Fatigue is one of the most pervasive symptoms of multiple sclerosis (MS), and has engendered hundreds of investigations on the topic. While there is a growing literature using various methods to study fatigue, a unified theory of fatigue in MS is yet to emerge. In the current review, we synthesize findings from neuroimaging, pharmacological, neuropsychological, and immunological studies of fatigue in MS, which point to a specific hypothesis of fatigue in MS: the dopamine imbalance hypothesis. The communication between the striatum and prefrontal cortex is reliant on dopamine, a modulatory neurotransmitter. Neuroimaging findings suggest that fatigue results from the disruption of communication between these regions. Supporting the dopamine imbalance hypothesis, structural and functional neuroimaging studies show abnormalities in the frontal and striatal regions that are heavily innervated by dopamine neurons. Further, dopaminergic psychostimulant medication has been shown to alleviate fatigue in individuals with traumatic brain injury, chronic fatigue syndrome, and in cancer patients, also indicating that dopamine might play an important role in fatigue perception. This paper reviews the structural and functional neuroimaging evidence as well as pharmacological studies that suggest that dopamine plays a critical role in the phenomenon of fatigue. We conclude with how specific aspects of the dopamine imbalance hypothesis can be tested in future research. Fatigue is a common symptom in multiple sclerosis (MS), with up to 90% of individuals with MS reporting fatigue . MoreoveFatigue is characterized by a lack of energy, feelings of exhaustion that are unaided by sleep, and the perception that one is unable to perform mental and physical activities . AlthougWe propose that fatigue arises due to a dopamine imbalance within the central nervous system (CNS). One of the ultimate goals of this review is to investigate the evidence that supports the dopamine imbalance hypothesis by examining studies showing structural and functional abnormalities in areas enervated by dopamine and clinical trials showing alleviation of fatigue after dopamine medication.non-motor functions of the basal ganglia” . Com et al. –45. TakeA potential difficulty in examining neural correlates of fatigue during task-related functional neuroimaging studies is the assessment of fatigue itself. In most of the clinical studies, fatigue is assessed with a self-report questionnaire, such as the FSS  or the Ml-carnitine, and placebo) showed amantadine to be successful in reducing fatigue , it is likely that it has a similar effect on fatigue. That is, fatigue might result from too much or too little dopamine in the brain.+ cells (a type of T cells) express the D3 receptor that contributes to the destruction of dopamine neurons in the SN and generate interferon-γ, a compound that proliferates inflammation and prevents dopamine synthesis , MS is considered to be an autoimmune disorder of the CNS. To a large extent, the immune system depends on the functioning of the leukocytes or white blood cells. T cells are a type of white blood cell that produce an immune response, i.e., they are activated when the body needs to fight an infection. In autoimmune diseases, including MS, T cells proliferate and attack healthy cells , passingynthesis , 70, 71.ynthesis , 17, 72.+ T cells through the blood–brain barrier. More importantly, recent findings also show that IFNβ treatment leads to increased production of dopamine  benzylamine to investigate dopamine storage capacity and serotonin transmission, respectively, in the brain of on-medication PD patients with and without fatigue (A few studies in clinical populations provide evidence in support of the serotonin hypothesis, suggesting that increased levels of serotonin might lead to fatigue –84. For  fatigue . This PEClearly, delineating the neurobiological processes underlying such a complex phenomenon as fatigue will not be simple. It is likely that the interaction of several neurotransmitters systems is involved in fatigue. Given the large body of evidence showing the mesocorticolimbic network and, in particular the basal ganglia involvement in fatigue, it is difficult to negate the involvement of serotonin neurons that also innervate the basal ganglia. However, recent studies show that decreased functioning of serotonergic receptors leads to increased functioning of dopaminergic neurons and dopamine release [for review see Ref. ]. TherefRecently, it has been suggested that inflammatory cytokines, compounds released by the cells of the immune system during inflammation, might be the cause of fatigue. The cytokine hypothesis developed based on the observation that fatigue co-occurs in individuals who have inflammatory biomarkers, such as tumor necrosis factor-α, interleukin-1, interleukin-6, and interferon-γ , 87–89.per se. Indeed, a recent study showed that methamphetamine, a psychostimulant that affects dopamine and, to a lesser extent, serotonin neurons, reduces frontocortical inflammatory cytokine levels (A large body of evidence in support of the cytokine hypothesis comes from animal literature , 89. Inde levels , while oe levels . Thus, sIn this review, we propose that fatigue arises due to an imbalance of dopamine, a modulatory neurotransmitter, in the CNS and the immune system. Based on the evidence cited above and building upon a previous framework on fatigue , we propGiven this framework, future research should be geared toward testing specific aspects of the hypothesis. While studies in cognition show that dopamine has a modulatory influence on cognitive performance, clinical trials in fatigue so far only show that dopaminergic medication decreases fatigue. Since fatigue and cognitive functions such as working memory and attention rely on a similar neural network, it is likely that dopamine has a modulatory effect on fatigue as well. Future neuroimaging and pharmacological research is needed to directly test whether this is the case. Thus, an important question is, does fatigue increase as dopamine levels increase above optimal levels? Another question that should be investigated is whether dopamine agonist administration decreases on-task or “state” fatigue in conjunction with performance improvement. This will help in linking objective measures of performance, which have been shown to be affected by dopamine, with subjective on-task fatigue ratings. Pharmacological studies should evaluate the effect of a dopaminergic medication not only in comparison to a placebo but also in comparison with non-dopaminergic medications, to show whether fatigue is differentially affected by a dopaminergic medication versus, for example, serotonergic medication.Neuroimaging studies should focus on manipulating mesocorticolimbic network activity in controlled experimental settings. This would allow researchers to observe network functioning in fatigued individuals and to answer specific questions about the time course of network activation in a controlled environment. Investigating the time course of network activation during task performance would reveal whether it correlates with on-task fatigue. It is also worth looking at whether the increased connectivity observed after dopamine medication, which has been shown to lead to an increase in working memory performance, is associated with fatigue reduction. Answering these questions will provide valuable evidence about the underlying mechanisms of fatigue, and will ultimately allow us to develop targeted treatments for fatigue.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fneur.2015.00052/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.


Phenotypic plasticity allows animals to modify their behavior, physiology, and morphology to adapt to environmental change. The global pest, the desert locust, shows two extreme phenotypes; a solitarious phase that is relatively harmless and a gregarious phase that forms swarms and causes extensive agricultural and economic damage. In the field, environmental conditions can drive isolated animals into crowded populations and previous studies have identified the biogenic amine serotonin as a key determinant of this transition. Here we take an integrated approach to investigate the neurochemical, physiological, and behavioral correlates defined by a laboratory based paradigm that mimics facets of swarm break down as gregarious locusts become isolated. Following isolation there was an increased propensity of locusts to avoid conspecifics, and show a reduced locomotion. Changes in choice behavior occurred within 1 h of isolation although isolation-related changes progressed with increased isolation time. Isolation was accompanied by changes in the levels of the biogenic amines dopamine, octopamine, and serotonin within the CNS within 1 h. Dopamine levels were higher in isolated animals and we focused on the role played by this transmitter in synaptic changes that may underpin solitarization. Dopamine reduced synaptic efficacy at a key central synapse between campaniform sensilla (CS) and a fast extensor tibiae motor neuron that is involved in limb movement. We also show that dopamine injection into the haemocoel was sufficient to induce solitarious-like behavior in otherwise gregarious locusts. Further, injection of a dopamine antagonist, fluphenazine, into isolated locusts induced gregarious-like behavior. This highlights that dopaminergic modulation plays an important role in the plasticity underpinning phase transition and sets a context to deepen the understanding of the complementary role that distinct neuromodulators play in polyphenism in locusts. The phenotype of an animal can be influenced and modified by cues in the external environment. These allow animals to adapt to changes in their environment and may represent a fundamental component of evolutionary change  taken from crowded and solitarious colonies maintained at the University of Southampton. Gregarious locusts were reared in a controlled environment room in metal cages (39 × 39 × 45 cm) at a density of approximately 50–100 locusts per cage, and were fed daily on seedling wheat and dry oats. For experiments, 10 four-day-old adult locusts, post final molt, were moved to a small cage (20 × 20 × 20 cm) to ensure continued crowded conditions. These locusts were housed within the same gregarious colony room at a temperature 30 ± 1°C under a 12D: 12L h (Dark: Light) cycle, and fed daily with fresh seedling wheat and oats.Experiments were carried out using adult desert locusts, 3/min.In a separate controlled environment room, maintained at a temperature 30 ± 1°C under a 12D: 12L (Dark: Light) cycle, 10 four-day-old adult gregarious locusts taken from the crowded colony were isolated in a way to prevent physical, visual and olfactory cues between locusts. Individual locusts were placed in plastic cages (20 × 20 × 20 cm), which were surrounded with patterned paper to prevent visual cues from other locusts and to provide each insect with a salient environment. Small holes were drilled in the bottom of the cage and a small hole (7 mm diameter) was drilled at the top of the cage. Plastic tubes (8 mm outside diameter × 1 mm thickness) were inserted into the top hole via Portex connectors. Tubes were fed to a Perspex air collection box from all cages and the air drawn from the room through the holes in the bottoms of the cages and out through the tube at the top and expelled from the colony using two air pumps  housed outside the colony room. Airflow rate was measured using a glass flow meter  at 126 ± 6.7 cmAn assay was designed to study the behavioral responses of individual gregarious or isolated locusts based on a number of measures described by Roessingh et al. , includiA locust was placed ventral-side-up in a cleft in a modeling clay platform. The legs were fixed using more clay, leaving the tibia of the right hind leg free. The cuticle of the ventral thorax and underlying air sacs were removed. The metathoracic ganglion was exposed and supported on a wax-coated silver platform and restrained with small pins to isolate it from the movements of the thorax. The posterior connectives of the metathoracic ganglion were cut while the anterior connectives and lateral nerves remained intact. During the dissection and recording process, the thoracic cavity was continuously flushed with locust saline. A pair of 50 μm copper wires, insulated except at the tips, was inserted through small holes in the femur into the extensor muscle of a hind leg to stimulate it, and to evoke antidromic spikes in the fast extensor tibia motor neuron (FETi). Microelectrodes were made from borosilicate glass capillaries (1 × 0.58 mm interior diameter) pulled on a pipette puller  and filled with 3 M potassium acetate, and with DC resistances of 50–80 MΩ. Before recording, the sheath of the ganglion was treated directly with protease (Sigma type XIV) for 1 min. A microelectrode was then inserted through the sheath and into the cell body of FETi. Intracellular recordings were made with the use of an Axoclamp 2A amplifier   as an organic modifier were added to the mobile phase solution. The flow rate was maintained at 0.7 ml/min. The chromatographs were acquired using the computer program PowerChrom . The supernatants of samples were injected directly onto the HPLC column. After the acquisition, they were processed to obtain the level of biogenic amines in the same sample by the ratio of the peak area of substances to the internal standard DHBA. We used a standard mixture for quantitative determination that contained amines, precursors and metabolites. Twenty compounds at 100 ng/ml each were DL-3, 4-Dihydroxy mandelic acid (DOMA), L-β-3,4-Dihydroxyphenylalanine (DOPA), L-Tyrosine (Tyr), N-acetyloctopamine (Nac-OA), (-)-noradrenaline (NA), 5-Hydroxy-L-tryptophan (5-HTP), (-)-adrenaline (A), DL-Octopamine (OA), 3,4-Dihydroxybenzylamine , 3,4-Dihydroxy phenylacetic acid (DOPAC), N-acetyldopamine (Nac-DA), 3,4-Dihydroxyphenethylamine (DA), 5-Hydroxyindole-3-acetic acid (5-HIAA), N-acetyltyramine (Nac-TA), N-Acetyl-5-hydroxytryptamine (Nac-5HT), Tyramine (TA), L-Tryptophan (Trp), 3-Methoxytyramine (3-MTA), 5-Hydroxytryptamine (5-HT), 6-Hydroxymelatonin (6-HM). Nac-OA Nac-DA and Nac-TA were synthesized by Dr. Matsuo . All other substances were purchased from Sigma-Aldrich.Individual locusts were quickly frozen using liquid nitrogen and the brain and metathoracic ganglion dissected out in cooled standard saline. Each ganglion was then placed in a micro glass homogenizer and homogenized in 50 μl of ice-cold 0.1 M perchloric acid containing 5 ng of 3, 4-dihydroxybenzylamine (DHBA) as an internal standard. After centrifugation of the homogenate , 40 μl of supernatant was collected. Biogenic amines in the sample were measured using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). The HPLC-ECD system was composed of a pump , an auto-sample injector  and a C18 reversed-phase column  heated to 30°C in the column oven. A glass carbon electrode  was used for electrochemical detection . The detector potential was set at 870 mV vs. an Ag/AgCl reference electrode, which was also maintained at 30°C in a column oven. The mobile phase containing 0.18 M chloroacetic acid and 16 μM disodium EDTA was adjusted to pH 3.6 with NaOH. Sodium-1-octanesulfonate at 1.85 mM as an ion-pair reagent and CHEL) of the tibia:F is the weight used, L is the length of the tibia between the holders, B is the tibial displacement and 48 is a constant.To measure cuticle stiffness the tibia was cut from the hind leg and fixed dorsal-side-up on steel holders using cyanoacrylate glue. Two weights (20 and 30 g) were hung sequentially from the middle of the tibia using cotton thread and the resulting bending distances of the tibia determined from digital images. The Beam bending equation was used to calculate the bending stiffness  thin film load cell was placed in contact with the tibia and was used to measure the kick force. A pair of 50 μm copper wires, insulated except at the tips, was inserted through small holes into the extensor muscle of a hind leg to stimulate the muscle and evoke a kick. All recordings were written directly to a computer with a CED 1401 A/D converter and displayed using Spike 2 software.For pharmacological analysis dopamine hydrochloride and two dopamine receptor antagonist, chlorpromazine hydrochloride and fluphenazine dihydrochloride, were obtained from Sigma-Aldrich, (UK) and dissolved to final concentrations in standard locust saline. Dopamine was tested at concentrations of 1, 5, and 10 mM, chlorpromazine at 0.1, 1, and 5 mM, and fluphenazine at 100 μM, 500 μM, and 5 mM at concentrations used in previous studies  was dissolved in standard locust saline to make a 422 mM solution. 4 μl of this solution was injected directly into the thoracic haemocoel of each locust via the ventral abdomen between the second and third abdominal segments using a microsyringe , following drug concentration, volumes, and methods published elsewhere   = 12.61, P = 0.0003, n = 16 gregarious, n = 19 isolated]. Following isolation for 4 h, 9 of 30 locusts were attracted to the stimulus group, 16 avoided the stimulus group, while 5 remained in the release chamber. By comparison, 17 of 29 age-matched gregarious locusts were attracted to the stimulus group, while 12 avoided the stimulus group. Statistical analysis again showed that there was a significant difference in the choice behavior between the two groups after 4h in isolation   = 13.47, P = 0.0002, n = 20 gregarious locusts, and n = 18 isolated locusts]. Finally, following isolation for 72 h 1 of 10 isolated locusts avoided the stimulus group while 9 remained in the release chamber. By contrast, 5 of 10 age-matched locusts were attracted to the stimulus group, four avoided the stimulus group and one remained in the release chamber. Statistical analysis again showed that there was a significant difference between behavioral choices of the two groups . The greater the time in isolation the greater the number of locusts remaining within the release chamber for the 10 min test period   compared to gregarious locusts  (Students ) Figure .T-test, P = 0.58, n = 13 gregarious and n = 11 isolated locusts). Analysis of walking showed that gregarious locusts had significantly higher mean . These differences in walking velocity reflect the times the two groups took to cross the arena with gregarious locusts reaching the end line in significantly shorter times of 42.5 ± 11.2 s compared to 120.8 ± 28 s in isolated animals  and isolated locusts covering 234 ± 20 mm  leg by the metathoracic ganglion). Given the importance of the hind leg, and exteroceptors on it, in initiating gregarisation . Interestingly, the levels of serotonin (5-HT) were also significantly higher in locusts isolated for as little as 1 h and, as for dopamine, this elevation in levels was maintained for up to 72 h in isolation    Figure . Pair-wi) Figure . Pair-wi) Figure . Pair-wi) Figure .Changes in the absolute and relative levels of these four amines were also found in the brains of animals isolated for 24 h although in this neural structure the serotonin levels were reduced in the animals subjected to isolation  Figure . FETi isn = 12) in gregarious locusts was reduced to 1.98 ± 0.22 mV (n = 12) in isolated locusts . While isolation had no effect on spike height it led to a significant reduction in spike duration (measured from the start of the spike to where it returned to the same voltage) following 72 h in isolation, from 32.92 ± 1.7 ms (n = 12) compared to 41.22 ± 2.7 ms in gregarious locusts    Figure . Althougin vivo. In gregarious locusts EPSP amplitude decreased significantly after 5 min perfusion of dopamine with increasing concentrations causing greater reductions in amplitude  . Dopamine also significantly reduced the amplitude of the CS-FETi EPSP in locusts isolated for 72 h  by superfusion over the ganglion  Figures . The effL. migratoria by the Kang group [15]. However, we found that CPZ had a deleterious effect on locusts, by rapidly abolishing neural activity, with many locusts dying, even at concentrations previously used elsewhere  = 3.15, P = 0.034]. The co-application of dopamine and fluphenazine antagonized the increase in EPSP amplitude caused by dopamine alone . The effects of dopamine were reversed following a 5 min wash in normal saline. Further analysis of the effect of fluphenazine on FETi spike duration showed that 500 μM fluphenazine alone had no effect   Figure .These results show that dopamine applied to the thoracic ganglia leads to changes in EPSP amplitude and spike duration in FETi, similar to those caused by isolation. The results we obtained using the dopaminergic antagonist suggest there is an intrinsic modulatory role for dopamine in the metathoracic ganglion.in vivo, we then asked how dopamine affected the behavioral choices of long-term crowded locusts isolated for 3 days. Dopamine (4 μl of 422 mM dopamine) or saline (as a control) were directly injected into the thoracic haemocoel and locusts placed in isolated or crowded colonies for 1 h before experimentation. Results showed that for gregarious locusts 60% of control animals injected with saline were attracted to the stimulus group (33 of 55) while 40% avoided them (22 of 55)  = 0.004, n = 11 control groups, 5 dopamine groups (each group five locusts), P > 0.05]. Post hoc statistical analysis showed that there were significant differences in choice behavior between the control and dopamine treated gregarious locusts . Moreover, there was also a statistically significant difference between the control and dopamine treated isolated locusts , with statistically more dopamine treated animals avoiding the stimulus group . Post-hoc analysis showed there was no significant difference between the control and the treatment animals within gregarious locusts , while there were significant differences between the isolated locusts that were injected with fluphenazine and control locusts . There were also significant differences between the control gregarious locusts and the control isolated locusts   Figure .Thus the presence of the agonist caused a significant shift in the proportion of animals that exhibited a tendency to avoid the cage of locusts in the test arena, consistent with the idea that dopamine acts to drive solitarious-like behavior expressed in this paradigm. In a similar way, the injection of the dopamine antagonist fluphenazine was sufficiently efficacious to drive animals into a solitarious-like state despite being housed in a continuing crowded environment Figure . These rPhenotypic plasticity in locusts is a complex process in which key sensory modalities are activated to drive characteristic behavior in the natural environment. Considerable insight into the biological basis of phenotypic plasticity and the formation of swarms has been achieved at the level of field investigation, however recent attention has utilized laboratory-based manipulation to mimic the environmental conditions that trigger and mediate the plasticity. These investigations have highlighted how the expression of behavioral plasticity is underpinned by physiological, neurochemical and molecular mechanisms (Matheson et al., Physiological changes are known to accompany gregarisation in desert locusts (Matheson et al., While direct injection of dopamine into the thoracic haemocoel was sufficient to cause gregarious locusts to show solitarious-like behavior, it is clear from our biochemical analysis of a number of key amines in the thoracic nervous system that a suite of changes underpins phase transition. The changes we find are also consistent with previous physiological observations made against these amines that can now be discussed in the context of the biogenic amine dependence of phenotypic plasticity. Parker , for exaAnstey et al.  showed tC. elegans dopamine has been shown to control and reconfigure patterns of locomotory behavior (Chase et al., Dopamine plays an important role in regulating and controlling many physiological and behavioral processes Homberg, . In cockDifferent environmental stressors such as temperature (Hirashima et al., in vivo by the injection of the dopamine antagonist, fluphenazine, which completely abolished the solitarious-like behavior of locusts isolated for 1 h. Treated locusts exhibited behavior similar to gregarious animals, implying that endogenous dopamine underpinned the change in behavior caused by isolation. It is therefore clear that in the desert locust serotonin and dopamine each play key roles in initiating different transitions of phase. It is also clear from these experiments on desert locusts that, in contrast to the migratory locust, dopamine is not the main driver of the transition from the solitary to gregarious phase suggesting that different neurochemical mechanisms are likely to underpin or differentially act upon the same transition in different species.Dopamine is produced in several areas of the central nervous systems of locusts, including over 6000 neurons in the brain that are dopaminergic Roeder, . Watson The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The problem of antimicrobial resistance is so dire that people are predicting that the era of antibiotics may be coming to an end, ushering in a ‘post-antibiotic’ era. A comprehensive policy response is therefore urgently needed. A part of this response will require framing the problem in such a way that adequately reflects its nature as well as encompassing an approach that has the best prospect of success. This paper considers framing the problem as a slowly emerging disaster, including its potential benefits and difficulties, from a conceptual and policy perspective. On 7 April 2011, World Health Day, the World Health Organization (WHO) announced that urgent actions were necessary if the effectiveness of antibiotics was to be ensured in the future. Failure to confront the rising problem of antimicrobial resistance (AMR) would result in the loss of the ‘miracle cures’ offered by antibiotics, the WHO’s Director-General Margaret Chan announced .1 These AMR is not, however, merely a future threat—the present human and economic costs of AMR already amount to thousands of casualties each year, increase the infectious disease burden around the world and leads to billions of dollars in direct health costs and lost productivity . What exThe problem of AMR is so dire that people are predicting that the era of antibiotics may be coming to an end, ushering in a ‘post-antibiotic’ era in which we will be as vulnerable to bacterial infections as we once were before Fleming’s discovery . Accordiprima facie, to conceptualize AMR as a type of disaster; in particular, as a slowly emerging disaster. We examine some reasons in favor of doing so, as well as some tensions and problems involved in such a conceptualization. In examining the conceptual question of how we should go about understanding which circumstances deserve the label of disaster, we seek to highlight that events or processes, such as AMR, are notably different from what are thought of as ‘typical disasters’ and how this might require us to think more deeply about how we should go about defining disasters, as well as why we attach the label of disaster to particular circumstances. Whether or not we have overwhelming reason to include AMR within the category of disaster, we may still have pragmatic reasons for approaching the problem of AMR as if it is some kind of disaster. We then proceed, secondly, to outline different policy-based considerations that might give us reason to approach AMR as if it is a disaster. These conceptual and policy considerations should be analyzed separately, even though there are many connections between them. It is very important that the question of ‘what is a disaster?’ should be addressed independently from the question of ‘how should we respond to a disaster?’, even though the former will bear on the latter without necessarily determining it. Nevertheless, it is still fruitful to investigate how framing certain problems as disasters can shape our approaches to them in a way that allows practitioners and policy makers to import existing knowledge and infrastructure to AMR, which will be our primary concern.The aim of this article is two fold. We begin, firstly, by exploring whether it is plausible, at least It is indisputable that AMR is a major threat to individual and public health. In recent years, much progress has been made in raising awareness for this fact and the problem is widely recognized by clinicians, scientists, national governments and global health agencies . It is aprima facie plausible that it might qualify as a disaster. How does it comport with our current ways of defining what constitutes a disaster?Unfortunately, the label of disaster—or even terms such as epidemic and pandemic—are often arbitrarily attached to large-scale health problems in an effort for them to receive more governmental funding, policy prioritization, media attention or public awareness. For instance, even among clinicians, scientists, politicians and journalists, problems as varied as vision care , autism a sudden, calamitous event that causes serious disruption to the functioning of a community or a society causing widespread human, material, economic and/or environmental losses which exceed the ability of the affected community or society to cope using its own level of resources : 43.an urgent and critical situation of a temporary nature that (i) seriously endangers the lives, health or safety of Canadians and is of such proportions or nature as to exceed the capacity or authority of a province to deal with it, or (ii) seriously threatens the ability of the Government of Canada to preserve the sovereignty, security and territorial integrity of Canada and that cannot be effectively dealt with under any other law of Canada : §3.(i) an event or situation which threatens serious damage to human welfare in a place in the United Kingdom, (ii) an event or situation which threatens serious damage to the environment of a place in the United Kingdom, or (iii) war, or terrorism, which threatens serious damage to the security of the United Kingdom  understand the term.responding to such events. For instance, even if the concept of disaster does not currently count AMR as a disaster per se, it does not mean AMR does not present an extremely serious threat that government policy and individual action should take measures to mitigate or prevent right now. Concomitantly, it also means that the fact that AMR should be a top priority that requires urgent action need not automatically qualify it as a disaster either. Even if we should endorse the stronger claim that disasters are not the kinds of events that can last for decades or centuries, it must be understood that this need not entail the normative implication that these circumstances are less urgent or less important to respond to. Indeed, this is why it is so important to keep the labeling question separate from the response question—the application of the label of disaster should not be thought to wholly settle the question of whether or to what extent we respond to particular circumstances. The situational features that constitute a disaster will shape both conceptual and normative considerations; however, we must be careful about how we understand and use these considerations in our deliberation and debate about such circumstances.To be clear, in differentiating the temporal boundaries of a disaster, it need not have any normative implications for how we ought to go about existing for a long duration and (ii) an event taking a long time to come into existence. In the case of the former, a disaster comes into existence, but that circumstance stays in existence for long duration. In the case of the latter, while there will be a number of causal antecedents that could be traced or identified, it would not be until the instantiation of the event itself that a disaster would exist. The notion of a slowly emerging disaster falls into this latter category.Part of the reason why some people may be tempted to conceive of disasters as having such massive temporal boundaries is because they are making the mistake of conflating the antecedent conditions that contribute to the instantiation of a disaster as being part of the disaster itself. The two are inextricably linked and extremely important for our understanding of disasters, but they should be kept distinct. We must be careful to distinguish between an event that takes a long time to occur and an event that has a long duration. That is to say, there is a difference between (i) an event X at time T can be pinpointed as the initiation of a disaster. This is certainly the case with AMR. While many microorganisms are becoming resistant to available preventative and therapeutic medicines, it is clear that AMR is not an all-or-nothing phenomenon. Unlike an event such as an earthquake or a terrorist attack, the emergence of AMR is not a distinct, specifiable event that occurs and immediately causes us to go from a time of normalcy to a time of disaster. Moreover, there can be different levels of resistance within a specific class of microbe. For instance, we distinguish between multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis.Furthermore, given the complexity of most disasters and the difficulty in predicting their moment of instantiation, there is often no clear, determinate point in time in which factor Moreover, a related concern is that if we retain the claim that what has become normal can no longer be a disaster, then there is a possibility that certain slowly emerging events might never constitute a slowly emerging disaster because each progressive period of time will be viewed as normal until it reaches its end point.per se—that is to say, we are not currently experiencing the disastrous consequences of a post-antibiotic era—it certainly is the kind of event described above as a disaster that takes a long time to come into existence. It is, in this sense, better understood as a slowly emerging disaster, since the antecedent conditions that can lead to a post-antibiotic era are currently in place and are proceeding in such a way that conceivably will lead to what would end up as a commonplace disaster.For the purposes of this article, however, we do not need to define the conditions under which we can legitimately conclude that we have entered a post-antibiotic era. We are here merely concerned with the question how we should conceive of such an event. In particular, we are interested in the question of whether it makes sense conceptually to understand widespread AMR and a widespread loss of antibiotic effectiveness as a disaster. While AMR currently need not qualify as a traditional disaster As noted earlier, many of the features of AMR can allow it to qualify as a disaster under many of the accepted public policy definitions used. One noted exception is the feature of being temporally bounded. If we waited to fulfill such definitions strictly, we would not be justified in labeling the threat of AMR a disaster at present—only when the post-antibiotic era has been achieved. This would be problematic, as by the time the actual disaster of the post-antibiotic era had been instantiated, the significant risk of harm, loss or burden will have already been actuated and our options to respond will have become constrained and ineffective. The idea of a slowly emerging disaster provides a middle way in thinking about public health threats. It does not attach the label of a full-fledged disaster; instead, it emphasizes the connection between the antecedent conditions and the resulting event—thereby placing the focus on how the antecedent conditions contribute to the disaster taking a long time to come into existence, as well as how this establishes response options to those antecedent conditions.Another advantage of understanding AMR as a slowly emerging disaster is that it need not completely conform to our commonplace view of full-fledged disasters, in which we go from normalcy to disaster back to normalcy. Our actions to combat AMR will never completely succeed to eliminate all drug resistance. Bacteria will continue to take advantage of the natural and anthropogenic conditions that allow them to mutate, making them resistant to the available antibiotics in use . In a reprima facie plausible to conceptualize it as a possible type of disaster. Further, we may still have good policy-based reasons for approaching the issue of AMR as a slowly emerging disaster.While the notion of a slowly emerging disaster itself is not without its problems, it is still So far, we have discussed the conceptual plausibility of describing AMR as a slowly emerging disaster. It will now be important to consider if and why such a conceptualization would be useful for the identification of suitable solutions to the problem. In this article, we wish to argue that understanding AMR as a slowly emerging disaster, rather than an acute medical problem, can account more comprehensively for the scale of the challenge and its societal implications.ceteris paribus, existing strategies fail to halt or reverse the trend towards more extensive drug resistance. In addition, understanding AMR as a predictable and unavoidable slowly emerging disaster—for which we can only mitigate consequences, rather than eliminate its causes—should lead us to rethink the often implausible rhetoric of combating and eradicating infectious diseases. However, mitigation strategies are less likely to succeed in those regions of the world that suffer from a high burden of infectious diseases and where the availability of effective antibiotics is consequently of particular importance used for the putative normative implications disasters are thought to bring about. In particular, there is a strong presumption that what may be immoral or illegal during times of normalcy could be rendered permissible during times of disaster.Concerns such as these should be taken seriously and weighed against the potential benefits of mobilizing a public response to AMR by describing it as a slowly emerging disaster. However, even if the concept of AMR as a slowly emerging disaster is found to be unsuitable for wider public communication, we believe that it may still carry weight for policy makers and health care professionals. For these groups, the classification of AMR as a slowly emerging disaster may serve less as a call to action and more as an opportunity to apply and transfer experiences collected during previous disasters, which approximate the scale and complexity of the challenge that AMR presents. And while AMR undoubtedly presents unique and novel challenges, components of a comprehensive response will be comparable to the reaction to other public health disasters. Framing AMR as a slowly emerging disaster underlines this point and would help us to build a response on the knowledge, practices and infrastructure that have proved to be successful in the past.


Finding quality consumer health information online can effectively bring important public health benefits to the general population. It can empower people with timely and current knowledge for managing their health and promoting wellbeing. Despite a popular belief that search engines such as Google can solve all information access problems, recent studies show that using search engines and simple search terms is not sufficient. Our objective is to provide an approach to organizing consumer health information for navigational exploration, complementing keyword-based direct search. Multi-topic assignment to health information, such as online questions, is a fundamental step for navigational exploration.We introduce a new multi-topic assignment method combining semantic annotation using UMLS concepts (CUIs) and Formal Concept Analysis (FCA). Each question was tagged with CUIs identified by MetaMap. The CUIs were filtered with term-frequency and a new term-strength index to construct a CUI-question context. The CUI-question context and a topic-subject context were used for multi-topic assignment, resulting in a topic-question context. The topic-question context was then directly used for constructing a prototype navigational exploration interface.F1 measure of 0.782, using a reference standard of 278 questions with manually assigned topics. Compared to NetWellness’ original topic assignment, a 36.5% increase in recall is achieved with virtually no sacrifice in precision.Experimental evaluation was performed on the task of automatic multi-topic assignment of 99 predefined topics for about 60,000 consumer health questions from NetWellness. Using example-based metrics, suitable for multi-topic assignment problems, our method achieved a precision of 0.849, recall of 0.774, and Enhancing the recall of multi-topic assignment without sacrificing precision is a prerequisite for achieving the benefits of navigational exploration. Our new multi-topic assignment method, combining term-strength, FCA, and information retrieval techniques, significantly improved recall and performed well according to example-based metrics. The Internet provides an important source of consumer health information to patients, caregivers, families, and laypersons. The proliferation of online health information from government agencies, non-profit organizations, for-profit companies, and chatting and social networking sites presents myriad of challenges for information access. For example, a study reported in JAMA  found thOne approach to addressing such challenges is to complement direct search with mechanisms for navigational exploration, one of the two basic modes for information access -4: Direct search, where a user, knowing what to look for, comes with specific pieces of information about a disease, procedure, or medication, or other related description and tries to retrieve a corresponding set of responses. For example, direct search is most effective for looking up the side effects of medications such as Warfarin, using the drug name as a key search string.1. Navigational exploration, where the goal is to explore and see “what is there.” For example, finding answers to questions such as “Other than prescribed medications, what are alternatives that may help with depression” involve more exploring than searching. In this mode, the consumer may not be able to easily and effectively formulate a descriptive direct search, and must rely on navigational menus or “facets” .The category-topic context consisted of 12 categories and 99 topics. The concept lattice of this context consists of 71 concept nodes  was achieved with no sacrifice of precision.Table F1 measure (0.933), and “Diet and Nutrition” had the lowest example-based F1 measure (0.625).Table The category-topic context and topic-question context dynamically drive the iCOACH prototype interface to allow the consumer multiple paths to quickly narrow down to relevant questions Figure . Based oMulti-topic assignment is a challenging topic. When each question is assigned to one or several topics among nearly a hundred candidates, the probability of getting even a small fraction of tens of thousands of questions correct at random is virtually zero.F1 measure of 0.782, using a reference standard of 278 questions with manually assigned topics. Our approach is non-standard. It is not a typical machine learning approach since we do not start with a training set for supervised learning. Neither is it a completely unsupervised approach since we do take advantage of existing knowledge in NetWellness. Our approach is perhaps best characterized as a hybrid version using a combination of information retrieval techniques and FCA.Our multi-topic assignment method combines semantic annotation using UMLS concepts (CUIs) and FCA and attained an example-based precision of 0.849, recall of 0.774, and Organizing consumer health information according to a conjunctive structure determined by FCA allows content items to be located from multiple pathways. It achieves a better “organizational precision and recall,” in the sense that items, if found, are in their right place (precision) and an item is located at where it should be .Our prototype interface iCOACH overcomes one of the disadvantages of the existing body of work on FCA-based menu-design and organization -24. One iCOACH is distinct from faceted search , where eiCOACH is a generalization of the conjunctive exploratory navigation interface CENI  which alSimilar to CENI, iCOACH reused NetWellness’ existing health topics, which may not represent the best choices of potential health topics. In separate work , a semanF1 measure  achieved the best example-based recall and re Table . For there Table , the topPerformance of categorizing questions into multiple topics is affected by a number of factors including the key CUIs selection algorithm, the quality of the topics to be assigned, and the quality of the questions themselves. To improve the performance of the basic step of tagging a question by relevant CUIs, we performed manual error review which indicated potential for further improvement. For example, two questions (Q1 and Q2) both asking about strong smell have the same key CUI-set {“C0442821: Strong”, “C0037361: Smell” }, which were correctly identified. However, the CUI-set did not completely determine the topic in this case, since Q1 asked about strong smell in diaper and assigned the topic of “Children’s Health,” while Q2 was more about a smell disorder related to the topic of “Ear, Nose, and Throat Disorders.” Further CUI-set selection including those tagged for answers may help address this issue. The quality of NetWellness contents naturally affects performance. Some NetWellness topics are too general. Some NetWellness questions are too short to be informative, and some too long. These represent the source of typical false negatives or false positives. An area of immediate opportunity is to redesign a collection of consumer health topics by refining and expanding the existing set of 99 topics.Our evaluation has been focused the on the multi-topic assignment problem. The iCOACH interface has not been directly evaluated by external users. However, its simplification CENI  was evalEnhancing the recall of multi-topic assignment without sacrificing precision is a prerequisite for achieving the benefits of navigational exploration. Our new multi-topic assignment method, combining term-strength, FCA, and information retrieval techniques, significantly improved recall and performed well according to established metrics. iCOACH provides an environment for organizing about 60,000 existing questions in NetWellness for navigational exploration. By organizing consumer health information sources such as NetWellness at the levels of categories, topics, and questions (contents), multiple entry points are offered for the consumer to explore information of interest, even though the precise terms for searching such information may be non-trivial or difficult to formulate.x1,x2,x3,x4 and their topic assignments in the reference standard Yi and predicted topic assignments Zi.We present a brief example to help illustrate the differences between example-based evaluation measures and traditional evaluation measures. Table P), recall (R), and F1 measure (F1) are computed using Eq. 1, Eq. 2 and Eq. 3 in Subsection “Evaluation metrics.” A precision of 0.875, recall of 0.775, and F1 measure of 0.792 are obtained as indicated in the last row.Table F1 value (0.584), where TP, FN, and FP represent numbers of true positives, false negatives, and false positives, respectively. The following steps show how the traditional evaluation metrics are calculated resulting a lower recall (0.438) and x4) with an unusually large number of labels to dominate the values of evaluation metrics (which is undesirable), while example-based metrics have been designed to avoid undue influence of such questions.As illustrated above, using traditional methods allows the performance on a few questions  of the category-topic context is indicated. Even though the category “Disease, Syndrome and Disorder” is not selected by the user, it is an “implicant” of the selected category due to the logic of FCA. Figure S3. User selects the second category, “Anatomy and Body System,” in the iCOACH prototype interface. The corresponding Health Topics relevant to both categories are automatically displayed (this is an updated list). Figure S4. The arrow in this figure points to the corresponding node in the diagram of the lattice after selecting the indicated categories in Figure S3. Again, even though the category “Drugs, Medication and Substance” was not selected by the user, it is a logical consequence of the selected categories, inferred by FCA. Figure S5. Finally, user selects the third category, “Population and Subgroups,” in the iCOACH prototype interface. The corresponding Health Topics now narrows down to only three that are relevant to all the selected categories. Figure S6. The arrow in this figure points to the corresponding node in the diagram of the lattice after selecting the indicated categories in Figure S5. Note that as more categories are selected, the corresponding concept node moves further down in the lattice hierarchy, covering fewer health topics. This demonstrates the duality in FCA with respect to attributes (in this case Categories) and their corresponding objects : more attributes serve to narrow down to fewer objects with all the relevant attributes.Click here for file


Current visualizations of molecular motion use a Timeline-analogous representation that conveys "first the molecule was shaped like this, then like this...". This scheme is orthogonal to the Pathline-like human understanding of motion "this part of the molecule moved from here to here along this path". We present MoFlow, a system for visualizing molecular motion using a Pathline-analogous representation.The MoFlow system produces high-quality renderings of molecular motion as atom pathlines, as well as interactive WebGL visualizations, and 3D printable models. In a preliminary user study, MoFlow representations are shown to be superior to canonical representations for conveying molecular motion.Pathline-based representations of molecular motion are more easily understood than timeline representations. Pathline representations provide other advantages because they represent motion directly, rather than representing structure with inferred motion. Few proteins are as conformationally rigid as their "structures", accessible through the Protein Data Bank (PDB), would imply. To be sure, PDB structure files often contain confidence-level information detailing how much uncertainty is present in the coordinates of the individual atoms, or contain several alternative structures implying that the protein can transition between them. However, canonical representations for molecular structures and their mobility convey none of this information to the user in an intuitive fashion. The atoms of proteins are represented as having single discrete locations and colored by confidence, or the molecule is represented as having a set of discrete, perfectly-known poses, with no representation of transition between them.Fluently reading these representations requires considerably user training and experience because they are highly reliant on convention. Molecular biologists who live with them daily do develop facility, but the end-customer biologist is often left bewildered because the representations eschew sensory cues.In this manuscript we introduce a sensory-based representation for molecular motion that is aimed at the end-customer basic bio/life-sciences researcher who wants to understand "how did my molecule move", and who wants to be able to make intuitive comparisons between different molecules, without becoming deeply experienced with quantitative molecular biology tools. We also introduce a web resource that enables users to construct these representations from their own data, in a 2D projection form, a 3D virtual model form, and also a 3D-printable form.As more detail is uncovered about protein structures and their flexibility, it is often found that the dynamics of conformation changes - not simply the alternate conformations themselves, but the molecular motion, the paths taken between conformations and the frequency with which different molecules take different paths - play a part in modulating the molecular functionality . This phThese users specifically need:• To quickly acquire a qualitative overall understanding of a molecular motion: "It moved like this".• To quickly identify regions of significant motion and of little motion: "This moved, that didn't".• To differentiate (largely) ordered motion from (largely) unordered motion. "It moved from here to there", versus "it jiggled about".• To understand timing and speed: "It spent a long time, here, then quickly moved there".• To understand the similarities and differences between different molecular motions : "These all moved similarly, while that mutant takes a different path".Addressing these needs requires a representation that promotes motion to the primary feature being conveyed, rather than representing conformations as the primary feature and requiring interpretation to recognize motion.Nuclear Magnetic Resonance imaging (NMR) or Molecular Dynamics simulations (MD) can yield snapshots of molecular conformations that represent changes in the structure of a molecule over time. Traditionally, visual representations of molecular motion have consisted simply of displaying these snapshots as a series of static images, often overlaid upon each other, similar to stroboscopic multiple-exposure pictures of motion . In suchSuch representations can be used in full 3D interactive modalities, as well as in 2D modes, and can easily be animated by simply playing each pose in sequence. Unfortunately, they are not well suited for conveying molecular motion and variations in molecular motion to users. Instead of representing motion, they represent poses - motion must be inferred. If a color gradient is used to convey sequential position in the molecule (as is commonly done in single-conformation images), then conveying temporal order for the poses becomes problematic. If color is used to convey temporal order, then sequential position in the molecule is lost. Even more problematically, if the motion is dramatic, some parts of the molecule may move so far between poses that the visual ordering becomes disjoint despite coloring, and understanding the motion between sequential poses becomes difficult for the viewer despite the temporal coloring scheme.et al. [We propose that a large part of the problem with this pose-based representation is that it is effectively orthogonal to the most intuitive representations for conveying motion. The pose-based representation is a direct equivalent to Timeline representations of fluid flow. As an alternative, effectively time-lapse representation, we propose using an analog to Pathline representations for molecular motion. While Wang et al.  identifimotion into a structure that is directly represented, rather than requiring the user to infer motion from representations of molecular structures. This has both immediate consequences for understanding, as well as larger consequences for possible extensions to the representational paradigm.Of critical importance, this simple transformation from sequential poses of the structure to integral paths for atoms over time converts MoFlow, our web-based system for generating time-lapse molecular-flow images and motion structures, accepts multi-pose Protein Data Bank (PDB) files, and creates an interactive WebGL visualization of the molecular motion, a renderable POV-Ray scene file, and a color-per-vertex VRML file that is suitable for printing on a 3D printer.In this manuscript we introduce the MoFlow system, which was developed to explain the movements of a particular molecule to a collaborator working on gene-therapy constructs, and present an initial user study comparing user-understanding for MoFlow molecular representations compared to typical pose-based representations, with a broad-based user population.http://www.mathmed.org/moflow/The MoFlow webserver can be accessed at: There is a clear parallel between Molecular motion and fluid motion, and importantly, between the results of molecular motion simulations and Computational Fluid Dynamics (CFD) simulations. This parallel strongly suggests that visualization methods that have been found successful for fluid flow data, may be similarly useful for visualizing molecular motion. There are however, subtle differences between molecular motion and fluid motion, and in the questions that one might wish to answer with each.The molecular motion with which we are interested is that of individual atoms in an individual molecule. At any given point in time, these atoms have one and only one location. These locations change over time, and so Pathlines for each atom can be traced. Likewise Timelines for the molecule correspond to the backbone pose conformation at different times. However, unlike fluid flow, there are no atoms "further along", or "following behind" the atoms of the molecule being examined. As a result, flow concepts such as Streaklines do not apply. Also, because each atom has only a single velocity, and exists at only a single point at any instant in time, Streamlines devolve to vector stream tangents, and do not imply a "bulk" motion as they do in fluid.An additional important difference derives from the fact that the atoms of interest in the molecule, have a distinct sequential ordering and connectivity that is meaningful to the user and must be represented.et al. [et al. [et al. [CFD visualization methods have previously been applied to some types of Molecular Dynamics simulation data. Schmidt-Ehrenberg et al.  generateet al.  used tim [et al.  used pat [et al.  also use [et al.  proposedUnfortunately for the researcher wishing to understand the motion of a macromolecule, such as a protein, undergoing a conformational change, none of these approaches deal with the composite trajectory of the backbone structure for a single molecule. Instead they have dealt with related concepts such as the paths of multiple molecules in solution, or the envelope of conformations that a particular molecule may adopt.Surprisingly, one of the most promising existing data analogs, and visualization approaches, is found in the analysis of dance, by Forsythe , where rWhile animation would seem an obvious means of addressing the problem of visualizing conformational change, a simple amalgamation of static poses into an animation makes the motion easy to understand, but makes it impossible to directly compare different stages of the motion. Non-sequential time points are difficult to compare due to change-blindness and inattention-blindness effects . The humMoFlow was originally developed to assist a collaborator who was trying to engineer novel cell-targeting motifs into the Adeno-Associated Virus Type-2 (AAV2) capsid protein. AAV2 is a nearly ideal candidate for a gene-therapy delivery vector, but suffers from several issues that make its direct application problematic; first, many people have already developed immunity to AAV variants, and second, AAV2 is reasonably non-specific about the cells it infects, making it difficult to use for delivering therapy constructs to targeted cell types. Removing epitopes that most peoples' immune systems recognize from the capsid  surface, and adding targeting motifs that recognize specific cell-surface receptors would be relatively straightforward, except for a confounding feature of the AAV2 capsid: it is both a packaging unit, and a machine with an enzymatic functionality required in the lifecycle of the virus.Research suggests that conformational changes of the capsid are a required feature of its lifecycle - specifically it appears that a conformational change induced by acidification of the endosome causes a phospholipase catalytic domain to be exposed, enabling the virus to escape the endosome. Therefore changes to the capsid, such as altering existing epitopes and adding new targeting factors, must be done without significantly affecting the ability of the capsid to undergo the conformational changes required to expose the phospholipase domain.Molecular dynamics simulations were conducted to identify how the AAV2 capsid protein moves during acidification, but while the results of these simulations were reasonably easy to understand for expert users of MD software, our gene-therapist collaborator who needed the information found all attempts to convey the results using typical molecular visualization software such as PyMol  and VMD Recognizing that the primary difficulty was due to the "timeline" representation of overlayed poses used by typical MD visualization software, we developed MoFlow as an alternative. The understanding gained from our alternative representation resulted in several successful engineered AAV2 capsid alternatives reported in Davis et. al. MoFlow accepts a time-ordered series of molecular "poses", typically produced by a molecular dynamics simulation, or by a conformational-morph prediction such as is produced by the Yale Morph server .From this ordered series of poses, MoFlow extracts the backbone alpha carbons and interpolates intermediate positions for each of these by constructing a natural cubic spline that passes through each of that carbon atom's predicted positions in order, over time. This spline is a smoothed Pathline for that atom. Different choices of temporal resolution in the input series of poses, and an interface control that enables the user to render only a subset of input poses, control the degree of smoothing.On-screen, and in our printable output, this spline is rendered as a piecewise-continuous cylinder extruded along the spline. Use of the alpha carbon trace of the molecule captures significant conformational motion, while effectively acting as a low-pass filter to eliminate the high-frequency motions of amino acid side-chains. However, the representation can also be used for other atoms in the molecule, revealing, for example, concerted motion in side-chains, rather than conformational motion in the backbone. A time-based color gradient  is applied to each pathline.For a single user-chosen time point in the simulation, the alpha-carbon trace for the protein backbone is also projected onto the pathlines. Spheres are positioned to represent the alpha carbons in a single pose (at a single timepoint), and cylinders are used to connect sequentially neighboring alpha carbons. This single molecular backbone is a single-point timeline for the molecule. This backbone provides structural context for the paths: for each atom, the pathline projects back into the past where the atom came from, and into the future where the atom is going.In the POVRay version, the linking-cylinder between every other sequential pair of atoms in the single molecular structure drags a fading transparent membrane behind it, similar to the integral-surface ribbons introduced by Hultquist . This meFigure Recognizing that this success derives from the fact that the spheres representing atoms have become so dense that they are visually interpreted as being continuous pathlines, we developed MoFlow to represent pathlines for each atom in a sufficiently sparse fashion as to enable the user to "see into" the motion, and to also enable the display of molecular backbone structure onto the pathlines. Figure Figure An additional visual cue we have added to the visualization is a set of fading, semi-transparent ribbons connecting alternate sequential atom pathlines. These ribbons "trail behind" the molecular structure backbone that is imposed on the pathlines, and provide a directional cue for the motion. By creating an oriented surface between adjacent pathlines, the direction of the motion along the pathlines becomes discernible.Figure Streptococcus mutans. Figure Figure Figure To aid in designing the final visual representation of motion, MoFlow allows the user to choose the number of "frames", or poses, of motion to include in the POVRay output. Figure To validate the utility of MoFlow outside our original design intent of aiding in engineering AAV2, we have conducted an initial user survey comparing the ability of users with minimal expertise in molecular visualization to correctly identify key features of molecular motion when presented with either canonical, or MoFlow representations for the motion The preliminary analysis was aimed at addressing the first user task described above, e.g. "it moved like this". This preliminary data will be used to help design a larger study and are included here as an exploratory example; further validation studies are planned. The survey forms are available upon request from the authors.The subject pool consisted of 12 subjects  aged between 13 and 48, from a variety of educational backgrounds and professions. All subjects had normal color vision. None had significant experience with molecular visualization.Images were generated either as a composite (non-MoFlow) multi-pose rendering or as a MoFlow time lapse rendering. Poses in the canonical rendering were manually colored to match, as near as possible, the temporal coloring applied to the MoFlow timelines .Each subject was shown, in random order, the individual images depicting conformational change in molecules, without being led or informed as to the differences between them (e.g. composite poses vs MoFlow). Users were told that the images represented a conformational change where the molecule moved from one conformation to another, the temporal coloring scheme was explained, and the subjects were asked to briefly study each and then complete a series of questions for each image. These questions were both task-oriented - designed to identify the level of understanding conveyed by the image  - as well as subjective . For each of three atoms in indicated at the midpoint of the molecular motion, a set of possible starting and ending points were labeled  and users asked to select the beginning and ending target for each indicated atom.To assess the utility of MoFlow we evaluated each users' position-mapping in a pair-wise fashion, where a correct mapping between the midpoint and any endpoint received a +1 score. The maximum score for each image was 6, as there were three atoms for which the users had to identify start and end points.While this preliminary survey was only intended to guide our development of a more meaningful survey, rather than to reach statistical significance, overwhelming feedback from users indicated that the MoFlow visualizations could be understood more easily, more completely, and more quickly. Users were more likely to score higher  on the MoFlow diagram than on the conventional overlaid poses diagram. Individual user scores ranged from between 0 and 4 (out of a possible 6) for both visualization schemes, with an average score of 1.5 for the traditional representation and an average score of 2.67 for MoFlow. Several of the users presented with the canonical representation after having seen the MoFlow representation reacted with considerable annoyance at having to try to interpret the "mess". Users presented with the MoFlow representation after the canonical representation instead expressed relief that the "rollercoaster" (pathlines) helped them understand what they had been trying to interpret in the previous image; one user wrote that the MoFlow diagram "...was super easy compared to the alternative". Indeed, many users expressed that the canonical representation failed to present any indication of motion, whereas the MoFlow pathlines were more intuitively representative of motion. Qualitatively, the preliminary survey suggested that MoFlow was generally easier to use, with an average ease-of-use score of 4.58/10 (where higher is better), as opposed to 2.83/10 for the canonical representation.We have presented a new method for rendering and visually exploring molecular motion that specifically takes into account the natural human intuition of movement over time. By indicating the motion of atoms as pathlines rather than the traditional orthogonal timeline representation, the eye is drawn naturally along the path of motion, providing an immediate understanding of the overall motion.As demonstrated in Figures MoFlow renderings are conveyed to the user as interactive WebGL objects as well as returned to the user by email in POV-Ray  format. As with many other applications, the ability to engage the user's senses with a tactile model greatly appears to increase understanding of the process involved - in this case, allowing the user to freely rotate the object and observe it from any perspective provides greater insight than a 2D - or even on-screen interactive 3D - visualization. Comparison of the benefit of onscreen versus 3D-printed MoFlow visualization is an ongoing project.In its current form, MoFlow satisfies the needs of the non-molecular-biologist end-user to qualitatively understand the trajectory features of molecular-motion data. The tasks as originally presented can be accomplished, and the visualizations were critical for the success of our AAV2 engineering project and aiding our gene-therapist collaborator to understand where to modify his molecule . MoFlow http://www.mathmed.org/moflow/ and requires a multi-pose PDB file as input. Output is provided as a WebGL response that can be saved from the browser, a POV-Ray file that requires rendering prior to visualization, and optionally as a 3D-printable color-per-vertex VRML file. The authors welcome any comments and feedback on this application.The MoFlow application is freely accessible via the web at The MoFlow can currently import and generate visualizations for large molecules undergoing complex conformational changes, however, there remain a number of improvements that are necessary to maximize convenience, and additional research is required to optimize some aspects of the visualization. For example, the user must currently manually balance model resolution with hardware limitations on the workstation - too many input poses, or requesting too-fine a tessellation in the WebGL version, can easily exhaust many system's available memory. In addition, it is not currently clear what the optimal temporal resolution for representing the motion in an MD simulation should be. Too fine a temporal resolution results in visualizations that are dominated by random perturbations of the structure and that fail to convey the overall conformation-change motion, while too coarse temporal resolution results in important aspects of the conformational change being omitted.Because the motion paths are interpolated between the atom positions in the input poses, the implied bond-lengths at interpolated positions are likely to be inaccurate, and in some cases implausible. For MD simulations where successive poses are separated by 1 to 2 bond-lengths, in practice this interpolation error does not appear to produce significantly inaccurate paths. In the MoFlow interface, the molecular backbone chain can be constrained to any of the input poses to maintain accuracy. Alternatively it can be placed at interpolated positions between the predicted poses, with the understanding that this interpolated pose is also subject to interpolation errors.The influence of thermal-motion noise in a simulation, coupled with the spline interpolation between input poses, can, in some circumstances, produce completely unexpected results. Typical brownian-motion effects appear as motion paths that "wander around" the atom positions, usually within 1 to 2 atomic radii, such as the paths shown in Figure At the same time, it is clear that viewpoint and subselection of regions of the motion-structure to visualize, can maximize, or significantly impede the utility of the MoFlow representation . We do not currently have an implemented solution for guiding the user to the most useful viewpoints and atom selections, however, preliminary attempts at maximizing the visually subtended surface area of the swept inter-pathline "ribbon" surfaces shows promise for selecting maximally informative views.All of these issues together highlight the fact that the use and interpretation of MoFlow representations requires a careful user. We have found no algorithmic solution that can eliminate all of the possible visualization flaws automatically, without also eliminating details that some users want to see: We cannot arbitrarily low-pass filter the data to eliminate thermal noise, without impeding users who want to see where thermal noise is greatest, and where it is least in their system; we can't eliminate regions of the motion to de-clutter the display, without confusing users interested in relative motion (or lack thereof) in those regions, etc.th atom, adjust the position of the backbone along the motion paths, and adjust the viewpoint of the visualization. Many additional parameters can be adjusted by modifying variables within the renderable and 3D printable files that MoFlow returns. In addition, we encourage users to contact us with suggestions, as MoFlow remains a continually-improving platform.As a result, MoFlow requires the user to provide input data at a temporal and spatial resolution that they consider appropriate, and through the MoFlow interface we provide tools to enable the user to simplify or focus the visualization on the features they consider relevant. Currently the MoFlow interface enables the user to limit the range of poses used to a subset of all uploaded poses, limit the range of atoms of the molecule that are shown, reduce the density of alpha-carbons shown such that a motion path is shown for every nmotion as a structure, it becomes possible to compare the structures generated for different motions. This enables visualization of properties such as the uncertainly and randomness present in molecular motions in a way that is impossible for representations that rely on molecular structures to infer motion.Moving forward, in addition to improving the visualization and interface, and increasing automation for the user, we are pursing new avenues for comparative studies of molecular motion that are made possible by MoFlow representations. Because MoFlow represents We have described here a new method for rendering and visual exploration of molecular motion that specifically capitalizes on the natural human intuition of movement over time. The representation has been successfully used for its intended purpose to facilitate protein engineering. It has been preliminarily validated to convey more accurate information, more easily, to untrained consumers of molecular-motion data. Simultaneously the fact that this system directly represents motion as a structure, rather than inferring motion, creates opportunities for new types of molecular comparisons. The core of this system is representing the motion of atoms as pathlines, as opposed to the orthogonal timeline representation of traditional molecular motion imagery and animation. This correction of the representation to align with the actual data of interest provides advantages in both perception and in the variety of questions that can be asked of the data.The authors declare that they have no competing interests.


Adolescence is associated with high impulsivity and risk taking, making adolescent individuals more inclined to use drugs. Early drug use is correlated to increased risk for substance use disorders later in life but the neurobiological basis is unclear. The brain undergoes extensive development during adolescence and disturbances at this time are hypothesized to contribute to increased vulnerability. The transition from controlled to compulsive drug use and addiction involve long-lasting changes in neural networks including a shift from the nucleus accumbens, mediating acute reinforcing effects, to recruitment of the dorsal striatum and habit formation. This study aimed to test the hypothesis of increased dopamine release after a pharmacological challenge in adolescent rats. Potassium-evoked dopamine release and uptake was investigated using chronoamperometric dopamine recordings in combination with a challenge by amphetamine in early and late adolescent rats and in adult rats. In addition, the consequences of voluntary alcohol intake during adolescence on these effects were investigated. The data show a gradual increase of evoked dopamine release with age, supporting previous studies suggesting that the pool of releasable dopamine increases with age. In contrast, a gradual decrease in evoked release with age was seen in response to amphetamine, supporting a proportionally larger storage pool of dopamine in younger animals. Dopamine measures after voluntary alcohol intake resulted in lower release amplitudes in response to potassium-chloride, indicating that alcohol affects the releasable pool of dopamine and this may have implications for vulnerability to addiction and other psychiatric diagnoses involving dopamine in the dorsal striatum. Adolescence is associated with high impulsivity and risk-taking behavior, making adolescent individuals more inclined to use drugs Drugs of abuse commonly act on the reward system and increase extracellular levels of dopamine in the nucleus accumbens acutely after intake Animal models are of great importance to our understanding of these mechanisms and the age window identified as adolescence in rodents is between postnatal day (PND) 28 and 50 The second objective of this study was to investigate the effect of environmental influence by voluntary alcohol intake during adolescence. The rationale behind this was that previous studies show that environmental factors during the adolescent period, such as intraperitoneally administered alcohol, increase basal extracellular levels of dopamine All animal experiments were performed under a protocol approved by the Uppsala Animal Ethical Committee and followed the guidelines of the Swedish Legislation on Animal Experimentation  and the European Communities Council Directive (86/609/EEC).ad libitum. The cages contained wood-chip bedding and paper sheets  and were changed once a week by animal care personnel. The animal room was kept at constant temperature (22±1°C) and humidity (50±10%) on a regular 12 h light/dark cycle with lights on at 06∶00 am. All rooms had a masking background noise to minimize unexpected sounds that could disturb the animals.Pregnant Wistar rats  arrived at the animal facility at gestation day 16. The animals arrived in batches over several weeks in order to accommodate the timing of the chronoamperometric recordings. The dams were single housed in macrolon cages (59 cm×38 cm×20 cm) with pellet food  and tap water An overview of the experimental outline can be found in Inactin, Nafion 5% solution, dopamine hydrochloride, L-ascorbic acid, potassium chloride, sodium chloride, sodium phosphate, calcium chloride and d-amphetamine sulfate were obtained from Sigma-Aldrich, LLC . Kerr sticky wax was obtained from DAB LAB AB . Carbon fibre microelectrodes  were purchased from Quanteon, LLC , the reference electrode silver wire  was purchased from A-M Systems Inc.  and glass capillaries (0.58 mm inner diameter) for the micropipettes were purchased from World Precision Instruments Ltd .Dopamine recordings were made at PND 28 (±1 day), PND 42 (±1 day) or PND 70 (±2 days). Surgery was performed immediately prior to the electrochemical recordings. A water-circulating heating pad  was used to maintain body temperature. The animals were anesthetized with Inactin 125 mg/kg intraperitoneally (i.p.) and placed in a stereotaxic frame . A hole in the skull was drilled overlying the recording site for the electrode, and another hole was drilled remote from the recording site for the placement of the Ag/AgCl reference electrode.in vitro in 0.05 M phosphate buffered saline to determine selectivity, limit of detection (LOD) and slope before use in vivoin vivo Ag/AgCl reference electrode High-speed chronoamperometric measurements  were performed using the FAST16-mkII recording system  according to a previously described procedure 2·2H2O) (pH 7.2–7.4) using a pipette-filling needle . The micropipette was affixed approximately 150–200 µm from the carbon fibre tip using sticky wax. The electrode was stereotactically placed in the dorsal striatum, AP: +1.0 mm, L: +3.0 mm from bregma, the incisor bar was adjusted according to age and weight A micropipette (10–15 µm inner diameter) was filled with isotonic potassium chloride solution  and after another 5 min release was again evoked every 10 min, producing peaks at 5, 15, 25, 35, 45, 55 and 65 min after the systemic