CHD). Animal studies have suggested that hypoxia results in cortical dysmaturation at the cellular level. New magnetic resonance imaging techniques offer the potential to investigate the relationship between cerebral oxygen delivery and cortical microstructural development in newborn infants with CHD.Abnormal macrostructural development of the cerebral cortex has been associated with hypoxia in infants with congenital heart disease . Regions of reduced cortical orientation dispersion index in infants with CHD were related to impaired cerebral oxygen delivery . Cortical orientation dispersion index was associated with the gyrification index .We measured cortical macrostructural and microstructural properties in 48 newborn infants with serious or critical CHD is impaired dendritic arborization, which may underlie abnormal macrostructural findings reported in this population, and that the degree of impairment is related to reduced cerebral oxygen delivery.This study suggests that the primary component of cerebral cortex dysmaturation in  Newborn infants with serious or critical congenital heart disease demonstrate abnormal development of the cerebral cortex at the microstructural level, assessed using diffusion magnetic resonance imaging.This study suggests that the primary component of cerebral cortex dysmaturation in congenital heart disease is impaired dendritic arborization.Impairment of cortical microstructural development was associated with reduced cerebral oxygen delivery measured in the newborn period.Identification of abnormal development of the cerebral cortex in this population provides insight into the mechanisms that may underlie poorer neurodevelopmental outcomes in congenital heart disease.The relationship between impaired cerebral oxygen delivery and abnormal cortical microstructure corroborates recent animal models investigating the role of oxygen tension in cortical development.Methods to quantitatively assess impairment of cortical development may enable more rapid assessment and iteration of novel interventions to improve the trajectory of brain development in congenital heart disease during pregnancy.Congenital heart disease (CHD) is the most common congenital abnormality, affecting almost 1% of newborns.The detrimental effect of CHD on early brain development can be observed via a faltering trajectory of brain growth in the third trimester of pregnancyLinking physiological changes in CHD to brain development is assisted by 4 recent findings. First, oxygen tension has been shown to regulate development of human cortical radial glial cells, with hypoxia exerting negative effects on gliogenesis by reducing the number of preoligodendrocytes while increasing the number of reactive astrocytes.In this study, we aimed to use high‐angular‐resolution diffusion imaging and NODDI to test the hypothesis that reduced cerebral oxygen delivery in CHD is associated with impaired cortical microstructural development. We predicted that infants with CHD would exhibit higher cortical FA and lower ODI when compared with a group of healthy matched controls, and that infants with the lowest cerebral oxygen delivery would exhibit the most severe impairment of cortical microstructural development.The project was approved by the National Research Ethics Service West London committee  and informed written parental consent was obtained before imaging. All methods and experiments were performed in accordance with relevant guidelines and regulations. The data, analytic methods, and study materials will be available to other researchers for purposes of reproducing the results or replicating the procedure on reasonable request.A prospective cohort of 54 infants with serious or critical CHDWe therefore studied 48 infants with CHD, born at a median gestational age (GA) of 38.8 weeks . A control group of 48 healthy infants was retrospectively matched to the CHD group by GA at birth and scan, born at a median GA of 38.5 weeks (38.1–38.9). Healthy infants were recruited contemporaneously from the postnatal ward at St Thomas’ Hospital as part of the Developing Human Connectome Project.T1‐weighted (T1w), T2‐weighted (T2w), diffusion‐weighted imaging (DWI), and phase contrast angiography magnetic resonance imaging was performed on a Philips Achieva 3 Tesla system  with a 32‐channel neonatal head coil and neonatal positioning device,T2w images were acquired using a multislice turbo spin echo sequence, acquired in 2 stacks of 2‐dimensional slices , using parameters: repetition time: 12 seconds; echo time: 156 milliseconds, flip angle: 90°, slice thickness: 1.6 mm acquired with an overlap of 0.8 mm; in‐plane resolution: 0.8×0.8 mm, scan time: 3:12 minutes per stack. The T1w volumetric magnetization prepared rapid acquisition gradient echo acquisition parameters were as follows: repetition time: 11 milliseconds, echo time: 4.6 milliseconds, TI: 714 milliseconds, flip angle: 9°, acquired voxel size: 0.8×0.8×0.8 mm, field of view: 145×145×108 mm, sensitivity encoding factor: 1.2, scan time: 4:35 minutes. DWI with 300 directions was acquired using parameters: repetition time: 3.8 seconds, echo time: 90 milliseconds, multiband: 4; sensitivity encoding E: 1.2; resolution: 1.5×1.5×3 mm with 1.5 mm slice overlap, diffusion gradient encoding: b=0 s/mm (n=20), b=400 s/mm (n=64), b=1000 s/mm (n=88), b=2600 s/mm (n=128) with interleaved phase encoding.T2w images were reconstructed using a dedicated neonatal motion correction algorithm. Retrospective motion‐corrected reconstructionMotion‐corrected T2w images were segmented into tissue type using an automated, neonatal‐specific pipeline,max=0, 4, 6, 8 for respective shells), with registration operating at a reduced rank=15.High‐angular‐resolution diffusion‐weighted imaging data were reconstructed using a slice‐to‐volume motion correction technique that uses a bespoke spherical harmonics and radial decomposition of multishell diffusion data, together with outlier rejection, distortion, and slice profile correction.−3 mm2 s−1. This is consistent with previous NODDI studies in neonates,−3 mm2 s−1) likely reflecting the higher water content of the neonatal brain.Nonbrain tissue was removed using FSL BET (Brain Extraction Tool).A multivariate group template was generated from both T1w and T2w images, using symmetric diffeomorphic normalization for multivariate neuroanatomy and a cross‐correlation similarity metric.We used an approach for aligning cortical data from multiple subjects into a common space to provide voxel‐wise spatial characterization of FA, MD, NDI, and ODI, as previously described.2 was measured at the time of scan using a Masimo Radical‐7 monitor  applied to the right hand.For infants with CHD, we calculated their cerebral blood flow using a previously described method.2) was calculated using the following formula:Cerebral oxygen delivery . All analyses were subject to family‐wise error correction for multiple comparisons, and thresholding for all analyses was at P<0.05. Linear regression was used to investigate the association between GI and diffusion metrics. To assess the relationship of GI and ODI independently of advancing brain maturity, GA at scan was included as a variable in the multiple linear regression model.The control group was retrospectively matched to the CHD group by GA at birth and at scan using an R implementationU test. All analyses of clinical variables were performed using SPSS V24 .Categorical clinical variables were compared using Fisher's exact tests. For continuous clinical variables, we determined medians and interquartile ranges, and compared groups using the Mann–Whitney The analysis included 96 newborn infants: 48 infants with confirmed serious or critical CHD scanned before surgery without evidence of arterial ischemic stroke, and 48 age‐matched healthy infants. Clinical characteristics of both groups are shown in Table Infants with CHD demonstrated widespread changes in cortical ODI, with the most significant reductions observed posteriorly in the posterior parietal cortex, insula cortex, cingulate cortex, primary motor cortex, supplementary motor area, and occipital regions Figure .Cortical FA was higher in infants with CHD, with effects seen in predominantly midline cortical structures  at time of scan was positively associated with cortical ODI across many regions of the cortex , with the most significant associations found in the bilateral temporal lobes, occipital lobes, cingulate cortex, and right insula cortex. To demonstrate this linear relationship, mean ODI data were extracted for each subject from significant voxels in the gray matter skeleton and plotted against CDO2 at the time of scan  and negatively with FA   but not for cortical FA . Cortical gray matter volume was significantly positively correlated with cortical ODI  but not with cortical FA . Results are summarized in Table P=0.005), and regional brain volumes were significantly smaller in those with CHD across all regions of the brain  Figure . The lin2. We speculate that hindered microstructural development underlies the abnormal macrostructural changes in brain development that have been observed through reduced birth head circumference,Long‐term neurodevelopmental impairment is a major remaining challenge for infants with congenital heart disease, yet our understanding of the underlying biological substrate remains limited. Our study suggests that the microstructural development of the cerebral cortex in infants with CHD is abnormal in the newborn period compared with healthy controls and, importantly, that the degree of impairment is related to reduced CDOWe found that cortical ODI was widely reduced in the CHD group, with associated but more sparsely distributed areas of higher FA. These findings suggest a hindered trajectory of normal brain development, with increased sensitivity to tissue changes using the more advanced NODDI model. As the brain matures in utero, cortical neurons migrate outward toward the pial surface, populating the cortexWhile reduced cortical folding complexity in newborns with CHD has previously been reported in our cohortChanges in cortical orientation dispersion were more pronounced posteriorly than frontally, which is consistent with a previously described sequence of cortical development maturing earlier in the occipital cortex and completing later in frontal regions.2 on development of cortical ODI. There was a widespread positive relationship between ODI and CDO2, supporting the hypothesis that impaired oxygen delivery to the developing brain may be associated with delayed cortical microstructural development. There was no relationship between ODI and either cerebral blood flow or preductal arterial saturation when considered individually, suggesting that both components of CDO2 are required to estimate oxygen delivery to the brain, and that when considered alone, neither component explains enough variance of ODI to achieve statistical significance. In the case of cerebral blood flow, this may additionally suggest that alternative proposed metabolic substratesHaving established group differences between infants with CHD and healthy controls, we investigated the effect of CDO2 was also measured in the postnatal period, while the most influential period on brain growth would have been in utero, and particularly during the third trimester. Despite this, we feel that postnatal CDO2 remains a useful surrogate for severity of cardiac circulatory compromise to date, taking into account both measures of cerebral blood flow and degree of hypoxia as a result of structural changes in congenital heart disease. Third, the underlying genetic basis of CHD is becoming increasingly better understood2 but also to intrinsic abnormalities in microstructural development of the brain. Future work in larger, more homogenous cohorts to characterize in utero flows, oxygen saturations, and CDO2 will allow further correlation of CDO2 or lesion type with measures of fetal brain development.There were limitations to our study. First, quantitative estimates obtained from microstructural studies are invariably model dependent, exhibiting biases and limitations that are related to model assumptions. Despite this, NODDI indices have been shown to correlate with histological changes in neurite geometric configuration2 is associated with impaired cortical maturation in this population supports the development of strategies to optimize fetal CDO2. The provision of supplemental oxygen to mothers during pregnancy may enable restoration of fetal cerebral oxygen tension to levels required to prevent or reverse abnormal corticogenesis.2 is most likely. In addition, the use of newer microstructural measures such as ODI may provide a crucial leading indicator in the postnatal period to assess the impact of novel interventions on cortical development before child neurodevelopmental outcomes can be assessed at a later age.There are currently no validated neuroprotective therapies available for infants with CHD. Our demonstration that reduced CDOThis research was funded by the British Heart Foundation (FS/15/55/31649) and Medical Research Council UK (MR/L011530/1). This work received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/20072013)/ERC grant agreement no. 319456 (dHCP project), and was supported by the Wellcome Engineering and Physical Sciences Research Council Centre for Medical Engineering at Kings College London (WT 203148/Z/16/Z), MRC strategic grant MR/K006355/1, Medical Research Council Centre grant MR/N026063/1, and by the National Institute for Health Research Biomedical Research Centre based at Guy's and St Thomas’ NHS Foundation Trust and Kings College London. Dr O'Muircheartaigh is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (206675/Z/17/Z). The views expressed are those of the authors and not necessarily those of the NHS, the National Institute for Health Research, or the Department of Health.None.Table S1. Differences in Mean Cortical Microstructure and Cerebral Oxygen Delivery Between Those With and Without Punctate White Matter LesionsTable S2. Differences in Mean Brain Volume, Regional Brain Volumes, and Mean Diffusion Measures, Between Those With Congenital Heart Disease (CHD) and Age‐Matched ControlsFigure S1. A, Mean orientation dispersion index (ODI) and (B) fractional anisotropy (FA) from significant cortical regions plotted against gestational age at scan, for both congenital heart disease  and control  groups.Figure S2. Infants with congenital heart disease  exhibit impaired orientation dispersion index compared with healthy age‐matched controls (n=37), overlaid on the mean orientation dispersion index (ODI) template.Click here for additional data file.


Myrmica rubra ant workers (i) detect and avoid fungus-infected substrates and (ii) excavate nest patterns that minimize their exposure to entomopathogenic spores. Small groups of M. rubra workers were allowed to dig their nest in a two-dimensional sand plate of which one half of the substrate contained fungal spores of Metarhizium brunneum, while the other half was spore-free. We found that the overall digging dynamics of M. rubra nests was not altered by the presence of fungus spores. By contrast, the shape of the excavated areas markedly differed: control nests showed rather isotropic patterns, whereas nests that were partially dug into a fungus-contaminated substrate markedly deviated from a circular shape. This demonstrates that the sanitary risks associated with a digging substrate are key factors in nest morphogenesis. We also found that M. rubra colonies were able to discriminate between the two substrates (fungus-infected or not). Furthermore, some colonies unexpectedly showed a high consistency in excavating mainly the infected substrate. This seemingly suboptimal preference for a contaminated soil suggests that non-lethal doses of fungal spores could help ant colonies to trigger ‘immune priming’. The presence of fungi may also indicate favourable ecological conditions, such as humid and humus-rich soil, that ants use as a cue for selecting suitable nesting sites.As entomopathogens are detrimental to the development or even survival of insect societies, ant colonies should avoid digging into a substrate that is contaminated by fungal spores. Here, we test the hypotheses that  These patterns of interactions are shaped, at least partially, by the spatial structure of the nest. Nest patterns result from stigmergic processes in which the built structure acts as a feedback on the digging behaviour of individuals, leading to adaptive, self-organized patterns, without the need for any template, centralized control or even direct communication between nest-mates –3. AlthoCoptotermes lacteus termites display an avoidance response or dig out shorter tunnels into substrates infected by Metarhizium brunneum fungus [Solenopsis invicta ant workers selectively avoid building their nest in nematode-infected soils [Formica selysi queens [Monomorium pharaonis ant workers [The topological features of a nest reflect both the intrinsic features of the colony and the characteristics of its environment. In the case of subterranean nests built by social insects, colony size ,4,12,13 m fungus . Likewised soils . Howeveri queens  and Mono workers  show a sMyrmica rubra ant colonies with soil patches infected by Metarhizium brunneum spores. We investigated whether the choice of the substrate, the digging dynamics, as well as the size and topology of the nest were influenced by the presence of potentially harmful pathogens. For this, we tested small groups of 50 M. rubra workers in a two-dimensional digging set-up [Metarhizium brunneum, while the other was spore-free. This allowed us to assess whether a contaminated substrate leads to a decrease of excavated soil as well as to a nest topology that minimizes the level of ants' exposure to fungus spores.In the present study, we challenged g set-up ,16 in wh2.2.1.M. rubra ants containing one queen, 200–300 workers and brood were used for the experiments. In the laboratory, each colony was reared in a plastic tray (Janet type: 47 × 29 cm) in which the floor was covered with plaster and the borders were coated with polytetrafluoroethylene  to prevent ants from escaping. A square 10 cm wide glass plate, placed 3 mm above the ground and covered with a red filter, was used as a nest ceiling. Each colony was fed with one mealworm (Tenebrio molitor) three times per week, while water and sucrose solution (0.3 M) were provided ad libitum. Laboratory conditions were kept at a 21 ± 1°C and 50 ± 5% humidity rate, with a constant photoperiod of 12 h per day.Eleven colonies of 2.2.Metarhizium brunneum fungus (Strain F52 from Novozymes) that is produced in the form of barley grains coated with fungal spores. This generalist entomopathogen fungus is known to kill more than 200 insect species [M. rubra ants [6 spores ml−1. In addition, the viability of conidia was determined by placing 5 ml of the final solution of spores on a thin layer of potato dextrose agar and by incubating it at 25°C for 4 days.We used a commercial strain of  species  and to pbra ants ,23. Fourbra ants . We esti2.3.6 spores ml−1) per 100 g of sand. As a control, spore-free substrate was made by adding 25 ml of solution containing 0.05% Tween 20 and 0.05% Triton-X per 100 g of sterilized sand. The used level of Metarhizium spores (25 × 104 spores per g soil) was of the same order of magnitude as the natural density of Metarhizium detected in soils . By cho [et al. , the dig2.4.N = 22) and the third one was used as control . The replication of the experimental condition allowed us to assess the effect of the mother colony on the digging response of ants to the contaminated substrate. Each group of 50 workers was dropped into a circular arena (55 mm diameter) to be tested 2 h later and was not fed until the end of the experiment to prevent them from being engaged in other tasks than nest-excavating ones. This starvation did not reduce the ants’ survival, because less than 2%  of the ants died at the end of the experiment. The nest sand plates were randomly placed in groups of four in a closed wooden box to simulate the darkness of natural nests. We started the experiment by connecting the circular arena hosting the group of 50 tested ants to a central hole made on the upper glass plate covering the digging area. The connection was made with a vertical plastic tube (3.5 cm) that was filled with clean sand to encourage ants to start digging. The digging process was followed for 40 h once the first ant reached the central hole of the nest sand plate. Snapshots of the digging area were taken under red light every 5 h using a Logitech camera (HD Pro C920) placed 20 cm below the glass plates. Image J software was used to automatically compute both the dug area (A) and the perimeter (P) of the nest for each snapshot. This allowed us to quantify the dynamics of digging activity and to compare nest patterns between the two halves of each nest plate.From each of the 11 colonies , we randomly sampled three groups of 50 workers: two groups were assigned to the experimental condition . Non-parametric tests with a significance level of α = 0.05 were used because all data did not meet the normality assumption. With regard to the digging activity, a generalized linear mixed-effects model (GLMM) was used to investigate the effect of treatment , colony and time on the area excavated by colonies. Colony and treatment were treated as categorical variables, whereas time was considered as a continuous variable. Moreover, time and treatment were specified as fixed effects, colony as a random effect and replicates as a nested random factor within the colony to account for the repeated measurements performed on mother colonies [U test to compare the final excavated volumes between control and experimental nests.Statistical analyses were performed by using Scolonies . Full moWithin each type of nest, we also used GLMM analyses to test for the effect of the side , colony and time on the area excavated by ants. Wilcoxon matched-pairs tests were used to compare the final excavated areas between the two sides in the experimental or in the control nests. To provide evidence of ants' preference for a spore-free substrate, we tested whether the number of ant colonies for which the most dug part was the clean half of the set-up differed from random by using a binomial test.A) and the perimeter (P) of a nest can be described by the linear equation log(P) = log(μ) + ω log(A), where the parameters’ values are μ = 2√π and ω = 0.5. For both the control and the experimental nests, the values of μ and ω were estimated from the intercept and the slope of regression lines that best fitted log-transformed values of final perimeters as a function of final areas. The slopes and intercepts of these linear fittings were compared between experimental and control nests by using F-tests.We characterized nest patterns by their level of digitation as well as by the anisotropy of their shape. As regards the level of digitation in control or in experimental nests, we assessed whether the perimeters of the final excavated areas significantly deviated from those expected from a circular shape, by using the Wilcoxon matched-pair test. In the case of a perfect circle, the relationship between the area . However, for both control and experimental nests, there was a highly significant effect of time  on the excavated area. Ants were the most active in digging during the 10 first hours with around half of the final total area being excavated . From 30 h onwards, the digging activity nearly ceased, and the excavated areas increased by only 9.9% for the control nests and by 7.5% for experimental ones during the last 10 h. Ultimately, a similar excavated area was reached in the control and experimental nests .We found no significant effect of treatment or time by treatment interaction on nest growth dynamics  and led to the same total area excavated .In control nests, the left and the right side of the sand plates were dug with similar growth dynamics , and reached similar final excavated areas .Similarly, the growths of dug areas over time in the infected and the clean sides of the experimental nests were not different (GLMM: time by side interaction: N = 11) of control nests did not differ from random . This confirmed that there was no bias due to external stimuli or substrate heterogeneities in our sand plates. The most dug part of control nests grew at a rate that was only slightly faster and not significantly different from the least dug one . The findings were quite different for the experimental nests. When considering the growth dynamics in the most dug part, the excavation increased at a significantly higher rate than the least dug part of the set-up . As a result, from 10 h onwards after the start of the digging activity, the excavated volume became significantly larger in the most dug part of the set-up compared to the other side . Unexpectedly, not all the ant colonies preferred to dig into the spore-free side of the experimental nests. Indeed, the proportion of colonies  that had mostly dug the clean half of the set-up did not differ from random . Furthermore, differences in the growth dynamics between the most and the least dug side were of the same magnitude, regardless of whether the most dug part was the clean side  or the infected side .With regard to the ants' preference for digging into one side of the set-up, the percentage of colonies that mostly dug in the left side , while ants' digging activity seemed more directional in experimental nests, leading to the emergence of long galleries extending preferentially in one side of the set-up .The anisotropy of nest patterns was estimated by the aspect ratio, i.e. the maximum over the minimum Feret values. These ratios were significantly higher for the experimental nests than for the control ones . Indeed, the final excavated areas were highly correlated between the two experimental groups that originated from the same mother colony . Surprisingly, a colonial effect was also observed in the ants' preference for a given type of digging substrate. In most cases (nine out of 11 colonies), each pair of experimental groups that came from the same mother colony chose to focus the main part of their digging activity in the same type of substrate . Indeed, the percentages of the total area that were dug into the infected side of the experimental nests were highly correlated between the two replicates  are less abundant inside nests, whereas others (Beauveria brongniartii) are more frequent inside than outside ant nests [Metarhizium brunneum fungus is known to be efficient at killing M. rubra workers [Pathogen avoidance is considered a first line of disease defence in animals. In the case of social immunity, insect societies should reduce exposure to sanitary risks by avoiding digging their nest in contaminated areas. However, pathogen prevalence is quite variable inside nt nests . In shar workers –35. Thes workers –41, poss workers ,39.Atta sexdens ants [Macrotermes michaelseni termites [Mo. pharaonis ant colonies that display a clear preference for infected sites when they migrate to a new nest [F. selysi are attracted to nest sites contaminated with Beauveria and Metarhizium pathogens [Previous studies on ens ants  and on Mtermites  showed tnew nest . Similarathogens , althougathogens .. However, host manipulation often results from a process of coevolution between the host and highly specialized parasites. This is not the case with Metarhizium fungus, which targets a broad spectrum of insect hosts [. Second, while being potentially a sanitary challenge for the ants, the presence of fungi may also be a cue associated with suitable nesting sites, indicating favourable ecological conditions, such as humid and humus-rich soil. Finally, regardless of substrate contamination, the similar death rates observed in all colonies after 40 h of digging indicate that the amount of conidia present in the soil was not a lethal threat for the ants. Previous studies found that contacts with a pathogen at non-lethal doses reduce the susceptibility of individuals to later exposure to the same pathogen [M. rubra ants and Metarhizium brunneum fungus naturally occur in the same habitats [From a functional perspective, the seemingly suboptimal preference shown by some colonies for a substrate containing live entomopathogenic fungus may be explained in several ways. First, the fungal pathogen may have manipulated the ants by luring them with odour cues in order to increase its probability to contaminate the whole ant colonyct hosts . Togethect hosts , a fungupathogen –44 or otpathogen . Althougpathogen ,47, thispathogen ,49. By ehabitats ,23.M. rubra colonies are able to discriminate between substrates on the basis of their pathogenicity, displaying either avoidance or attraction to the contaminated substrate. Mechanisms that underlie such discrimination remain unclear but lead to anisotropic nest patterns, thereby demonstrating the key role of soil biotic factors in nest morphogenesis. The preference for fungal-infected soils seems to be a colonial trait and may be associated with factors that are beneficial to the colony. Further investigations are still needed to understand whether these two distinct nesting strategies are based on genetic factors, are due to different features of ants' nesting biotopes or the outcome from differences in life-history traits of ant colonies such as their previous exposure to entomopathogenic fungi.Overall, we showed that To conclude, we found that the pathogen load of a digging substrate is a key factor of nest morphogenesis in ant societies. The presence of entomopathogenic spores in the soil does not alter the growth dynamics of excavated nests but makes their shape less isotropic with a few long galleries extending in the substrate. Quite unexpectedly, pathogen avoidance was not systematic as some colonies even showed the opposite preference of fungus-contaminated substrate. The relevance of this seemingly suboptimal preference remains to be investigated. The present study is a first report of pathogen-induced changes in collectively built nests, and more work is needed to understand this relatively unexplored area of disease defence in social insects.


Seizures occur in a recurrent manner with intermittent states of interictal and ictal discharges (IIDs and IDs). The transitions to and from IDs are determined by a set of processes, including synaptic interaction and ionic dynamics. Although mathematical models of separate types of epileptic discharges have been developed, modeling the transitions between states remains a challenge. A simple generic mathematical model of seizure dynamics (Epileptor) has recently been proposed by Jirsa et al. (2014); however, it is formulated in terms of abstract variables. In this paper, a minimal population-type model of IIDs and IDs is proposed that is as simple to use as the Epileptor, but the suggested model attributes physical meaning to the variables. The model is expressed in ordinary differential equations for extracellular potassium and intracellular sodium concentrations, membrane potential, and short-term synaptic depression variables. A quadratic integrate-and-fire model driven by the population input current is used to reproduce spike trains in a representative neuron. In simulations, potassium accumulation governs the transition from the silent state to the state of an ID. Each ID is composed of clustered IID-like events. The sodium accumulates during discharge and activates the sodium-potassium pump, which terminates the ID by restoring the potassium gradient and thus polarizing the neuronal membranes. The whole-cell and cell-attached recordings of a 4-AP-based in vitro model of epilepsy confirmed the primary model assumptions and predictions. The mathematical analysis revealed that the IID-like events are large-amplitude stochastic oscillations, which in the case of ID generation are controlled by slow oscillations of ionic concentrations. The IDs originate in the conditions of elevated potassium concentrations in a bath solution via a saddle-node-on-invariant-circle-like bifurcation for a non-smooth dynamical system. By providing a minimal biophysical description of ionic dynamics and network interactions, the model may serve as a hierarchical base from a simple to more complex modeling of seizures. In pathological conditions of epilepsy, the functioning of the neural network crucially depends on the ionic concentrations inside and outside neurons. A number of factors that affect neuronal activity is large. That is why the development of a minimal model that reproduces typical seizures could structure further experimental and analytical studies of the pathological mechanisms. Here, on a base of known biophysical models, we present a simple population-type model that includes only four principal variables, the extracellular potassium concentration, the intracellular sodium concentration, the membrane potential and the synaptic resource diminishing due to short-term synaptic depression. A simple modeled neuron is used as an observer of the population activity. We validate the model assumptions with in vitro experiments. Our model reproduces ictal and interictal events, where the latter result in bursts of spikes in single neurons, and the former represent the cluster of spike bursts. Mathematical analysis reveals that the bursts are spontaneous large-amplitude oscillations, which may cluster after a saddle-node on invariant circle bifurcation in the pro-epileptic conditions. Our consideration has significant bearing in understanding pathological neuronal network dynamics. A simple canonical mathematical model of epileptic discharges has been proposed by V. Jirsa et al.  based onWhile formulating the model assumptions, special attention was focused on two specific experimental observations. First, in a number of experiments, IDs were formed from a clustered number of short discharges –8. TheseThe proposed model consists of three subsystems that describe: (i) the ionic dynamics, (ii) the neuronal excitability, and (iii) a neuron-observer .The proposed population model consists of the following equations:K]o and [Na]i represent extracellular potassium and intraneuronal sodium concentrations, respectively; V(t) is the membrane depolarization; xD(t) is the synaptic resource; ν(t) is the firing rate of an excitatory population; and an inhibitory population firing rate is assumed to be proportional to ν(t). The dynamics described by these equations is driven ν(t), which is calculated with a sigmoidal input-output function:x]+ is equal to x for the positive argument and 0 otherwise. The input current u(t) includes the potassium depolarizing current, the synaptic drive, and the noise ξ(t), respectively:All the terms of these equations are explained in the next section. Here, only the main variables are introduced, which are as follows: o = 0.02mM, δ[Na]i = 0.03mM, δxD = 0.01; the Gaussian white noise ξ(t) has zero mean and unity dispersion, ⟨ξ(t)ξ(t')⟩ = τmδ(t−t'); the noise amplitude is σ/gL = 25mV; the maximum pump flux is ρ = 0.2mM/s; the volume ration is γ = 10 ; the poU(t) are as follows:gU = 0.4nS/mV, CU = 200pF, VT = 25mV, Vreset = −50mV, U1 = −60mV, U2 = −40mV; an initial condition is U = −70mV.A representative neuron was modeled with a quadratic integrate-and-fire neuron , 19. The The model states that the elevation of the extracellular potassium concentration [K]o plays a primary role in self-regenerating IDs. Before each ID, the extracellular potassium concentration accumulates after a series of IIDs. Each IID involves the activation of interneurons o tends to return to the bath concentration due to diffusion and glial buffering. These processes are expressed by the first term in the right-hand side (r.h.s) of τK. The potassium is pumped into the neurons by the ATP-dependent Na-K pump. In K]o through the second term in the r.h.s.Between IIDs, the potassium concentration [ The intracellular sodium concentration [Na]i increases due to the firing activity ν(t), as expressed by the final term in ν(t). The sodium is pumped from the neurons by the ATP-dependent Na-K pump. In Na]i through the second term in the r.h.s. The leakage and intracellular diffusion are taken into account by the first term in the r.h.s. of τNa. Describes a mean membrane depolarization due to the input u(t) using a single-compartment leaky neuron model. Not taking into account a spike generation, the voltage V(t) reflects a nominal, extreme level of membrane polarization. Together with τm bath = 8.5mM  increase. The generation of SB begins when the input current u(t) leads the depolarization V(t) close to the threshold VthgL. According to u(t) in accordance to xD(t) begins to vanish, the current u(t) decreases, and SB terminates. During each SB, a representative neuron generates a few spikes (u(t).The mechanism of the IIDs is shown in w spikes . The spiK]o and [Na]i increase at each SB and then relax during the interburst intervals (K]o relaxation (τK = 10s) compared with the characteristic interburst interval. This fast relaxation prevents any significant potassium accumulation during the train of SBs.The ionic concentrations [ntervals . The meaRegime with ictal discharges. The simulations of the model based on Eqs (K]bath = 8.5mM. Each ID is characterized by a high rate of activity (black line) for 30 seconds. It consists of SBs resembling IIDs  compared with the characteristic interburst interval. This slow relaxation leads to potassium accumulation at some point of the train of SBs. At some critical level of [K]o, depolarization due to the shift of VK(t) (term 1 in xD(t). It begins an ID via the positive feedback provided by the depolarizing effect of the potassium accumulation.The regime with IDs is obtained with relatively slow o and [Cl]in. K]o and [Cl]in during ID are similar. Presumably, this proportionality is explained by the activity of K-Cl-cotransporters, which evoke a potassium outflux in response to chloride accumulation within the neurons. seizure . The mostrations . Changesntenance . The cenK]o during a single SB results in moderate depolarization on about 1 mV according to δ[K]o provides the same level of potassium increase after each SB; the parameter τK defines the speed of potassium level relaxation, and the parameter gK,leak controls the level of polarization dependent on [K]o.The extracellular potassium concentration increases during a single SB up to 1–2 mM according to data from   and V(t) oscillate faster than the dynamics of [K]O and [Na]i, producing the SBs. Thus, the subsystem of Eqs Based on the simulations of the system of Eqs σ = 0) are shown in the phase space  that has the only attractor of the deterministic system was examined. A small amplitude noise that leads to small voltage fluctuations near 0 cannot excite the system. Indeed, small deviations from the equilibrium result in the monotonic convergence to the equilibrium; however, deviations larger than the threshold result in a large excursion before returning to the resting state. This implies a stochastic excitability. As the noise intensity increases, the time between two successive activations decreases. A random trajectory beginning from the stable equilibrium and declined by the noise to the zone of excitation is plotted in the phase plane  along wiK]O determines an additive current in K]O leads to an inward current. The inward current shifts the U-shaped nullcline left and down and shifts the straight line nullcline to the right  than Eqs τm and τD. These observations allowed for reducing the full model to a slow subsystem that is based on Eqs K]o. On the other hand, Eqs ν(t). In the simulations, the slow high-amplitude oscillations of [K]o and [Na]i did not follow the fast fluctuations of ν but were controlled by a slow component of ν. Therefore, to extract a slow subsystem, ν(t) was averaged, and the dependence of this average variable K]o was evaluated. This was done with the integration of Eqs K]bath (from 3 to 22 mM during 200s) with the other parameters taken from the basic parameter set. The obtained dependence was approximated as follows:K]o < 20 mM.To analyze an appearance of IDs, the full model was reduced to a slow subsystem. In the simulations , the dynν by K]bath = 8.5 mM, τK = 100 s, τNa = 20 s, δ[K]o = 0.02 mM, δ[Na]i = 0.03 mM, γ = 10, ρ = 0.2 mM/s, as in the basic parameter set.After the substitution of The reduced model based on Eqs K]bath = 3 mM, the system converges to a fixed point, which is a stable node. The other two equilibrium points are an unstable focus and a saddle. At some critical value of K]bath = 8.5 mM, as shown in K]o is observed, which controls the generation of SBs during IDs. Overall, the analysis explains the dynamics of the regime with ID generation due to oscillations of potassium and sodium concentrations, with each ID composed of clustered SBs.The slow dynamical system was then analyzed based on Eqs In the present work, a mathematical model of IDs and IIDs is proposed based on a consideration of only a few dominating processes that underlie the generation of the discharges. These processes have been described in the form of a simple mathematical model consisting of four ordinary differential equations written in terms of physically meaningful variables, referred to as Epileptor-2, as an alternative to the well-known but more abstract model Epileptor . Along wThe first assumption has been partially validated by experimental observations from the literature . It is aThe second assumption seems to be too strong if taking the sometimes crucial role of GABAergic interneurons in interictal discharges into account ,31,38,39The third assumption postulates an essential role of the extracellular potassium concentration. It corresponds to the Fertziger and Ranck hypothesis ; howeverThe fourth assumption has been validated based on the experimental data. More precise matching would require explicit consideration of excitatory and inhibitory populations synaptic components to fit experimental estimates of either cumulative excitatory and inhibitory synaptic conductances ,46 or AMThe fifth assumption is based on experimental evidences of a short-term depression of glutamatergic synapses ,48,49. AIn summary, the assumptions of the Epileptor-2 model are in line with a wide range of experimental evidence obtained in different models of epilepsy.Based on the simulations, both ictal and interictal discharges consist of elementary events, SBs. To the best of the authors’ knowledge, the IDs have not been modeled before as bursts of spike bursts. The SBs have been mathematically analyzed. They were found to be large amplitude stochastic oscillations. The second-order system of differential equations is probably the simplest model of these kernel elements of pathological discharges. This statement is an important prediction, which opposes the inherently stochastic mechanism of the discharge generation to an oscillatory mechanism. The latter explains a stochastic sequence of the discharges due to noise in a deterministic periodic process. In contrast, the former implies that the discharges are impossible without noisy fluctuations. Similar behaviors have been studied for a neuronal model .The original Epileptor model explains epileptic discharges in terms of bifurcations . In thisK]O as the control variable of fast oscillations, SBs. The variable [K]O shows slow oscillations with pulses  the amplitude of the fast oscillations is large  this large  due to t voltage  occurs dK]bath, similar to previous studies conducted with a single neuron model [K]bath just above the critical value and (ii) the amplitude of [K]O oscillations is always finite, which indicates a similar shape of IDs at different [K]bath. Both predictions are more or less consistent with the experimental observations. The whole system of the Epileptor is of fold/homoclinic type [The analysis explains the scenario of an origination of a regime of ID generation during a change in an external factor, an increase of [on model ,10. Usinnic type .The Epileptor-2 can not be classified in the same way. Instead, fast and slow dynamics are to be considered separately. In contrast to the Epileptor, the Epileptor-2 is a non-smooth, stochastic dynamical system. The non-smoothness is due to the kink in Several mechanisms of seizure recruitment have been tested in computational models with varying levels of mathematical abstraction ,52,53. TIn this paper, a simple model of epileptic discharges is proposed, and the fundamental mechanisms described by this model are illustrated. The model is qualitatively and in a sense quantitatively compared to a wide range of experimental recordings. Similar to the known Epileptor model, this model can be applied to investigate the pathological states in a network of coupled oscillators . It can All animal procedures followed the guidelines of the European Community Council Directive 86/609/EEC and were approved by the Animal Care and Use Committee of the Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences.2PO4, 1 MgSO4, 2 CaCl2, 24 NaHCO3, and 10 dextrose. The ACSF was aerated with carbogen (95% O2/5% CO2). Recordings were made at 30˚C. Pyramidal neurons in deep layers of the entorhinal cortex were visualized using a Zeiss Axioscop 2 microscope  equipped with a video camera  and differential interference contrast optics. Patch electrodes (3–5 MΩ) were pulled from borosilicate filamented glass capillaries  on a P-1000 Micropipette Puller . For current-clamp recordings, a potassium-gluconate-based filling solution was used with the following composition (in mM): 135 K-gluconate, 10 NaCl, 5 EGTA, 10 HEPES, 4 ATP-Mg, and 0.3 GTP (with pH adjusted to 7.25 with KOH). For voltage-clamp recordings, a solution based on cesium-methane-sulfonate (CsMeS) was used with the following composition (in mM): 127 CsMeS, 10 NaCl, 5 EGTA, 10 HEPES, 6 QX314, 4 ATP-Mg, and 0.3 GTP (with pH adjusted to 7.25 with CsOH). For cell-attached voltage-clamp recordings, a sodium-chloride-based filling solution was used. It had the following composition (in mM): 138.5 NaCl, 8.5 KCl, 10 HEPES, 5 EGTA (with pH adjusted to 7.25 with NaOH). Cell-attached voltage-clamp recording of neuron firing activity was performed as described [Experimental procedures were described previously in details in our recent paper . Shortlyescribed .Recordings were performed with two Model 2400 patch-clamp amplifiers , and an NI USB-6343A/D converter  using WinWCP5 software . The data were filtered at 10 kHz and sampled at 20 kHz. After formation of the whole-cell configuration, access resistance was less than 15 MΩ and remained stable (< 30% increase) during the experiments in all cells included.2PO4, 0.25 MgSO4, 2 CaCl2, 24 NaHCO3, 10 dextrose, and 0.05 4-AP. The flow rate in the perfusion chamber was 5–6 ml/min. The liquid junction potentials were measured as described [Epileptiform activity was induced with the pro-epileptic solution, containing the following (in mM): 120 NaCl, 8.5 KCl, 1.25 NaHescribed , and theThe new model is based on the previous biophysically detailed considerations of ionic dynamics during the pathological states of brain activity ,17,30,42https://yadi.sk/d/927UjbS-3QQhMW; the code and executive file in Delphi-Pascal are at https://drive.google.com/file/d/1AJhAFKLOjvgauBF_6SQzvol8zzmfMaDV/view?usp=sharing and https://drive.google.com/open?id=10ij-Nt780jROcMv9qniUm4rAJNr8WD2j, correspondingly.The simulations were performed in the Delphi-7 environment. The mathematical analysis of the stochastic oscillations was performed using Wolfram Mathematica 10 . The Euler-Maruyama explicit numerical scheme was applied for the integration of the stochastic ordinary differential equations. The typical value of a time step was 0.5 ms. The results were dependent on the numerical parameter in a similar extent as for different realizations of noise. The numerical realizations of the model are available from the websites: the code in Wolfram Mathematica is at S1 TextDisinhibition as a model of epilepsy. Depolarization block. Alternative model of a neuron-observer.(PDF)Click here for additional data file.


We correct the following error in our publication. The primary focus of the work is on the description of antiviral resistance-associated markers in the influenza PA endonuclease domain , which is responsible for the endonuclease activity of the virus. Figure S1 presents data from minigenome assays that measure cumulative viral polymerase activity with and without the endonuclease inhibitor RO-7. The construct used in these assays contained the I38T resistance substitution in the PA plasmid. After further sequencing of this construct, two additional unexpected changes, P224S and P295L, both outside the endonuclease domain, were detected. Neither mutation was described in the original manuscript.Volume 9, issue 2, e00430-18, 2018, In a summary of four independent experiments, we have subsequently determined that P224S/P295L in combination with I38T lower polymerase activity approximately 1.7-fold with the CA/04 virus. When only I38T is present, a statistically insignificant decrease was observed, suggesting that P224S and P295L do negatively impact polymerase activity, albeit mildly. P224S and P295L were not present in the PR/8 PA plasmid.Regardless of the impact on output from minigenome assays, the main conclusions of the work, that I38T is a critical determinant of endonuclease inhibitor resistance, remain unaltered. Importantly, the presence of P224S/P295L in conjunction with I38T does not impact resistance to RO-7. In the presence of 250 nM RO-7, the CA/04 constructs containing I38T, P224S, and P295L retain 71.6% of normalized polymerase activity compared to 72.6% of the I38T-alone plasmid. Further, we have confirmed that the P224S/P295L substitutions are not present in either the WT or the reverse genetics CA/04 or PR/8 viruses generated in this study and used to evaluate RO-7-resistant viruses (listed in Table 1).Figure S1 and its legend have been replaced online.


Saccharomyces cerevisiae,Homo sapiens, andSus scrofa genomes. The datasets were used as evidence in a project that investigated the history of genomic science. To design the datasets, we first retrieved all sequence submission data from the European Nucleotide Archive (ENA), including accession numbers associated with each of our three species. Second, we used these accession numbers to construct queries to retrieve peer-reviewed scientific publications that first described these sequence submissions in the scientific literature. For each species, this resulted in two associated datasets: 1) A .csv file documenting the PMID of each article describing new sequences, all paper authors, all institutional affiliations of each author, countries of institution, year of first submission to the ENA (when available), and the year of article publication, and 2) A .csv file documenting all institutions submitting to the ENA, number of nucleotides sequenced and years of submission to the database. We utilised these datasets to understand how institutional collaboration shaped sequencing efforts, and to systematically identify important institutions and changes in the structure of research communities throughout the history of genomics and across our three target species. This data note, therefore, should aid researchers who would like to use these data for future analyses by making the methodology that underpins it transparent. Further, by detailing our methodology, researchers may be able to utilise our approach to construct similar datasets in the future.This data note describes a unique two-step methodology to construct six linked datasets covering the sequencing of Medical Translation in the History of Modern Genomics (TRANSGENE); a project that explored the history of scientific collaboration around DNA sequencing. By investigating the interactions between different institutions in the determination and description of new DNA sequences, this project showed changing and varied configurations of genomic science between the 1980s and 2010s. These historical configurations and their dynamics were shaped by the objectives, organisation and development of research communities and their distinct target species (Saccharomyces cerevisiae), a farm animal that has been the object of agricultural genetics, immunogenetic research and commercial breeding programmes (the pigSus scrofa), andHomo sapiens (subsequently referred to as ‘human’).This data note describes the methodology used to construct six novel datasets for the European Research Council funded project,species . To docudatasets in the data repository at the University of Edinburgh  via automated routines and Application Programme Interfaces (APIs).Extracting data on sequence submissions to the2. Linking particular sequence submissions to peer-reviewed publications that first described these in the literature via API queries, which utilised sequence accession numbers to mine Europe PubMed Central and SCOPUS.We then discuss our approach to re-structuring and cleaning these data and offer a description of the content of each dataset. Finally, we reflect on the strengths and weaknesses of these datasets and methods.This project entailed a large and unique data collection exercise of over 13 million records, which were retrieved via 30 million API queries to three different databases. This involved a two-step process. First, we retrieved all sequence submission data from the ENA, including accession numbers associated with particular sequence lengths. Second, we used these accession numbers to construct API queries to retrieve peer-reviewed scientific publications that first described and linked to these sequence submissions in the scientific literature. S. cerevisiae (1980–2000),H. sapiens (1985–2005), andS. scrofa (1990–2015). The date ranges for each species were selected based on the history of science objectives underlying our project. The purpose was to capture submissions before, during, and after the completion of concerted efforts to comprehensively sequence the genome of each of the species. The search was conducted by making a series of calls to ENA’s API for each species and each year our project investigated. The query was constructed by specifying the taxon’s number(tax_eq) in the ENA index  and the sequence release date(first_public) to filter records that were released within a certain year. The search parameter offirst_public was specified as “greater than or equal to” 1st January and “less than or equal to” 31st December of the year. Additional parameters were used to specify search forsequence release records (result=sequence_release) and download the data in .XML format (display=xml). In cases where records per year exceeded the ENA’s limit of 100,000 records per API call, the pagination function (offset) was deployed.We retrieved sequence submission data from the ENA for each of the three species over defined periods –Software availability;This procedure allowed us to mine the ENA database based on the species and years relevant to our study and extract data on: 1) the number of nucleotides submitted for each of these species; 2) all accession numbers associated with these sequence lengths; 3) the date of submission; 4) the name of the submitting individual and/or their institutional affiliation (if available); and 5) papers in the scientific literature associated with each accession number (if specified by the submitter) . This linkage allowed us to identify a list of PubMed IDs (PMIDs) of the publications linked to these accession numbers  at 1000 publication records. We deployed a routine to automate the search for each accession number in our dataset. The routine’s procedure to compose and make an API call to Europe PMC using a list of accession numbers  has been made available in an online repository  API by using the ENA accession number as a parameter to search for associated publications (PMID) and utilising other default parameters such asapikey, apart fromview=complete to specify returning of full meta-data. The routines and R scripts used are also available . Although this correspondence between submission and first publication was not universal,VantagePoint (2017) v.10 by using a combination of fuzzy logic algorithms available in the software (i.e. “Fuzzy word matching” to make word comparisons at 95% or lower) and manual cleaning to standardise institution, author and country names according to a pre-specified protocol. The protocol specified ensured consistency in name conventions, fully spelling out acronyms and abbreviations, removing articles and legal entities, using proper case conventions, removing white spaces and ineligible characters, removing duplicates, and keeping school and department data if it appeared more than 50 times in the dataset. All our publication dataset and the main submitting institutions, as documented by the volume of DNA nucleotides registered in the ENA, were cleaned according to this protocol. Missing data, particularly regarding institutional affiliation, was filled manually by scrutinising the record on SCOPUS’ web front-end. To replicate this cleaning process, other open source software, such as OpenRefine, may also be used as an alternative.Once collected, researchers in our team cleaned these datasets viaIn total, each species has two associated datasets: 1) A .csv file documenting the PMID of each article describing new sequences, all paper authors, all institutional affiliations of each author, countries of institution, year of first submission to the ENA (when available), and the year of article publication, and 2) A .csv file documenting all institutions submitting to the ENA, number of nucleotides sequenced and years of submission to the database. While the data about yeast submissions is provided sequence per sequence with full dates and information about both submitting individuals and institutions, the pig and human submission datasets offer aggregate figures of both number of sequence submissions to the database and number of nucleotides sequenced per institution per year.Our corpus of publications includes all data necessary to construct co-authorship networks of collaboration between individuals, institutions and countries that were involved in the sequencing efforts.Our study reflects that the growing capacity in data infrastructure and the development of bioinformatics offers new opportunities not only for life scientists and molecular biology but also for social scientists and historians of science. The method outlined in this paper provides a novel source of evidence to evaluate the development and growth of collaboration in DNA sequencing and genomics research. It is also able to avoid placing a narrow focus on a number of key players based on previous studies or historical accounts. Our datasets show a diversity of countries and institutions involved in the sequencing of the human, yeast and pig genomes. Thus, they enable us to complement previous historical studies that have been focused on a limited number of large-scale sequencing centres e.g..This analysis is, however, limited by the data infrastructure that we have used. Its organisation and, especially, its absences can indeed shape and affect how and what we can know about the past; how and what information is being recorded, what is missing, what can and cannot be automatically retrieved, what is considered important (or not), and for what questions the information was expected to provide answers to. These processes, including storage and curation, were built into the databases and can have significant impacts on what we know and what we can study about collaboration in genomic sequencing. For instance, as noted above, a substantial proportion of accession numbers in the ENA did not have any further information about submitters. We need to consider these absences, along with their underlying meanings and power dynamics more carefully, especially when we use digital research methods and online data .For this reason, we argue that qualitative work should accompany digital research methods. In our project, we developed a mixed methods approach based on constant, bi-directional interactions between quantitative data and other qualitative evidence, such as documents stored in archives . This ap I would like to thank the authors for this read. It was a thorough and clear explanation of valuable methods for linking institutional and authorship metadata with submitted sequence data to the European Nucleotide Archive. Good clean metadata is the dream of all bioinformaticians, and public archives often fall short. This work is a nice illustration of linking together multiple public archives with a lot of manual effort to build a valuable metadata dataset for an interesting historical project.This is valuable work and I feel the abstract sells it short. Firstly, I would recommend a clearer problem statement or sentence about the larger project before diving into the methods. Also, you don't make enough of why you need to link in EuropePMC and SCOPUS. Some quick statistics on the number of ENA accessions missing the needed metadata would really illustrate why the link to the literature is such a good idea. i.e. 50% of Homo sapiens accessions are missing both submitted and publication metadataI assume it's due to the scope of the larger project, but a sentence mentioning why the choice of the three species would be nice.I'd just like to applaud the sharing of the code. Yay for open science.My one request that might take a few hours of work but would be a valuable addition: Your method uses the first publication that is linked to an accession as likely being the original authors. That is probably right. But a small validation would be really useful here. You could pick 10 (or more) random accession and see if that strategy worked. It doesn't have to be perfect, but good to see if it works okay. I have some minor comments and one request that I hope will not be too laborious.Are sufficient details of methods and materials provided to allow replication by others?YesIs the rationale for creating the dataset(s) clearly described?YesAre the datasets clearly presented in a useable and accessible format?YesAre the protocols appropriate and is the work technically sound?YesReviewer Expertise:Bioinformatics & biomedical machine learningI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.  on data curation  as it addresses many of the relevant issues raised in the limitations relating to curation and completeness of data specifically in the context of modern biology.This article describes methodologies used to construct datasets on sequence submissions and co-authorship relationships relating to genomic sequencing of three major organisms. The methodology is clearly described, as are its limitations and prospects for use by other scholars. I particularly appreciated the careful reflections on strengths and weaknesses of the approaches taken, and agree that these approaches have clear prospects for enriching our historical/sociological accounts given tendencies to focus on the strongest (or loudest!) research centres to the neglect of other participants particularly in genomic sequencing efforts. Would strongly suggest citing Leonelli's bookAre sufficient details of methods and materials provided to allow replication by others?YesIs the rationale for creating the dataset(s) clearly described?YesAre the datasets clearly presented in a useable and accessible format?YesAre the protocols appropriate and is the work technically sound?YesReviewer Expertise:History/philosophy of contemporary biological sciences I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your positive comments and excellent feedback. We are grateful for your time and appreciate your comments on the originality, significance, and timeliness of this paper. We have adopted your suggestion and cited Leonelli's (2016) seminal work in this area too.


