantum Design physical property measurement system. A channel width The data that support these findings are available from the corresponding author (L.E.H.) upon reasonable request.How to cite this article: Atxabal, A. et al. Spin doping using transition metal phthalocyanine molecules. Nat. Commun.7, 13751 doi: 10.1038/ncomms13751 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary References