Emerging evidence suggests that Parkinson's disease (PD) results from disrupted oscillatory activity in cortico-basal ganglia-thalamo-cortical (CBGTC) and cerebellar networks which can be partially corrected by applying deep brain stimulation (DBS). The inherent dynamic nature of such oscillatory activity might implicate that is represents temporal aspects of motor control. While the timing of muscle activities in CBGTC networks constitute the temporal dimensions of distinct motor acts, these very networks are also involved in somatosensory processing. In this respect, a temporal aspect of somatosensory processing in motor control concerns matching predicted (feedforward) and actual (feedback) sensory consequences of movement which implies a distinct contribution to demarcating the temporal order of events. Emerging evidence shows that such somatosensory processing is altered in movement disorders. This raises the question how disrupted oscillatory activity is related to impaired temporal processing and how/whether DBS can functionally restore this. In this perspective article, the neural underpinnings of temporal processing will be reviewed and translated to the specific alternated oscillatory neural activity specifically found in Parkinson's disease. These findings will be integrated in a neurophysiological framework linking somatosensory and motor processing. Finally, future implications for neuromodulation will be discussed with potential implications for strategy across a range of movement disorders. Purposeful motor behavior requires adequate spatio-temporal co-ordination of limbs and axial body parts. E.g., in catching a ball, joint movements, are arranged in such a way that the hand reaches the target's location at the correct time. These spatial and temporal characteristics of movement are also distinctively represented in the organization underlying cerebral motor control, embedded in both segregated and integrated neuronal circuitries , 2. Rega(i) planning “to be” at the right time at the right place and(ii) planning the serial order of required muscle contractions in order “to move” effectively. The first highlights engagement with a dynamic environment and underscores the relation between motor and perceptual timing. Given the relative slow cerebral processing time, such environmental engagement requires anticipation on near-future events. With regard to the temporal orchestration of muscle contractions, proprioception makes an additional sensory contribution to motor control, both in feedforward and feedback modes .The dynamics of an environment, i.e., the succession of events, is perceived as a sequence of spatial changes. Segregated analysis of the various features of  stimuli, such as shape, color, and visual motion, along parallel processing streams with varying synaptic delays, implies that the initial stimulus time is dispersed within the brain, thus losing an absolute  measure of time , 5. MoreThe concept of minimal intervals of spatial change finds particularly support from a variant of the “flash-lag illusion,” i.e., a “flash-lead” illusion. This phenomenon implies that a moving object is perceived to be behind a spatially concurrent stationary flash before the two disappear . DisappeOne of the most remarkable features of disturbed temporal processing in PD is noticed when assessing the defining feature of PD; bradykinesia. When asked to make regular finger, hand and arm movements, PD patients show specific difficulty in maintaining sufficient velocity and regularity/rhythm. Furthermore, there is a vast amount of experimental evidence that shows that temporal processing in the perceptual domain is abnormal in PD –21. ThisThe pathophysiological mechanism of the disrupted temporal processing in PD are not yet fully elucidated. The fact that there is a dopaminergic depletion in PD and that dopaminergic drugs (and DBS) restore alterations in temporal-processing made initial hypothesis about the its origin go to a “dopaminergic clock” . This isStereotactic and functional neurosurgery for movement disorders have provided a unique opportunity to directly record neural activity in the basal ganglia and cortex from either the single neuron, using micro-electrode recordings (MER), or from populations of neurons, using local field potentials (LFP). Furthermore, with advanced signal processing techniques it is currently also possible to extract EEG characteristics, that are specific for movement disorders.variability of beta-power inversely correlated with symptom severity in PD  oscillations is found throughout the motor system. Up to now, the origin of these oscillation in patients is not yet established but recent animal studies have shown that dopaminergic depletion leads to increased firing of striatal spinal projection neurons of the indirect pathway which in turn become prone to being recruited for exaggerated beta oscillations  These bety in PD . This isvolatility of beta oscillations (beta modulation) is involved in the sequencing of complex sensorimotor processes including repetitive movements  oscillatory activity to disturbed temporal processing in PD. However, beta oscillations have been involved in many other processes beyond movement. Transient beta oscillations play an important role in the successful stopping of actions , 43. Furovements  as well ovements . Based oovements ] represeovements  And consovements ].Although DBS is an established treatment for refractory movement disorders, it has its limitations in terms of efficacy and side-effects. One of the reasons for this is that not only pathological but also physiological neural activity is suppressed. Given the natural fluctuation of the severity of Parkinsonian symptoms, e.g., due to dopaminergic medication and fatigue, DBS might, in PD, only be effective when pathological neural activity is present, and symptoms are present . This ievents when pathological activation occurs. Such approach has been clinically applied for almost a decade in epilepsy patients  and recs  aligneds  and diss [e.g., . In lines [e.g.,  and corts [e.g., , which mDystonia is another movement disorder in which temporal processing has been extensively tested. Temporal discrimination thresholds have been found to be elevated across both the visual and somatosensory domains . InteresCompared to cerebellar ataxia, as seen in cerebellar degeneration, PD patients show a selective disturbance in rhythmic temporal prediction, and not in single interval prediction , 21. TheIn order to further elucidate the true nature of disturbed temporal processing and its potential therapeutic consequences in PD, several new avenues are currently being explored. Testing temporal processing draws on psychophysics, and there are some drawbacks of the different psychophysical paradigms which have been previously applied. Since most of these paradigms rely on self-report, they can be subject to bias. Standard staircase methodology in which the separation between two stimuli is slowly increased or decreased in a predictable manner allows the obtained thresholds to be readily biased by a decision strategy unrelated to temporal discrimination ability . For exaThe analysis of psychophysical paradigms has also progressed hugely since the methods of limits were established and their limitations acknowledged in the 1960s. By using reaction time data as well as well as accuracy of response inferences can also be made about the decision making components integral to the TDT. Such factors have begun to be explored in PD. For example, computational modeling has revealed that timing deficits in PD cannot be solely attributed to perceptual temporal distortions, but are also associated with impulsive decision strategies that bias patients toward premature responses . SimilarAlthough such studies increase the complexity of interpretation of simple TDT threshold embracing such techniques and analysis may prove its utility in the future. We are still far from having disease specific psychophysical markers for temporal processing. By better estimation of the true psychophysical deficit psychophysical deficits unique to particular disease states may be found. This yields greater power to researchers to discover the corresponding neural correlate which could be used for aDBS. This especially involves the experiments in which state of art neuromodulation, neurophysiology and psychophysics are simultaneously applied.Temporal processing is disturbed in PD while the severity of the movement disorder is sometimes correlated with the magnitude of changes in perceptual temporal processing. It is not yet established whether there is a causative link but pathological neural oscillatory activity might play a role. Furthermore, DBS improves motor performance and perceptual temporal processing and reduces pathological neural oscillatory activity. These observations provide indirect evidence that temporal processing is similarly affected by the same pathological neural oscillatory activity. As we move toward an era of more effective adaptive DBS finding neural correlates of temporal processing abnormalities may allow DBS to be dynamically titrated in response to a wider range of pathophysiological parameters. By bringing together neuromodulation, advanced neurophysiology and psychophysics, these hypotheses can be tested.BdJ and AS respectively wrote the 1st and 6th + 7th sections of the manuscript. MB wrote the first draft of the manuscript. MB. BdJ, AS, and ME revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We investigated what factors may foster or hinder physicians’ cancer screening risk literacy–specifically the ability to understand evidence regarding screening effectiveness and make evidence-based recommendations to patients. In an experiment, physicians in training (interns and residents) read statistical information about outcomes from screening for cancer, and had to decide whether to recommend it to a patient. We manipulated the effectiveness of the screening  and the demand of the patient to get screened (demand vs. no demand). We assessed participants’ comprehension of the presented evidence and recommendation to the patient, as well as a-priori screening beliefs , numeracy, science literacy, knowledge of screening statistics, statistical education, and demographics. Stronger positive a-priori screening beliefs, lower knowledge of screening statistics, and lower numeracy were related to worse comprehension of the evidence. Physicians recommended against the ineffective screening but only if they showed good comprehension of the evidence. Physicians’ recommendations were further based on the perceived benefits from screening but not on perceived harms, nor the patient’s demands. The current study demonstrates that comprehension of cancer screening statistics and the ability to infer the potential benefits for patients are essential for evidence-based recommendations. However, strong beliefs in favor of screening fostered by promotion campaigns may influence how physicians evaluate evidence about specific screenings. Fostering physician numeracy skills could help counteract such biases and provide evidence-based recommendations to patients. Physicians with higher numeracy and less positive screening beliefs had better comprehension, perceived less benefits from screening, and were less likely to recommend it; perceived harms were not significant predictors of recommendations. The effect of positive screening beliefs in particular was further mediated by knowledge of screening statistics  tended to rate harms “very small” whereas participants with lower numeracy (medium and lowest tercile) rated harms as “small”.Related to the above-mentioned effect of numeracy and despite the lack of significant relationship between perceived harms and recommendations, there was also an indirect effect of numeracy on recommendations via perceived harms, This study demonstrated the importance of understanding evidence about screening effectiveness for preventing biased and misleading physician recommendations. It also identified (a) a-priori positive beliefs about screening as markers of low cancer screening risk literacy, (b) physician numeracy as a specific skill that can foster comprehension and help counteract biases, and (c) knowledge of screening statistics as specific knowledge required for the correct evaluation of screening effectiveness.We found similar comprehension and knowledge gaps as those documented in previous research with physicians in training and experienced physicians in other countries  ,13,43. MFortunately, a recent study showed that even 90 minutes of training can dramatically improve the risk literacy of medical professionals in training  . The traPositive a-priori beliefs about screening were related to stronger recommendations in favor of screening via knowledge of screening statistics and comprehension of the evidence. In other words, physicians who tended to view screenings very positively were not aware of what statistics should be consulted to rate screening effectiveness, which contributed to their lower comprehension of the presented evidence and stronger recommendations in favor of screening. This suggests that participants who already had a strong positive opinion about the value of screening in general may have been less likely to examine the statistical questions and evidence critically and thoroughly, leading to wrong answers. Another possible explanation is that participants with stronger positive beliefs were previously exposed to misleading statistics regarding screening or were never exposed to information about screenings with little or no effectiveness. This could have helped generate their strong positive beliefs about screenings and contributed to their inability to properly evaluate the evidence presented. Whatever the mechanisms, making recommendations to patients based on general beliefs when the evidence at hand is at odds with these beliefs represents a bias, and the extensive training received did not protect the physicians in our study from such misguided judgments that are also found in laypersons . Future The specific knowledge about what screening statistics are relevant to assess screening effectiveness was a unique predictor of correct comprehension of the evidence, suggesting that this knowledge should be part of medical curricula for specialties where screenings for diseases such as cancer are relevant.In addition, consistent with previous research in experienced physicians , high nuA process model similar to that found in laypersons ,17 showeAlso similar to results obtained in laypersons, perceived harms were much less predictive of decisions . It is pWhereas the current results show that numeracy and better statistical knowledge can help counteract the detrimental effects of previous beliefs, science literacy did not emerge as an important factor. However, the scale used in the current research was brief and also easy for the surveyed population. Future research should investigate the role of science literacy using more appropriate and elaborate instruments.Similarly, the patient demand manipulation did not show the expected effect on recommendations. On one hand, it is possible that the manipulation was not strong enough to produce an effect–in the demand condition the patient was said to want to undergo the screening but nevertheless requested the physician’s opinion. On the other hand, patient demand may not be important among inexperienced physicians in artificial scenarios. However, it is often mentioned by physicians themselves as a determining factor and thus is likely highly important in real clinical situations –37.Participants of the current study were enrolled in one of the top-ranking medical programs in Peru and in Latin America. Given that cross-cultural differences in risk literacy have been documented , it is nDespite a rich literature on patient risk literacy, not many studies have addressed what influences physicians’ risk literacy. Given the multiple nuances and challenges of doctor-patient communication, research on physician risk literacy beyond artificial scenarios and in actual interaction with patients is needed. The current results demonstrate that in the context of cancer screening, a-priori positive beliefs about the goodness and desirability of screening, likely reinforced by multiple screening campaigns, and low physician numeracy can be important precursors of low physician risk literacy and biased, misleading recommendations.S1 Table(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Socioeconomic Action Plan. It is time to raise the socioeconomic design of nutrient policies to the same level of sophistication as the ecological foundations of the BSAP.This paper analyzes the main weaknesses and key avenues for improvement of nutrient policies in the Baltic Sea region. HELCOM’s Baltic Sea Action Plan (BSAP), accepted by the Baltic Sea countries in 2007, was based on an innovative ecological modeling of the Baltic Sea environment and addressed the impact of the combination of riverine loading and transfer of nutrients on the ecological status of the sea and its sub-basins. We argue, however, that the assigned country-specific targets of nutrient loading do not reach the same level of sophistication, because they are not based on careful economic and policy analysis. We show an increasing gap between the state-of-the-art policy alternatives and the existing command-and-control-based approaches to the protection of the Baltic Sea environment and outline the most important steps for a Baltic Sea  The greatest environmental challenge in the Baltic Sea is eutrophication and the ecological risks it causes . The reduction targets of the BSAP are 11% for N and 43% for P for the riverine loads, so the distance to the goal is still considerable. The area of anoxic bottoms has been increasing, however , environmental directives, and the Baltic Sea Region Strategy.There is a fundamental difference between nutrient load policies aimed at point sources, such as waste water treatment plants (WWTPs) and industry, and those targeting nonpoint sources, such as agriculture and forestry. Point sources release nutrient loads via definite points, pipes, so their loads can be easily measured and directly subjected to regulation. Nonpoint loads, in contrast, are diffuse, coming from surface and drainage; they are stochastic due to varying weather and often associated with a delay between action and releasing loadings. Therefore, it is not possible to register the exact amounts of nutrients that are released from a given field parcel or forest plot, making it impossible to levy policy instruments directly on loads (Shortle and Dunn Table −1 and € 223 (kg P)−1 (Hyytiäinen and Ollikainen −1 and a 95% reduction in phosphorus costs € 17 (kg P)−1 This discussion provides a lesson: cost-effective abatement with equalization of the marginal abatement cost should be the guiding principle of nutrient policies toward point and nonpoint sources, because this principle reflects best the technological and economic possibilities to reduce loads the most.Abatement of nutrients in WWTPs and industrial point sources provides the backbone of any well-designed policy for the Baltic Sea protection. Abating both nutrients in the WWTPs is certain and less costly relative to other sectors, albeit reducing nitrogen requires a high initial but a long-lasting investment. The best available technique facilitates higher emissions reductions than the abatement rates in the above example, as experience in many countries has demonstrated. Hautakangas and Ollikainen  provide The main weakness of nutrient policies toward point sources is that both the EU’s Urban Waste Water Directive and HELCOM recommendations are inattentive relative to current abatement possibilities and abatement costs, and we propose that they should be scaled up accordingly.Another weakness of policies toward point sources is their reliance on command-and-control instruments only. Countries should make further efforts by financing research and providing result-based schemes, to create better incentives to extend abatement beyond conventionally known abatement technologies. These efforts include economic incentives not only to increase abatement but also to find new and innovative ways to treat wastewater, such as extracting phosphorus from sewage water for new products and using the abatement process and sludge to produce energy in the spirit of the EU’s circular economy initiative.A topic not so often discussed in the literature is abatement in industrial point sources. It is a drawback that HELCOM does not report separate data on industrial point sources’ contributions to nitrogen and phosphorus loads at the Baltic Sea level but provides only an aggregate number for all point sources. Databases for improving water policies toward industrial point sources should be developed.As the setting of optimal taxes or quantitative limits on runoff from fields is infeasible, many countries have agri-environmental schemes that rely on farmers’ voluntary participation and pay for taking conservation measures among the given a set of measures targeting nutrient loads. Designing an effective voluntary agri-environmental program faces three basic challenges: (i) how to make it effective for water protection, (ii) how to invite the farmers that could contribute the most to the goals of the program  contains cross-compliance conditions, which require farmers to undertake environmental measures to be eligible for the single farm payment. Figure Participation rates in voluntary agri-environmental programs vary enormously, with Denmark, Poland, Latvia, and Lithuania having low participation rates. Caution should, however, be used when assessing the role of high participation rates in producing water quality improvement. There is a trade-off between participation and ambition. For instance, Lankoski and Ollikainen  demonstrThe impact of the measures included in agri-environmental schemes on nutrient loads depends on their environmental effectiveness and local conditions. Most programs offer a set of measures for farmers to choose from, such as buffer strips and buffer zones, reduced fertilization, catch crops, conservation or restoration of wetlands, grassland management, set-aside and cover crops  subsidies to incentive-based instruments, and increasing environmental targeting (result-based measures) by introducing environmental benefit indexes to both flat rate policies and incentive-based schemes. Both avenues facilitate improved environmental targeting, and the incentive-based instrument helps by using government budget money more efficiently.G and the area payment be s per ha. Suppose for simplicity that each farmer supplies a field parcel of size one hectare (denoted by i) to the program and implements some of the listed water quality measures on this parcel. The costs of these measures, ci, differ between farms. Figure Latacz-Lohmann and van der Hamsvoort  demonstrs*5 = G (the budget is exhausted). The last parcel (5) receives a compensation that exactly matches its farmer’s opportunity costs but the others receive “overcompensation” , because their costs are lower than the payment. This overcompensation is information rent, as it results from the authorities’ incomplete information on farmers’ costs. Because of overcompensation, only 5 parcels are enrolled in this example, and parcels 6 and 7 remain out of the program.In Fig. In environmental tendering, the authorities announce the environmental goals (a reduction nutrient runoff) and invite farmers to offer their fields with assigned water protection measures to the program. While under a discriminatory tendering, each enrolled field parcel receives exactly its bid; under a uniform tendering, a uniform compensation is paid for all winning parcels . As the government saves money, more fields can be allocated in the program. In Fig. Figure Both tendering and flat rate policies can be improved by using a performance-based approach, such as environmental benefit indexes (EBIs), which assess the environmental performance of the chosen measures. An EBI is simply a number, scaled, for instance, between 0 and 1 or 0 and 100. An EBI is a product of chosen features drawing on their modeled impacts on water quality and weighted by their relative contribution to it. For instance, such features include slopes of fields, sizes of the buffer strips, or soil phosphorus reserves. How well the constructed EBI describes factors determining nutrient runoff depends directly on the state of scientific knowledge. EBIs also help to differentiate fixed payment rates and target more efficiently environmental outcomes, as is done in the Conservation Reserve Program (CRP)  find large differences in the load reductions from manure investments between the countries. While the potential is modest in Denmark, which already has mandatory and high requirements regarding utilization of nitrogen in manure, the effects are higher in Sweden, Finland, Poland, and the three Baltic states. Sufficient storage capacity is important for farmers to be able to apply the manure on fields during the period when the crops grow and utilize the nutrients from the manure in spring and early summer  to 17 273 million LSU). The number of pigs has decreased in all countries, with a total decline of 14%. Poultry production has increased in almost all countries, with a total increase of approximately 21%. The strongest trend, however, lies in the number of livestock farms: it has decreased by almost 40%, indicating also an increasing farm size and manure concentration Eurostat . The samThe structural change in livestock farms has important implications for the availability of land for manure applications. Larger farms sizes entail higher risks that manure will be overapplied on the fields closest to animal facilities. Solutions to tackle the problem vary across countries. In many countries, the Nitrate Directive or phosphorus fertilizer limits command that expanding livestock farms have enough manure spreading area. Clearing peat land forest to create fields in Finland has been very detrimental, because new fields have increased deforestation, GHG emissions, and nutrient runoff. Denmark has promoted biogas production, which provides climate benefits but does not alleviate the transportation cost problem unless nutrient separation techniques are adopted.Thus, current policies have not adapted to the rapid increase in the size of livestock farms . The water quality targets must not be compromised when climate policy is given more attention  Do current policies provide sufficient incentives for innovation? (ii) If not, how can the incentives be improved? (iii) Will the novel technologies be adopted by the intended users?Markets suffer from under-provisioning of innovations: innovators’ net gains from innovation are small in comparison to the overall gains, because innovations could be copied by other firms  and city or an industrial point source could offset their loads by buying reduction from another agent that can reduce loads at lower social costs. Moreover, municipal WWTPs could be willing to promote water protection if an equivalent sum of their investment could play for higher reductions elsewhere in the drainage basin. Therefore, pursuing nutrient neutrality should be promoted by creating transparent and clear systems for nutrient compensations. To provide an example, four Finnish WWTPs located on the coast of the Gulf of Finland compensated for their P loads by investing in abatement in Vitebsk, Belorussia.More importantly, nutrient compensations may have a much higher status in the future. Water protection within the EU is unified by the Water Framework Directive (WFD). The recent Weser ruling of the European Court of Justice (C-461/13) strengthened the legal status of WFD-specific water quality standards, which will be reflected in environmental permitting processes also around the Baltic Sea. Under a strict interpretation, an environmental permit cannot be given to an economic activity if it increases the pollution of elements critical to water quality standards.To prevent the emergence of unintended constraints from well-intended regulatory changes, some flexibility should be built into environmental instruments. One option is utilizing nutrient offsets in the permitting process. In our example, the facility would create nutrient credits by decreasing the nutrient loading risk from the livestock facilities it collects the manure from. These credits would be taken into account when determining the net effect of the new facility on nutrient pollution. Similar practices could be used for many economic activities as long as the basic condition is met: the new or expanding economic activity together with the offsetting credit generates a net decrease to total nutrient loading to the respective water body.We must ensure that the regulation is keeping pace with not only the structural change and the challenges it imposes but also new innovations that help mitigate nutrient loading.Our analysis has identified weaknesses and possibilities for improvements in Baltic Sea protection policies. For point sources, the key weaknesses include too lax regulatory policies toward WWTPs and missing incentives for developing new and novel abatement solutions, for instance, promoting a circular economy. For nonpoint sources, current CAP policies prevent using performance-based policies. Inefficient regulation of livestock farming and missing incentives for promoting technological developments belong to other key weaknesses. The invented novel policy principles and instruments facilitate improving the environmental and economic efficiency of Baltic Sea policies. They provide polluting agents with stronger incentives to protect the sea, to promote environmentally friendly innovations, and to engage voluntary citizens in useful work for the Baltic Sea, and they constitute a sound standpoint to meet the challenges that climate change brings to the Baltic Sea region in coherence with water policy requirements.Cost-effective abatement with equalization of marginal abatement cost should be the guiding principle of nutrient policies toward point and nonpoint sources, because this principle reflects best the technological and economic possibilities to reduce loads the most.Both the EU’s Urban Waste Water Directive and HELCOM recommendations are inattentive relative to current abatement possibilities and costs in WWTPs. They should therefore be scaled up accordingly to promote cost-effective abatement.The rigid EU CAP policy toward agriculture should be changed to facilitate modern, incentive-based, and performance-oriented agri-environmental policy instruments instead of the current measure-based approach.Tighter regulation of large livestock farms and promoting industrial-scale treatment of manure in the food processing sector provide possibilities for new technological solutions and business opportunities.Promoting industrial-scale treatment of manure by vertically integrating the food processing sector would provide one possible solution to farm manure management.There are ample possibilities to create coherent nutrient and climate mitigation measures in agriculture that should be utilized.Regulation must keep pace with economic development and tightening environmental standards by utilizing flexible and innovative instruments, such as nutrient offsets.The research community can now provide key solutions to reduce loading and fit the long-term economic growth to the ecological limits of the sea with all stakeholders engaged to determined actions. A Baltic Sea Socioeconomic Action Plan is called for to systematically update and strengthen nutrient policies in the Baltic Sea region countries. As the first steps toward developing this plan, we suggest that the following features should be the backbone of such a plan.


Visualization of algal viruses has been paramount to their study and understanding. The direct observation of the morphological dynamics of infection is a highly desired capability and the focus of instrument development across a variety of microscopy technologies. However, the high temporal (ms) and spatial resolution (nm) required, combined with the need to operate in physiologically relevant conditions presents a significant challenge. Here we present a short history of virus structure study and its relation to algal viruses and highlight current work, concentrating on electron microscopy and atomic force microscopy, towards the direct observation of individual algae–virus interactions. Finally, we make predictions towards future algal virus study direction with particular focus on the exciting opportunities offered by modern high-speed atomic force microscopy methods and instrumentation. Considering the sheer diversity and abundance of algal viruses ,2, and tVirus structure knowledge took a leap forward in the year 1956. Crick and Watson suggested the hypothesis that ‘rod’ and ‘spherical’ viruses could be built from regularly packed identical sub-units. The basis for this idea was the high degree of order seen in X-ray photographs of crystallized plant viruses . These tPost-1956, research included the probable icosahedral structure of Tipula iridescent virus using EM with double shadowing , direct The main technique for ultrastructural studies in this early era was electron microscopy (EM) having been invented in 1935 . This de2 . Using only basic electron micrograph and X-ray diffraction data the pair were able to generate a theory on virus structure using geometric rules, later proven correct by direct visualization. EM and cryo-EM data has been iteratively improving since its inception and use in icosahedral viral analysis, increasing resolution for PBCV-1  methods for virus samples  for an ir PBCV-1  three-foEM techniques are not only useful for intact virus structural study but can also be used in investigation of viral dynamics and the analysis of individual components. For example, when using TEM and its various derivatives such as scanning transmission electron microscopy tomography, immuno-electron microscopy and whole-cell focused ion beam-scanning electron microscopy ,25. ThesAnother form of microscopy and virus structure analysis tool is atomic force microscopy (AFM). Invented in 1986 by IBM scientists , the AFMAlthough a cryo-atomic force microscope was developed in 1996 , this teFor the next twelve years the California group published extensively their research into AFM study of viruses (reviewed in ), with tIt is of note that a 2002 study by the California structural group performed a similar viral infection experiment to the IBM scientists from 10 years previous , however−1  sho shoSacchEmiliania huxleyi in natural and modified forms. Also, discovery of specific membrane areas involved in algal viral adhesion, and fusion entry and exit should be possible.To measure these algal host-virus interactions, there are currently two main approaches that could be pursued. One is taking advantage of the speed of HS-AFM to truly visualize and gather data on dynamic infection processes. To achieve this, taking advantage in improvements in live cell imaging techniques and using a HS-AFM with the resolution and frame rate necessary to observe fast biological processes is key. The second avenue is an aspect of AFM not thus far mentioned. Atomic force microscopes can be operated in a point-measurement mode that enables them to collect force-distance curves at different locations on the nanoscale surface. These curves can then be processed to obtain data about the material properties of the sample surface. With these methods it may be feasible to quantify lipid domains/sphingolipid raft size structure and characteristics with conceivable inference of sphingolipid production rates in examples such as Having the sensitivity to measure piconewton interaction forces enables AFM to study the interaction forces between single biomolecules  and to pThere are other imaging modes available for AFM  that havC. variabilis cells and AFM of osmotic shock treated virus was used to examine single virions and their emerging DNA with associated proteins (AFM, in its basic form is fundamentally a surface imaging technique similar to rudimentary SEM. SEM in truth does have an interaction volume, giving information about electronic and elemental characteristics. AFM can provide physical and mechanical characteristics of samples such as friction and stiffness. However, in the context of this section SEM and AFM are considered as surface imaging techniques. The idea that AFM in this respect provides insight into the internal features of various biological samples seems counter-intuitive considering the mechanism of data acquisition. Indeed, few studies have explored this idea. With current technology, techniques including Cryo-ET, confocal and fluorescence based microscopy arguably produce better results with less effort. However, some intriguing progress has been made into using AFM to examine the structures hidden below the surface of viruses. As eluded to above, in the case of virus structural study, the disruption of the capsid shell and in some cases external lipid membrane either by accident or on purpose, often exposes the features not immediately available by AFM or SEM as seen in proteins b. FurtheThe focus of algal virus study has followed the same paths as generally pioneering studies into medically relevant, human pathogenic viruses. This being the initial interest into the unseen structure of these nanoscale biological particles, how their structure relates to and follows geometric rules, the structure of the individual building blocks and evenually insight into infection dynamics and strategies. The principles outlined early in this review of theory and orthogonal instumentation and methodology have been key in every stage of the improving understanding. Theory suggests hypotheses to later be proven by improved instrumentation, and the improved instumentation providing data from which more detailed theory can be hypothesised. Key to this process, and outlined in part in this review, is the constant iteratively refining methodology for analysing these host-viral systems on such a small scale. We have covered the basic history of some of the methods available for use in experimental studies at this time, with a particular focus on different forms of microscopy and especially atomic force microscopy. Atomic force microscopy, we believe, has potentially been overlooked as purely a structural tool, but has showed recent promise as a method for dynamic observations. We have confidence that with reducing costs and increased availablity and when applied in parallel/correlatively with other virology approaches; these techniques can be effectively functional in the study of algal viruses to determine replication mechanisms and to collect evidence for existing theory. Hopefully, as is usually the case, once methods are established they become far easier to perform and more widely available, leading to a far wider range of algal viruses being studied in greater detail when compared with the limited test systems being used in recent histroy and current work.What is the limit of resolution when using these techniques? For EM, we are currently at near atomic resolution  and for


Aging-related changes challenge older adults’ experience of social connectedness and increase their risk of perceived isolation. As seniors cope with these changes, they often face the question of whether to age in place or move to a senior living facility. While many studies explore these scenarios separately, there is little research comparing these two living arrangements. Therefore, this study examined the following questions: how do older adults experience social connectedness and perceived isolation? How does this experience vary between older adults living alone in private homes and those living in assisted living facilities? I conducted 16 qualitative interviews with middle-old and old-old women (ages 75+) who lived alone in a private home or in an assisted living facility in southeast Michigan. The themes that emerged revealed differences in three aspects of social connectedness; interactions, relationships and belonging. Community dwelling respondents’ interactions were characterized by intentionality, with minimal investment in forging new or deeper relationships and an emphasis on belonging to the world in terms of awareness, contribution, and cognitive ability. Assisted living respondents’ experiences where characterized by availability of interactions and casual relationships within the facility and an emphasis on belonging to the facility community, while positioning oneself between the status of resident and staff. Surprisingly, neither group reported a notable degree of perceived isolation. Both had adapted their social connectedness expectations to reflect their current situation. These findings have meaningful implications for older adults facing decisions about where to age, as well as for the communities that serve them.


Abnormal eye movements in children, including nystagmus, present a significant challenge to ophthalmologists and other healthcare professionals. Similarly, examination of supranuclear eye movements and nystagmus in children and interpretation of any resulting clinical signs can seem very complex. A structured assessment is often lacking although in many cases, simple clinical observations, combined with a basic understanding of the underlying neurology, can hold the key to clinical diagnosis. As the range of underlying diagnoses for children with abnormal eye movements is broad, recognising clinical patterns and understanding their neurological basis is also imperative for ongoing management. Here, we present a review and best practice guide for a structured, methodical clinical examination of supranuclear eye movements and nystagmus in children, a guide to clinical interpretation and age-appropriate norms. We also detail the more common specific clinical findings and how they should be interpreted and used to guide further management. In summary, this review will encourage clinicians to combine a structured assessment and a logical interpretation of the resulting clinical signs, in order to recognise patterns of presentation and avoid unnecessary investigations and protracted delays in diagnosis and clinical care. The function of eye movements is to bring visual stimuli to the fovea and hold them there, during head movements or movement of the stimuli themselves. Examination of these eye movements in children, in an outpatient setting, can present a clinical challenge. However, the information gained from an effective examination can help the clinician discern benign from pathological disease, localise neuropathology and non-invasively monitor a variety of neurological disorders. Hereafter, we outline methods of eye movement examination, the most important findings and their clinical implications for clinicians without access to a dedicated eye movement laboratory.Human supranuclear eye movement pathways can be conceptualised as providing solutions to the following visual demands: to compensate for large head movements (the vestibulo-ocular reflex (VOR)), to use visual information to fine-tune the VOR and to return the eyes to the central position after full excursion (the optokinetic reflex (OKR)), to track a moving visual target without moving the head , to place a visual target onto the fovea (saccades) to maintain eccentric eye position (gaze holding) and to allow binocular co-ordinated eye movements to achieve and maintain binocular foveation (vergence movements). Table As with all clinical assessments in children, a detailed history from the parent and child is important and can direct the clinician to certain aspects of the subsequent examination. Table When examining supranuclear eye movements in a child, it is important to assess each separate eye movement control system in a systematic way, with an understanding of what to expect . ‘NormalA three-neuron arc, including the vestibular ganglion, vestibular nuclei and the oculomotor nuclei Fig.  2] oper oper2] oA mismatch between the speed of head movement and the speed of the VOR movement (VOR gain) can be tested in older children by measuring visual acuity during slow head movements (which should not change). However, this test is rarely used in the clinic as it is not possible in preliterate children and isolated abnormalities of VOR gain are rare.‘Dolls Head Manoeuvre’  or Spinning baby tests are the standard tests for VOR in the clinic.The infant’s head is held with both hands and rapidly, but gently, moved horizontally and vertically to test both VOR, respectively. The eyes should stay fixed in position despite the movements. The rotations can be small and should not be performed in children with cervical spine problems or downbeat nystagmus Fig. .Fig. 2HoThe examiner stands and holds the infant at arm’s length. In the case of a very young infant the back of the head is supported as shown. The examiner rotates to the right with the infant through 2–3 revolutions, whilst observing the infant’s eyes. This induces a per-rotatory nystagmus (driven by VOR and OKR) in the infant with fast phases to the examiners left. The rotation is then abruptly stopped inducing a post-rotatory vestibular nystagmus in the infant, with fast phases beating to the examiners right. The procedure is repeated in the opposite direction and in small infants can also be used to test vertical VOR by laying the infant flat in the examiners arms. In older children this test can be modified by the child sitting on an examiners lap and facing them while both rotate on a swivel chair. Older children can also be examined by sitting alone in a swivel chair and spun. Normal findings include a large per-rotatory nystagmus (unless the child is fixing on the examiners face) but importantly, only two to three post-rotatory nystagmus beats in the opposite direction . This can be seen as ‘Locking up’ during spinning .During doll’s head testing, a catch-up saccade, when testing in one direction only, suggests a specific semi-circular canal pathology.In the presence of acute vertigo in older children, abnormal VOR in one direction, lack of a skew deviation and unidirectional nystagmus suggest a peripheral vestibular  rather than central cause .After spinning, post-rotational nystagmus lasting more than two to three beats is a sign that the VOR is not being supressed by visually guided reflexes. This is either an indication of very poor vision or a cerebellar disorder .The VOR reflex may be decreased or absent in children with the CHARGE syndrome  , in typeSome authors consider the pathway for OKR, tested with an OKR drum or tape in the clinic, as the same as the smooth pursuit pathway, involving striate and extra-striate cortices with descending connections via the internal capsule to the brainstem pontine nuclei, cerebellum (floccular region), vestibular nuclei and the oculomotor nuclei . Most clOKR drum or tape is held at 50 cm from the child’s face and rotated slowly in both horizontal and vertical directions whilst observing the child’s eye movements. It is performed both monocularly and binocularly. The examiner would expect to see slow eye movements in the direction of the stimulus with a fast movement seen in the opposite direction , but may also be seen in drowsy infants. Interestingly, a full-field OKR can be elicited in some patients with DVM even where a small field OKR is absent. It has been hypothesised that using a full-field stimulus may elicit an OKR reflecting a more primitive, subcortical OKR subtype called OKNd , howeverVertical OKR can be demonstrated in young infants with horizontal nystagmus as a superimposed waveform if there is moderate vision. It can be used (prior to EDTs) to reassure that there is unlikely a profound visual disorder. There has been debate about whether horizontal OKR is lost, reversed or altered by shifting null zones in congenital nystagmus. By clinical observation alone, it is rare to observe robust horizontal OKR in the presence of horizontal nystagmus but where clearly demonstrable, possibly suggests that an ‘idiopathic’ or albinism related cause is less likely.Quick phases of the OKR are intermittently absent in SIF (see disorders of saccades below). Similar to VOR testing, it can result in a lack of OKR or in deviation of the eyes in the direction of the slow phases because of a mismatch between the slow phase movement and the insufficient corrective saccade.Asymmetry in response to a right vs left moving OKR stimulus, when tested binocularly, indicates a unilateral cortical or pontine lesion. Asymmetry of OKR when tested in this way may occur in some patients with early-onset poor vision in one eye.A vertical binocular OKR response which is worse than horizontal response indicates a midbrain lesion.th nerve nucleus as well as contralateral third nerve nucleus via the medial longitudinal fasciculus (MLF)  which projects to the pons and midbrain nuclei. In the pons and midbrain, the neurons from the ventral limb input into a local control system for saccadic firing. This comprises excitatory burst neurons (EBNs), which fire to generate saccades, inhibitory burst neurons (IBNs), which are activated by EBNs and inhibit firing of neurons to contralateral extra-ocular muscles and pause units. IBNs tonically fire to inhibit the EBNs and therefore continuously inhibit spontaneous saccades. Horizontal saccades are mediated by the ipsilateral pons and contralateral frontal eye field (FEF) and parietal eye field (PEF); vertical saccades are generated in the midbrain. The EBNs project to the ipsilateral 6LF) Fig. .Fig. 5SaFrom a clinical stand point, it is important to remember that horizontal and vertical saccades are localised separately in the ponto-medullary junction and the midbrain respectively and that the cerebellum controls the metrics for both.spinning baby test, includes a saccadic component and can be the most informative assessment in younger infants. In older children, a brightly coloured target is presented in front of the child at a large angle  and eye movements are simply observed for direction, accuracy and symmetry (test monocularly if cross-fixating). The examiner should observe the presence of saccades to the right, left, up and down in both eyes. When looking for asymmetry  fixing on the bridge of the nose can aid the examiner.Clinically, saccades can be tested in many ways according to age. The previously described Saccadic latency in infants is up to 1 s for the primary saccade (200 ms in adults) and gross abnormalities are usually seen in SIF.SIF may be congenital with no recognisable pathology or associated with many underlying disorders. It is usually horizontal only and intermittent; if vertical it suggests a midbrain lesion or metabolic disorder such as Niemann–Pick disease type C or Gauchers disease type 3 .Isolated dysfunction of vertical saccades is due to a midbrain lesion such as haemorrhage/Niemann–Pick disease type C. It is usually caused by lesions of the riMLF which is the centre for vertical saccadic control. As upgaze movements are double innervated, abnormalities of downward saccades are often observed first.Lesions of the interstitial nucleus of Cajal also cause dysfunction of vertical saccades but usually in addition to a vertical gaze-evoked nystagmus (GEN).Lesions of the posterior commissure cause dysfunction of vertical saccades which may be accompanied by convergence-retraction nystagmus and lid retraction in parinaud dorsal midbrain syndrome. Pressure on the posterior commissure from raised intracranial pressure is also thought to give rise to the ‘setting-sun’ sign (eye seemingly fixed in downgaze) which is unique to infants and children .Lesions of the cerebellar oculomotor  vermis or fastigial nucleus can cause saccadic dysmetria. Hypermetric saccades are seen in lesions of the fastigial nucleus  and hypometric saccades with lesions of the dorsal vermis.Large visual field defects may also cause hypometric saccades into the area of loss and they are also seen in resolving DVM type 1.Isolated dysfunction (slowing ±hypometria) of horizontal saccades is due to pontine lesion of the PPRF, e.g., bleeding/glioma/Gaucher disease type 3.Square-wave jerks are to one side of fixation only (<5° from fixation), last 200 ms and are most commonly seen in spino cerebellar atrophies. They are an exaggeration of normal microsaccadic fixation movements and are seen in 90% of infants at up to 3/min and are rarely of key clinical significance.Opsoclonus is characterised by intermittent, multidirectional, back-to-back saccades and is best viewed after changing fixation or on lid closure. Ocular flutter describes the same anomaly but confined to horizontal eye movements. They share a common aetiology, which is thought to be loss of cerebellar inhibition of spontaneous saccades. Opsoclonus–myoclonus syndrome describes the combination of opsoclonus, myoclonus and ataxia and is most commonly associated with neuroblastoma, other para-neoplastic states and CNS infections . Ocular Anti-saccades are voluntary saccades generated to the opposite direction of a target introduced into the peripheral visual field. They represent a voluntary saccade overcoming a reflexive saccade and may be misdirected  in children under seven, schizophrenia, ADHD and Tourette syndrome. They are rarely of clear diagnostic value in young children.not nystagmus (as there are no slow phases). It can be simulated in many healthy subjects and clues to the diagnosis include: miosis, lid flutter and an inability to sustain the movements for more than 30–45 s.‘Voluntary nystagmus’ is an infrequent but persistent cause for referral in children. It is characterised clinically by back-to-back saccades , usually induced by convergence effort and often accompanied by mild lid closure. It is therefore Gaze holding is controlled by a group of neural structures which differ for vertical/torsional and horizontal meridians. These distinct groups of structures are collectively termed the horizontal and vertical ‘neural integrators’. The horizontal neural integrator includes the nucleus prepositus hypoglossi (Pons), vestibular nuclei (Pons and Medulla) and vestibulo-cerebellum (Cerebellum). The vertical/torsional neural integrator is the Interstitial Nucleus of Cajal (Midbrain) although an intact flocculus (Cerebellum) is also necessary. Both neural integrators have multiple neural connections and their primary role is to coordinate tonic discharge to the extra-ocular muscles in order to maintain eccentric gaze.Observe the eyes in primary gaze and in vertical and horizontal eccentric gaze either by turning the patient’s head, or by presenting a large stimulus in eccentric view. Abnormalities of gaze holding are usually characterised by nystagmus, due to a slow drift towards primary position and a corrective saccade into the attempted position of eccentric gaze (GEN). Examination of nystagmus is detailed below.Typical signs of damage to the neural integrator, whether vertical, torsional or horizontal, include GEN (due to loss of eccentric gaze holding) and rebound nystagmus (nystagmus beats seen in the opposite direction to the eccentric gaze when the eye is returned to primary position). Abnormalities of VOR suppression  and OKR are also commonly seen together and reflect underlying cerebellar pathology.GEN can also be seen with the use of anti-convulsants, sedatives and other drug intoxications.GEN in all directions implies a cerebellar lesion, particularly the flocculus and has many causes.Isolated horizontal GEN is often caused by an isolated pontine lesion. This is because vestibulo-cerebellar disorders tend to yield additional oculomotor signs.Purely vertical GEN is due to a lesion of the vertical/torsional neural integrator which is the interstitial nucleus of Cajal (midbrain).The presence of rebound nystagmus may indicate chronic, rather than acute cerebellar disease.The neuroanatomy underlying vergence eye movements is poorly understood. Neurons within the mesenchephalic reticular formation (midbrain) and adjacent rostral superior colliculus have been identified as being related to vergence movements. These include neurons whose discharge is proportional to vergence angle  and burst neurons. Subsets seem to exist for convergence and divergence activity but detail is currently lacking.Vergence movements are binocular co-ordinated movements in opposite directions in order to move the point of fixation towards, or away from the subject. Convergence movements can be observed in infants using a large visual target, moved slowly towards the nose in primary gaze from approximately two months of age. They are thought to be initially driven by accommodation but after 4 months they are driven by retinal disparity which coincides with the development of fusion and stereopsis . From arVergence abnormalities are rare in children unless associated with strabismus.Neuropathology associated with isolated anomalies of vergence is very rare.convergence spasm can occur in association with organic and non-organic disease. However, organic pathology is rarely found in an otherwise well child with isolated convergence spasm [Numerous reports and case studies have suggested that ce spasm , 13.Convergence insufficiency describes a remote near point of convergence, variable exophoria for near and reduced fusional convergence at near. It is not associated with underlying intracranial lesions and when occurring in isolation, usually in adolescents, may be treated with orthoptic exercises and prisms [d prisms –16.Convergence paralysis is characterised by complete loss of convergence and crossed diplopia. It is highly suggestive of intracranial pathology resulting from space occupying lesions of the midbrain, infection, trauma, demyelination or toxins [r toxins –19. It iDivergence paralysis is rare in both paediatric or adult practice. It is characterised by complete loss of divergence, esotropia for distance and normal convergence. Bilateral sixth nerve palsy should be excluded. Causes include intracranial infection and lesions of the brainstem and periaqueductal grey matter [y matter –22.Divergence insufficiency is a more controversial entity and is characterised by an esotropia for distance, normal convergence and variable or reduced fusional divergence [vergence . It has vergence –25. In tThe neuroanatomical pathways underpinning smooth pursuit movements have been studied extensively and many pathways and nuclei are known to play a part. The following is a simplification of the core circuit. Visual information from V1 is conveyed to area V5  and then descends to the pontine nuclei. They are joined here by additional inputs from descending fibres from the frontal eye field (Brodman area 8). Fibres are relayed from the pontine nuclei to the flocculus and ventral paraflocculus of the cerebellum. Floccular Purkinje cells then encode eye position, velocity and to a smaller extent, acceleration. Projections then extend to the vestibular nuclei and on to the oculomotor nuclei within the brainstem , 26, 27.Observing suppression of post-rotational nystagmus after the spinning baby test.Observing suppression of VOR while spinning baby and encouraging fixation on examiners face.Observing suppression of VOR using a target which moves with the head and encouraging horizontal and vertical head movements. The target can be the patients finger held in front of the face or a target on a tongue depressor held in the patient’s mouth.Testing should be performed both horizontally and vertically using a large, slow moving, brightly coloured target or a slowly rotating mirror. The examiner should observe the character of the smooth pursuit movements (jerky or smooth) and identify asymmetric smooth pursuit which can be aided by focussing on the bridge of the patient’s nose. An OKR drum or tape can be used if examining an uncooperative infant. Suppression of VOR and smooth pursuit are closely related oculomotor functions with similar underlying neurological substrates. Therefore, abnormalities in both systems commonly occur together. VOR suppression can be assessed by:Lesions of the flocculus or cerebellar pathways cause saccadic pursuit and loss of VOR suppression by visual fixation in addition to down beat nystagmus (DBN).Reduced smooth pursuit in all directions indicates a ‘general’ cerebellar flocculus disorder .Asymmetrical loss of smooth pursuit  suggests a structural lesion of the ipsilateral cortex or cerebellum. It may also indicate early-onset poor vision in one eye.Nystagmus is a disorder of eye movement which can be defined as ‘a repetitive, to-and-fro movement of the eyes that is initiated by slow phases’ . It is iClassification of nystagmus has been inconsistent in the literature and in the following section, we use the term infantile nystagmus syndrome (INS) and fusion maldevelopment nystagmus syndrome (manifest latent nystagmus or FMNS) according to the CEMAS classification of 2001 . It is iObserve the child at play to identify any consistent visual behaviours such as head positions and head shaking , in addition to signs of systemic neurological features such as ataxia.In addition to a routine, age-appropriate history, orthoptic examination and refraction, children’s eye movements should be observed in five positions of gaze and note taken of the direction of the fast phase of nystagmus (if jerk is seen), the amplitude of the nystagmus (fine or coarse) and the effect of monocular viewing and removal of fixation.Where MLN or FMNS is suspected, rotating the child to induce post-rotational nystagmus and then occluding each eye separately can make identification easier. The induced post-rotational nystagmus to the right or left will either add to the existing MLN or reduce it, depending on which eye is covered, yielding a clear inter-ocular difference in nystagmus intensity.Abnormal eye movements should be observed for at least 5 min  to examine for reversal of nystagmus direction and any alteration in head posture. Inconsistencies in history/examination/previous clinical notes may suggest an alternating head posture often due to periodic alternating nystagmus (PAN), which itself can be congenital or aquired.An examination of saccades/smooth pursuit/gaze holding/VOR/vergence should also be performed as described above.An ocular examination should be performed with the best age dependant equipment available specifically looking for signs of photophobia, retinal dystrophy, retinal/choroidal hypopigmentation, iris transillumination and anterior segment anomaly e.g., cataract.Ocular coherence tomography (OCT) of the macular should be performed where possible to identify foveal hypoplasia and other abnormalities of retinal morphology that would give an indication of retinal origin for the nystagmus.Where possible, electro-diagnostic tests should be performed including: an electroretinogram (ERG) and a visual evoked potential (VEP) to identify retinal and post-retinal pathology such as a retinal dystrophy, or abnormal decussation at the chiasm in albinism.PAX6 gene disease) and retinal dystrophies .Where possible, parents should be examined for the presence of subtle nystagmus, iris transillumination and foveal hypoplasia  or related to identifiable lesions of the cerebellar nodulus/uvula. It alternates less regularly in congenital forms when it tends to be more asymmetrical and less predictable ).DBN is often more pronounced in downgaze but also in horizontal gaze. It can be accompanied by an alternating skew mimicking a superior oblique over-action. Some children adopt a resulting chin down posture which can mimic tonic upgaze. DBN is typically caused by lesions of the cerebellar flocculus bilaterally  and can also be found less commonly in ‘idiopathic’ patients and those with retinal dystrophy.DBN may be responsive to treatment with aminopyridines and UBN and PAN also to Baclofen treatment.Vertical and multidirectional nystagmus can be caused by intracranial lesions/metabolic disease but also retinal dystrophies. We would advocate electro-diagnostic testing (where available without significant delay) prior to neuroimaging for this reason in children with apparently isolated vertical nystagmus.Up beat nystagmus (UBN) is usually due to lesions of the midbrain or medulla, e.g., tumours or multiple sclerosis in adults.See-saw nystagmus is characterised by a cyclic movement of both eyes. In one half of the cycle, one eye will rise and intort and the other eye will fall and extort; then, in the next half cycle, the vertical and torsional components are reversed. It is most commonly caused by parasellar masses but has been described in many other conditions. It is rare in children.Vertical nystagmus may be seen as a side effect from systemic medications, commonly antiepileptics, such as phenytoin.Alexander’s law states that nystagmus intensity increases when the eyes are moved in the direction of the fast phase. Originally, described for peripheral vestibular nystagmus it also holds true for central vestibular nystagmus and possibly other forms. Torsional nystagmus is often vestibular and is rare in primary position when it is usually medullary in origin .In this section, we discuss some of the more common clinical dilemmas faced by clinicians.This is a common dilemma. The following features help the clinician to differentiate end-point nystagmus from GEN. In the authors’ experience, using the following criteria, these patients rarely pose a significant diagnostic dilemma Table .Table 4DA full orthoptic examination looking for presence of early-onset manifest strabismus (which is often seen in FMNS) can provide a clue towards an FMNS diagnosis.A full examination of the anterior and posterior segments of the eye can identify monocular cataract or optic nerve hypoplasia leading to FMNS. Bilateral pathology may cause INS, FMNS or both.INS can include an FMNS component but is not always affected by monocular occlusion unlike isolated FMNS in which the direction of the fast phase always changes towards the viewing eye. INS with an FMNS component may result in an increase in frequency and amplitude with monocular occlusion, without a change in the fast phase direction.Null positions and head postures are frequent in INS and much less common in FMNS unless the child is cross-fixating. However, some children with very asymmetrical visual acuity and FMNS may demonstrate a head posture to utilise adduction to dampen the FMNS in the seeing eye.INS onset is often earlier (<6–12 months) than FMNS.This is a frequent question for clinicians and many cases will have some features of FMNS and other features suggesting INS. However, the most clinically relevant distinction to make is between those cases with INS/FMNS and those with additional features suggesting a neurological cause, requiring additional investigation and management.Opsoclonus/flutter have characteristic, seemingly random, fast eye movements with no slow component (unlike nystagmus) and no inter-saccadic interval. When only horizontal they are described as flutter.They are often intermittent (unlike nystagmus) and induced by changes in fixation or blinking.Because opsoclonus/flutter is typically acquired and intermittent, children are often visibly troubled during periods of abnormal eye movement (unlike early-onset nystagmus).Onset of abnormal eye movements can be noted at any age in opsoclonus/flutter; however, for most children with INS, the abnormal eye movements are noted between 3 and 6 months of age and worsen before stabilising.Eye movement tics and voluntary nystagmus in older children has been confused with ocular flutter. However, as stated above, opsoclonus and ocular flutter are more common with changes in fixation or blinking and importantly, can be seen under closed eyelids which is rarely seen with tics and voluntary nystagmus.Pathological square-wave jerks are rarely seen in children but have been described in association with various underlying pathologies and medications. The key distinction is that they comprise of only fast (saccadic) movements rather than the anomalous slow movement seen in nystagmus.Opsoclonus/flutter can occasionally be confused with nystagmus especially in younger children. Making the distinction is important for clinical management and a few key differences exist:In most tertiary referral practice, children with clinical features consistent with INS , otherwise normal neurological development, normal posterior segment examination and ERG/VEP would not undergo neuroimaging in the first instance. Should subsequent examinations yield other eye movement findings, additional developmental issues or new ophthalmic findings (such as subtle optic nerve hypoplasia) neuroimaging would be sought. A similar approach would be taken in a child with typical FMNS (MLN) and early manifest strabismus or either INS or FMNS in a child with an underlying condition known to be associated with nystagmus such as Down’s syndrome.Where a clear underlying cause for the nystagmus is identified through clinical examination, OCT and ERG/VEP, such as early-onset retinal dystrophy or features of albinism, neuroimaging would not be initially sought.Unilateral nystagmus (or significant inter-ocular asymmetry) or supranuclear eye movement anomalies besides nystagmus would be joint managed with paediatric neurologists and neuroimaging would form a part of a broader set of urgent investigations in all cases.For most clinicians, this is a major concern when faced with a child who has (often striking) abnormal eye movements. Practice varies widely and is often governed by access to clinical tools, equipment, necessity of general anaesthesia for brain imaging studies and varying opinion on risk of general anaesthesia in young children. The following is a guide based on the authors own experience and practice.Isolated abnormalities of specific supranuclear eye movements in children are rare. For most, a pattern will emerge from gaining some information about each of the systems described above, in addition to an orthoptic workup, refraction, good history, electro-diagnostic tests (where available), examination of the family and basic neurological examination. FMNS (MLN) is common, as is INS related to albinism or an eventual ‘idiopathic’ diagnosis. Additional information can be gleaned from eye movement recordings and hand-held OCT devices but in many cases the clinical diagnosis does not rely solely on these scarce resources. Similarly, the role of genetic testing in these patients is evolving but at present, hampered by the resources, financing and infrastructure required for sequencing and variant interpretation. It is likely that in coming years these technologies will become a part of routine clinical practice but even then, a methodical and detailed clinical examination will remain paramount in assessing and managing this complex group of patients.


Metformin is the most widely prescribed drug to treat patients with type II diabetes, for whom retrospective studies suggest that metformin may have anticancer properties. However, in experiments performed with isolated cells, authors have reported both pro- and anti-apoptotic effects of metformin. The exact molecular mechanism of action of metformin remains partly unknown despite its use for over 60 years and more than 17,000 articles in PubMed. Among the various widely recognized or recently proposed targets, it has been reported consistently that metformin is capable of inhibiting mitochondrial respiratory chain Complex I. Since most of the effects of metformin have been replicated by other inhibitors of Complex I, it has been suggested that the mechanism of action of metformin involved the inhibition of Complex I. However, compared to conventional Complex I inhibitors, the metformin-induced inhibition of Complex I has unique characteristics. Among these, the most original one is that the concentrations of metformin required to inhibit Complex I are lower in intact cells than in isolated mitochondria. Experiments with isolated mitochondria or Complex I were generally performed using millimolar concentrations of metformin, while plasma levels remain in the micromolar range in both human and animal studies, highlighting that metformin concentration is an important issue. In order to explain the effects in animals based on observations in cells and mitochondria, some authors proposed a direct effect of the drug on Complex I involving an accumulation of metformin inside the mitochondria while others proposed an indirect effect (the drug no longer having to diffuse into the mitochondria). This brief review attempts to: gather arguments for and against each hypothesis concerning the mechanism by which metformin inhibits Complex I and to highlight remaining questions about the toxicity mechanism of metformin for certain cancer cells. Metformin is a drug with pleiotropic effects. It takes part in glucose homeostasis, mainly by inhibiting liver glucose production . It alsoin vivo or with intact cells, to results obtained with isolated mitochondria or isolated Complex I. In this last case, authors tend to assume that metformin accumulates in mitochondria, here we will discuss evidence supporting or not this assumption. Finally, since pro- and anti-apoptotic effects of metformin are observed in intact cells, we will examine the role of metformin concentrations as a potential cause of these conflicting observations.However, the mechanism by which metformin affects the activity of Complex I remains debated. In order to clarify whether the different conclusions found in the literature may be due to methodological differences, this review compares results obtained Metformin is a hydrophilic compound charged positively at physiological pH. Its hydrophilicity limits its permeability through lipid membranes. Metformin enters and leaves cells by the presence of several transporters including Organic Cation Transporters (OCTs) and multidrug and toxin extrusion (MATE) transporters . This leThe pharmacological inhibition or the genetic ablation of OCTs reduce the distribution of metformin to the liver, small intestine and kidney –32 while−/− hepatocytes  affect the metabolic effects of metformin is not systematically reported in the literature. On the one hand, metformin failed to reduce fasting plasma glucose concentration in OCT1-knockout mice submitted to a high-fat diet for 8 weeks and failed to suppress glucagon-stimulated glucose production in OCT1atocytes . On the atocytes . A broadatocytes , 36, othatocytes , 38.To the best of my knowledge, no study correlating metformin concentration in tissue (or cells) and metformin-induced Complex I inhibition was ever published.in vivo  is different from that of the cytosol. In order to maintain such a different metabolite composition, the inner membrane is impermeable to almost all hydrophilic molecules which enter or leave the mitochondria through specific transporters. Among the numerous recognized mitochondrial carriers, no specific carrier for metformin has been identified yet.Despite this, many authors have hypothesized that metformin accumulates in mitochondria , 15, 44.From a theoretical point of view, this hypothesis is plausible. Indeed, because the mitochondrial respiratory chain transfers protons from the matrix to the intermembrane space, mitochondria build up and maintain an electrical mitochondrial membrane potential that drives the accumulation of positively charged molecules into mitochondria, provided the molecule crosses the membrane. In these conditions, Nernst equation indicates that for a physiological mitochondrial membrane potential of −180 mV the thermodynamic equilibrium is reached after a 1,000-fold accumulation of a positively charged molecule if the molecule has one charge. Since metformin is a positively charged molecule and assuming the presence of a still unknown carrier for metformin in the inner membrane, its mitochondrial concentration could reach the millimolar range despite a cytosolic concentration within the micromolar range . Such an accumulation is 2 orders of magnitude higher than that measured by several groups , 42, 43.Thirdly, a large mitochondrial accumulation is not compatible with the low volume of distribution of metformin and its short half-live (see above).14C] metformin, the radioactivity was not found to accumulate in liver mitochondria of rats treated orally with metformin  have been synthesized , 48 in oar range , 48, whiAlthough this may seem odd, it has been reported by several different laboratories that the concentrations required to inhibit Complex I are lower for intact cells than for isolated mitochondria , 13, 19.50 ranging from 19 to 79 mM depending on laboratories  inhibition of Complex I with an “apparent” ICThis observation is not easily reconcilable with the proposal that metformin accumulates in mitochondria. Indeed, assuming that metformin did accumulate in mitochondria, Complex I inhibition would have been observed at micromolar concentrations of metformin (corresponding to millimolar concentrations inside mitochondria), which has not been reported. One could argue that at millimolar concentrations of metformin, the inhibition of Complex I would depolarize mitochondria, preventing metformin accumulation. However, it has been shown that millimolar concentrations of metformin did not depolarize isolated mitochondria .In these particular conditions of incubation (isolated mitochondria exposed to millimolar concentrations of metformin), it has to be noted that the inhibition of Complex I is observed almost exclusively during ATP synthesis  and disappears when mitochondria are depolarized (uncoupled State) or at rest  , 15. SucIt has been proposed that the reason why the inhibition is not observed after uncoupling might be due to the fact that the driving force for metformin accumulation within mitochondria disappears in these particular conditions. Although, as stated above, the accumulation of metformin in mitochondria is not supported by any evidence, this hypothesis does not explain the lack of inhibition in State 4, a situation in which the driving force  is higher than in State 3.Curiously, it has been reported that the inhibition of oxygen consumption in isolated mitochondria is accompanied by NADH oxidation . This ob+ ratio (as assessed by the Lactate/pyruvate and 3-hydroxybutyrate/ acetoacetate ratios) (The incubation of intact cells in the presence of metformin leads to a slower inhibition of Complex I depending on metformin concentration (hours are required for micromolar concentrations of metformin) , 50. Con ratios) , 5. Impo ratios) , 11, 19. ratios) , 14 or wIn intact cells the inhibition of oxygen consumption is strictly located on Complex I. This conclusion comes from the observation that metformin has no effect on oxygen consumption when electrons feed the respiratory chain downstream Complex I (using succinate for example) regardless of the respiratory State  , metformin acts rapidly but the effect requires millimolar concentrations. The inhibition does not only affect Complex I and Complex I inhibition is not observed in all the respiratory states.The respiratory chain is a sequence of redox reactions which couple an electron flux with a vectorial transfer of protons. Mammalian respiratory chain complex I is a large protein complex with at least 45 subunits. It includes a hydrophobic part embedded in the inner membrane involved in proton transfer and a hydrophilic part protruding into the matrix in which electrons pass from NADH to ubiquinone via a succession of redox reactions. Complex I inhibitors rotenone and piericidin bind at, or close to, the ubiquinone binding site, inhibiting both electron flux and proton extrusion. Using artificial electron acceptors, a rotenone-insensitive NADH oxidation which is not coupled with proton pumping  can occur in Complex I.Using isolated Complex I and millimolar concentrations of metformin, it has been shown that metformin does not inhibit NADH oxidation due to artificial electron acceptors, behaves as a non-competitive inhibitor of the physiological electron pathway and preferentially binds Complex I when the enzyme is in its “deactive” conformation . HoweverTo account for the fact that the concentration of metformin required to observe the inhibition of Complex I on whole cells is lower than the concentration required to observe the inhibition on mitochondria, two hypotheses have been proposed in the literature  proposes that omplex I . Althougomplex I , while romplex I , 54. HowThe second hypothesis necessarily involves an accumulation of metformin in the mitochondria that would be driven by mitochondrial membrane potential. Although proposed by several authors, this hypothesis is not yet supported by any evidence (see above).Apparently contradictory effects are found in the literature regarding the effects of metformin on cell death. Some authors have put forward its protective effects against cell death  while otThe permeability transition pore (PTP) is a channel located in the inner membrane normally closed in order to maintain a high mitochondrial membrane potential required for ATP synthesis. Once permanently opened, the membrane potential collapses , leadingAs there are several signaling pathways involved in cell death, there are many factors activating these pathways. To discriminate whether a given condition leading to cell death involves PTP opening or not, experiments are performed in the presence or absence of a recognized PTP inhibitor . Using this approach, it has been reproducibly observed that PTP opening occurs when cell death is triggered by calcium overload or oxidative stress .The molecular nature of the PTP has long been a subject of dispute but recent and compelling data from different laboratories suggest that the PTP might involve ATP synthase , 64. SurKnowing that metformin partly inhibits Complex I, we tested whether it also inhibited PTP opening and related cell death. We found that, metformin was less potent than rotenone but also inhibited PTP opening . SuggestAlthough PTP opening irremediably leads to cell death, PTP opening is not mandatory to kill cells as cells can die with a closed PTP. Although Complex I inhibition prevents PTP opening-related cell death (see above), it can also induce cell death in several models. Indeed, it has been repetitively reported that rotenone  or bigua−/− cancer cells in the presence of glucose, while it induces cell death in case of glucose deprivation  and are thus not supposed to be very sensitive to mitochondrial poison. But is it so simple? As soon as cells consume oxygen at the mitochondrial level, they are supposed to produce mitochondrial ATP. Thus, even if the proportion of mitochondrial ATP production is reduced in cancer cells, this mitochondrial ATP production exists and its reduction could be toxic. Supporting this proposal, it has been reported that metformin inhibits the proliferation of HCT116 p53rivation . Moreovease NDI1 , very elThe suggestion that metformin's toxicity is related to an energetic stress raises several questions: Why is metformin less toxic in non-cancer cells that are yet more dependent on mitochondrial ATP production? How can metformin protect against PTP-induced cell death despite its effect on ATP production? In other words, what triggers that a same inhibition of Complex I either prevents or induces cell death?in vitro, micromolar concentrations are sufficient to prevent PTP-opening induced cell death. Although it has been shown that cellular energy status is inversely correlated with metformin concentrations  . It is ain vivo and those used in vitro. On the other hand, two different laboratories that attempted to measure such an accumulation put forward a total absence of metformin accumulation in mitochondria  in order to confirm if metformin is found in large amount in mitochondria of cells exposed to metformin. Currently, the published evidence does not support the generally accepted hypothesis of metformin accumulation in mitochondria.Facing the facts, one must admit that there is either a technical mistake in the studies that did not find metformin accumulation in mitochondria or there is absolutely no experiment performed at millimolar concentrations of metformin that reflect what occurs The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Leishmania parasites (Kinetoplastea: Trypanosomatidae). Given that this finding raised a strong negative reaction from leishmania experts and the subsequent discussion did not lead to a clear resolution, we decided to analyze wild gorilla (Gorilla gorilla gorilla) and chimpanzee (Pan troglodytes troglodytes) fecal samples collected from the same area in Cameroon as in the original study. Fecal samples, used to circumvent the difficulties and ethics involved in obtaining blood samples from endangered wild apes, were screened by three different PCR assays for detection of Leishmania DNA. We did not detect any leishmania parasites in analyzed feces; however, sequencing of SSU rRNA revealed an unexpected diversity of free-living bodonids (Kinetoplastea: Bodonidae) and parasitic trypanosomatids (Kinetoplastea: Trypanosomatidae) other than Leishmania. A single detected Phytomonas species, found in chimpanzee feces, most likely originated from animal plant food. On the other hand, the presence of four free-living bodonid species and four parasitic insect monoxenous trypanosomatid, including two possible new species of the genus Herpetomonas, could be explained as ex post contamination of feces either from the environment or from flies (Diptera: Brachycera).Charismatic great apes have been used widely and effectively as flagship species in conservation campaigns for decades. These iconic representatives of their ecosystems could also play a role as reservoirs of several zoonotic diseases. Recently it was demonstrated that African great apes can host   •Leishmania parasites.We analyzed wild gorilla and chimpanzee fecal samples, from the area where a previous study detected •We detected leishmania DNA neither in great ape fecal samples nor in feces of experimentally infected rodents.•Leishmania.We revealed unexpected diversity of bodonids, monoxenous and dixenous trypanosomatids other than  Today, these highly intelligent, charismatic species are threatened by many factors, including destruction of their forest habitats, hunting for bushmeat and the illegal pet trade, and disease. Circulation of pathogens between free ranging primates and local human population attract a lot of attention, as primates might be reservoirs for several diseases of man and vice versa.Leishmania major parasites in feces of wild western lowland gorillas originating from southern Cameroon. The detection was based on PCR and fluorescence in situ hybridization. This highly unexpected finding elicited a negative reaction by a group of experts with extensive experience of working in the field of leishmaniasis research  are detectable in the feces of great apes. The original findings of Trypanosoma and Leishmania (belonging to the same family Trypanosomatidae). However, there is no evidence that leishmaniasis and/or trypanosomiasis are present in the resident human population living in the study area  and western lowland gorillas  were collected from September to October 2014 at the northern periphery of the Dja Faunal Reserve in Cameroon around the research site La Belgique with approval of the Ministry of Scientific Research and Innovation and the Ministry of Forests and Wildlife, Cameroon. Feces were collected noninvasively from within gorilla night nests and from the forest floor under chimpanzee night nests, early in the morning , thus ensuring that we collected only fresh feces. Samples were extracted from within the core of the feces and placed in collection tubes containing RNAlater. Samples were kept at room temperature for a maximum of one month before transport to the laboratory, where they were subsequently stored at −70 °C until further processing. DNA isolation was performed from approximately 3g of fecal homogenate using the QIAamp DNA Stool Mini Kit. A stool sample from a human volunteer was used as a negative DNA control; and two positive controls were prepared from two human volunteer stool samples each mixed with approximately 100 cells of cultured Trypanosoma ranarum or Leishmania tarentolae and kept for one week in RNAlater at −70 °C before DNA isolation.Fecal samples of free-ranging central chimpanzees (Four different polymerase chain reaction (PCR) assays were accomplished according to previously-established protocols. While quantitative PCR (qPCR) used kinetoplast DNA (kDNA) as the target , conventEach sample was independently subjected to each of the aforementioned PCRs at least twice, and more often three times. The results of the above mentioned different PCR protocols have always been consistent, as reported elsewhere e.g., .Trypanosoma brucei brucei, four mice by a cutaneous strain of Leishmania major, and three hamsters by a viscerotropic strain of L. infantum. The fecal pellets were collected weekly up to two month after infection and analyzed by above-mentioned PCR protocols.In order to test whether it is possible to detect leishmania and/or trypanosome parasites in feces, we infected five BALB/c mice by 3Trypanosoma or Leishmania was detected in any of the analyzed samples by any of the used PCR assays. On the other hand, we detected using nested PCR targeting SSU rRNA parasitic trypanosomatids other than Leishmania/Trypanosoma, as well as free-living bodonids (both being members of the order Kinetoplastea), in the great ape fecal samples. Five gorilla samples contained three species of bodonids, Bodo aff. caudatus , Parabodo nitrophilus (D50) and Neobodo aff. designis (D67), and two chimpanzee samples were found positive for two bodonid species, Neobodo aff. designis (D55-Cu34) and Neobodo sp. 1 (D08-Cu11).No parasite DNA belonging to the genera Herpetomonas. Of four Herpetomonas-positive fecal samples (two of each studied great ape species), sufficiently long (∼2000 bp) SSU rRNA sequences were obtained from one gorilla sample  and one chimpanzee sample  and the subsequent phylogenetic analysis revealed two possible new herpetomonad species designated as new typing units, TU299 and TU300  within the as yet unnamed cosmopolitan genus of monoxenous trypanosomatids , an entirely new lineage (and thus a possible new genus)  contained a parasite DNA which represents a possible new species (TUnus) see  within tTrypanosoma brucei brucei develops heavy infection in blood with serious symptoms and experimental mice had to be killed seven days after infection. Mice infected by Leishmania major developed skin ulcerating lesions on injected ears, while L. infantum parasites developed in the spleen and liver and caused only moderate symptoms in infected hamsters. While the trypanosome infections were detectable in the feces of three out of five infected mice using the above-mentioned PCR protocols, we did not detect leishmania DNA in any fecal sample of the experimentally infected rodents.Experimental infection of rodents succeeded in all cases. 4Leishmania spp. in gorillas and chimpanzees by detecting of parasite DNA in great ape feces coming from the same area as in the original study , which are the principal hosts of these parasites. Flies use feces as the main food source for their larvae and can penetrate ape fecal boules within minutes after deposition. The feces were collected from unhabituated apes, and therefore not immediately after defecation , thus resulting in a strong likelihood of contamination by fly feces containing Herpetomonas parasites. The second potential explanation is that the apes consumed food items, for example fruit, that was previously contaminated by feces of flies infected with herpetomonads.In the last decades, it has been demonstrated that monoxenous trypanosomatids are very common parasites or commensals of insects and the number of newly described species or typing units (TUs) is constantly increasing see . EntirelPhytomonas is transmitted by a phytophagous insect vector (Heteroptera) and infects a variety of plant species. Because of its life-cycle, contamination of the feces from the surrounding environment is not probable, and thus the detected parasites are more likely to have passed through the chimpanzee digestive system; we therefore assume that the parasites found in the feces originated from chimpanzee food items. This is different to the above-mentioned case of free-living bodonids and monoxenous insect trypanosomatids in great ape feces, whereby it is highly likely that they were contaminated by items from the external environment . Though it cannot be confirmed, we believe that the detection of phytomonad DNA in the analyzed great ape feces could demonstrate that the methods employed are suitable for the detection of dixenous trypanosomatid parasites (including leishmania) in this type of material.The dixenous genus Phytomonas in the fecal samples of great apes from the northern periphery of the Dja Faunal Reserve. However, these microorganisms most likely enter the feces via contamination from the external environment or by passive passage through the digestive tract of the host. Since we detect leishmania DNA neither in great ape fecal samples nor in feces of experimentally infected rodents, we are unable to support the findings of L. major parasites.To summarize, we found an unexpected diversity of free-living bodonids, insect monoxenous trypanosomatids and a member of the genus All authors: No reported conflicts.