injection, see The electrodes were cut off and left in place after the finished experiment and the brains were frozen. The placement was verified by sectioning of the frozen brains. From the 12 animals at PND28, 1 was excluded due to wrongful placement, and 2 because of recording errors. For the 12 animals at PND 42, 1 animal was excluded due to wrongful placement. For the 16 animals at PND 70, 3 were excluded due to recording errors. For the 16 ethanol-drinking animals at PND 70, 2 were excluded due to recording errors. Recording errors include pipette clogging and electrical disturbances such as power cuts and disturbances of the general power supply to the recording unit.The maximal amplitude of the evoked peaks and the time for the peak to decline to 80% of its amplitude (T80) were calculated using the FAST Analysis software version 4.4 , see Differences in reference amplitudes between the age groups are shown in No effects of age , time  or time and age  were found for the reference T80 values. The mean ± standard error of the mean (SEM) reference T80 values were 17.3±1.3 for the PND 28, 19.5±0.9 for the PND 42 and 20.5±1.0 for the PND70.Differences between the age groups in amplitude response to amphetamine are shown in The T80 response to amphetamine is shown in 2 = 9.80; p = 0.08] towards differences driven by the intake during the second week (PND 35–37), which was slightly higher than the following weeks. A Friedman ANOVA of the preference showed an increase over time , mainly as a result from increases over the first three weeks, see Ethanol intake data for the 14 rats that were used in the chronoamperometric recordings are shown in Differences in reference amplitudes between the ethanol- and water-drinking groups are shown in No effects of drinking group , time  or time and drinking group  were found for the reference T80 values. The mean ± SEM reference T80 values were 20.5±1.0 for the water-drinking rats, and 19.1±1.3 for the ethanol-drinking rats.The response to amphetamine in the ethanol- and water-drinking groups is shown in For the T80 values, Age-dependent effects on dopamine release and uptake was investigated under basal conditions and in response to amphetamine in early and late adolescent, as well as adult, rats. The impact of alcohol drinking during adolescence was also examined and is, to our knowledge, the first study investigating release and uptake in voluntary drinking adolescent rats with a chronoamperometric technique.2 density The age-dependent differences in reference amplitudes are in agreement with a previous study using voltammetry in combination with electrical stimulation, which showed that adult rats released more dopamine upon stimulation than young rats max should therefore not be reached. Using the linear part of the peak curve to calculate uptake rate under these conditions will only produce uptake rates dependent on amplitudes The uptake measure, T80, did not reveal any differences between the ages in the current study, whereas Stamford (1989) found that the rate of uptake was higher in adult rats. This can be due to methodological differences in the measure of uptake; T80 includes both the linear and the curvilinear part of the curve, whereas Stamford used the linear part of the curve The greater potassium-evoked release in adulthood could be due to a larger releasable pool of dopamine No age-dependent effects on T80 after amphetamine was found, which indicates that amphetamine exerts similar effects on dopamine uptake in all ages. This is again supported by the results from Stamford (1989), showing no differences in the degree of uptake blockade after nomifensine between age groups. There are also studies suggesting that age-related differences in dopamine transporter structure and function are related to the cocaine-binding site on the transporter, but not the amphetamine-binding site Voluntary adolescent alcohol intake for six weeks resulted in lower reference amplitudes compared to water-drinking controls. The amplitudes were similar to those seen in early adolescent rats. Since the effects were seen in amplitudes and not uptake time, it is conceivable that alcohol affects factors controlling the releasable pool of dopamine rather than the dopamine transporter and there are data supporting unaffected uptake after adolescent alcohol When treated with amphetamine, there were no significant differences between the alcohol- and water-drinking groups in amplitudes or T80. However, there was a trend towards an effect on amplitudes, due to the increase displayed by the alcohol-drinking group. There is also more variation in the response to amphetamine in the alcohol-drinking group, which could be due to a variation in alcohol intake, although this variation is not correlated to the response (data not shown). This also points to a limitation with this study, namely that blood alcohol levels were not measured. The study was based on undisturbed access for 24 h and for blood alcohol levels to be measured the access would have had to be limited and the stress involved in blood sampling would have risked to disturb the intake patterns of the animals. Thus, correlations between response and individual blood alcohol levels cannot be ruled out. However, the intake data presented in this study is similar to other studies, showing neurobiological effects of alcohol, using Wistar rats in similar ages or intake paradigmsNo differences in uptake time after amphetamine suggests that adolescent alcohol has no effect on dopamine transporter function in response to amphetamine, but would also benefit from investigation by application of exogenous dopamine Furthermore, two interesting observations were made. Firstly, the reference amplitudes after alcohol intake are similar to those seen in animals at the beginning of the alcohol intake period, i.e. PND 28. Secondly, the size of the increase in amplitudes after amphetamine in the alcohol-drinking animals is similar to the late adolescent rats, i.e. PND 42. Whether these findings relate to altered development of the releasable pool and the storage pool of dopamine in the neurons remains to be elucidated. The current study did not include a group of adult alcohol-drinking rats so conclusions about the possibility of age-specific effects cannot be drawn. However, indications of age-specific effects can be found in the discrepancies between studies of adolescent alcohol-exposed rats that show unaffected dopamine uptake The data show a gradual increase of evoked dopamine overflow with age, supporting previous studies suggesting that the pool of releasable dopamine increases with age. In contrast, a gradual decrease in evoked overflow with age was seen in response to amphetamine, supporting a proportionally larger storage pool of dopamine in younger animals, making them potentially more sensitive to dopamine-releasing drugs. Adolescent alcohol intake resulted in overflow lower than in water-drinking controls., indicating that alcohol affects the releasable pool of dopamine and this may have implications for vulnerability to addiction and other psychiatric diagnoses involving the dopamine system in the dorsal striatum.


It is well established that dopamine neurons in the SNc innervate not only the striatum, which is the main target, but also other basal ganglia nuclei including the two segments of globus pallidus and the subthalamic nucleus (STN). The role of dopamine and its depletion in the striatum is well known, however, the role of dopamine depletion in the pallidal complex and the STN in the genesis of their abnormal neuronal activity and in parkinsonian motor deficits is still not clearly determined. Based on recent experimental data from animal models of Parkinson’s disease in rodents and non-human primates and also from parkinsonian patients, this review summarizes current knowledge on the role of dopamine in the modulation of basal ganglia neuronal activity and also the role of dopamine depletion in these nuclei in the pathophysiology of Parkinson’s disease.Parkinson’s disease (PD) is a neurological disorder characterized by the manifestation of motor symptoms, such as akinesia, muscle rigidity and tremor at rest. These symptoms are classically attributed to the degeneration of dopamine neurons in the  Furthermore, it is well established now that dopamine neurons in the SNc innervate not only the striatum but also other basal ganglia nuclei including the two segments of globus pallidus, the external part  and the internal part , as well as the subthalamic nucleus  is a neurological disorder characterized by the manifestation of motor symptoms such as akinesia, muscle rigidity and tremor at rest. These motor deficits are classically attributed to the degeneration of dopamine neurons in the Dopamine cell degeneration in the pathophysiology of PD is considered as the main hallmark of the disease (Agid and Blin, In normal physiological conditions, dopamine has long been known to be a crucial neuromodulator of striatal processing of cortical informations carried by glutamatergic synapses on medium spiny neurons, which represents the principal projection neurons of the striatum. Dopamine excites medium spiny neurons of the “direct” pathway through dopamine D1 receptors, while it inhibits striatal neurons of the “indirect” pathway through dopamine D2 receptors (Alexander and Crutcher, The pallidal complex and the STN are innervated by nigral dopamine fibers, by separate fiber system and also by collaterals of nigrostriatal fibers. This has been shown in rodents (Lindvall and Bjorklund, Dopamine, through D1 and D2 family receptors, in the pallidal complex and the STN may modulate the motor circuit and consequently dopamine depletion in these structures may play a role in the pathophysiology of PD. Lesions of dopamine neurons in the SNc in rodents and monkeys have been shown to reduce dopamine levels in GPe, GPi and STN in addition to the striatum (Parent et al., Rajput et al.  have recStudies on the expression of dopamine receptors in the pallidal complex of parkinsonian brains reported conflicting data. While some studies found no difference in dopamine D1R expression in the GPe and GPi (Rinne et al., The contribution of pallidal dopamine in the pathophysiology of PD has also been demonstrated in rodents. Activation of D1 or D2 dopamine receptors in the GP induced movement facilitation (Sañudo-Peña and Walker, in vitro (Tiberi and Caron, in vitro rat brain slices (Baufreton et al., in vivo study, in which we have shown that local microinjection of an inverse agonist of D5 receptors, flupenthixol, reduced burst activity of STN neurons and therefore improved the motor deficits in the 6-OHDA rat model of PD (Chetrit et al., STN holds a pivotal position in basal ganglia circuitry exerting an excitatory drive on the output structures of the system (Albin et al., While the degeneration of the dopaminergic nigrostriatal pathway is the hallmark of PD, there is strong evidence about the key role played by dopamine loss at extrastriatal sites, especially in the pallidal complex and the STN, in the pathophysiology of the disease. Activation of dopamine receptors in these important basal ganglia nuclei modulates their neuronal activity, and consequently participates in the beneficial effects, and may be in the adverse effects, of the classical dopaminomimetic antiparkinsonian drugs. Together, we can assume that dopamine transmission within the globus pallidus and the STN is necessary to achieve normal motor control.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.