Methylmalonic acidaemia with homocystinuria type C (cblC defect) is an inherited error of cobalamin metabolism. Cobalamin deficient processing results in high levels of methylmalonic acid and homocysteine. The latter is considered to be a risk factor for multiple sclerosis (MS). We report on the first case of a patient with comorbid cblC defect and MS.MMACHC  and another splicing variant in PRDX1 (c.1-515G > T) that cause the silencing of the wild-type MMACHC allele, so confirming the diagnosis of cblC defect. Although cblC treatment was effective, when 17-year-old he experienced a relapse of neurological symptoms. Further imaging and laboratory studies eventually supported the diagnosis of MS.This young male presented at the age of 14 with a relapsing-remitting neurological disorder associated with imaging alterations suggestive of MS. Treatment resulted in a partial clinical improvement with vanishing of white matter lesions. Later on, the emergence of unexpected clinical features led to a metabolic work-up, revealing a cobalamin intracellular defect. Genetic analysis disclosed a single variant in While the comorbid association of MS and cblC in our patient may remain anecdotic, we suggest measuring Hcy and MMA levels in young patients with a relapsing-remitting demyelinating disorder, in order not to miss a cblC defect, that requires a specific and effective treatment. While the majority of patients become symptomatic during the first year of life, adolescents and adult patients mainly present with psychiatric symptoms, dementia, myelopathy, peripheral neuropathy and thrombosis . High le2MMACHC gene (NM_015506) revealed a heterozygous variant in exon 4 , inherited from his father. The lack of other pathogenetic variants on the MMACHC gene prompted us to sequence the PRDX1 gene (NM_001202431) that revealed a c.515-1G > T splicing variant, inherited from the mother, and confirmed the diagnosis of cobalamin disorder type C [This 17-year-old male is the first child of nonconsanguineous healthy parents. His younger sister is healthy. He was born after a pregnancy complicated by rubeola infection: at birth anophthalmia of the right eye was detected and brain imaging showed a congenital arachnoid cyst in the left temporal lobe. Familial history revealed a father's 1st cousin diagnosed with MS in adulthood. When 14-year-old, the patient complained a decline in school performance and a loss of strength of the inferior limbs. A year later, he was admitted to a Neurology Unit due to sudden visual loss (3/10 in the left eye) and the subacute emergence of a spastic paraparesis. A brain magnetic resonance imaging (MRI) showed multiple subcortical and periventricular white matter lesions suggesting the diagnosis of MS. The treatment with steroid improved visual acuity (8/10), but it was ineffective on lower limb spasticity. Subsequently, a treatment with interferon 1-b was started. On follow-up, a brain MRI showed a complete vanishing of the white matter lesions. However, at the age of 16, he suffered from a sudden episode of lethargy, loss of ambulation, bilateral upper limb dystonia and urinary incontinence. Brain and spinal MRI were normal. A partial spontaneous recovery was observed during the following weeks. A comprehensive clinical evaluation revealed a hypertrophic cardiomyopathy and proteinuria. A few months later, on examination he showed spastic paraparesis, pale optic disc, and large-amplitude horizontal left beating nystagmus. A further metabolic work-up revealed high Hcy  and low methionine  plasmatic levels and a marked increase of urinary MMA . Molecular analysis of DRB1*13–15 haplotype. The diagnosis of relapsing-remitting MS was definitively made in comorbidity with cblC defect. The patient has received treatment with natalizumab for 10 months in addition to the specific treatment for cblC. During the follow-up we observed no relapses nor novel lesions at MRI and no progression of cardiac nor renal dysfunctions. Written informed consent of patient and his parents was obtained.The treatment with betaine (6 g/day), carnitine (6 g/day), hydroxy-cobalamin , and folinic acid (15 mg/day) resulted in a remarkable lowering of plasma Hcy (27 μmol/L) and urine MMA (47 mmol/mol creatinine) as well as in an improvement of spastic paresis with restoration of autonomous gait. Unexpectedly, at the age of 17, he complained an acute loss of strength and paresthesia in the right arm. A brain and spinal MRI showed asymmetrical and multifocal cerebral white matter lesions, some of them with an open-ring enhancement, and a C2-C3 medullary lesion . Oligocl3The patient here reported suffered from a relapsing-remitting demyelinating disorder resulting from an early onset MS and a concomitant alteration of cobalamin metabolism. While he showed a complete remission of white matter lesions under steroid treatment, which is a relatively common feature in pediatric-onset MS , other nMMACHC gene product is involved in the regeneration of S-adenosylmethionine, the most important methyl donor, from Hcy. Several MS risk loci have been found in this metabolic pathway, such as SCL19A1, SHMT, MTHFR, CBS, suggesting a major role of methylation processes in MS pathogenesis [MMACHC adjacent gene PRDX1, was demonstrated to cause MMACHC gene silencing by methylation of the promoter through a mechanism called “epi-cblc” [PRDX1 encodes the peroxiredoxin-1, an enzyme induced by oxidative stress to protect the integrity of the blood brain barrier; and b) vascular peroxiredoxin-1 immunoreactivity is upregulated in active MS lesions in brain tissues [ogenesis . Recentlpi-cblc” . The pre tissues . So far  tissues .While the comorbid association of MS and cblC in our patient may remain an anecdotic observation, given the higher prevalence of MS than intracellular cobalamin defects in this age group, we suggest measuring Hcy and MMA levels in young patients with a relapsing-remitting demyelinating disorder, in order not to miss a cblC defect, that requires a specific and effective treatment.None of the authors have any disclosure to declare.Data availability not applicable.All the reported investigations and a specific permission for the publication of the results was obtained through a written informed parental consent.


The global health system is currently facing the new SARS-COV 2 pandemy. This exceptional situation requires, from our African health systems, to reorganize and readapt the usual protocols when they were carried out before the crisis and/or their urgent implementation otherwise. As imaging is one of the pillars of the diagnosis of infection with this emerging virus, it was essential to rethink the imaging department organization so as to dedicate a unit to COVID-19 activity while maintaining the usual emergency activity within the Ibn Sina university hospital in Rabat. The protection of exposed personnel and the bio-cleaning of radiology equipment and rooms also became an evidence. The active involvement of the administration, the Clinical Pharmacy Department and the Nosocomial Infections Control Committee is a key to the success of this reorganization. The global health system is currently facing the new SARS-COV 2 pandemic. This exceptional situation requires, from our African health systems, to reorganize and readapt the usual protocols and their urgent implementation. As imaging is one of the pillars of the diagnosis of infection with this emerging virus, it was essential to rethink the imaging department organization so as to dedicate a unit to COVID-19 activity while maintaining the usual emergency activity within the Ibn Sina university hospital in Rabat. The protection of exposed staff and the bio-cleaning of radiology equipment and rooms also became an evidence. The active involvement of the administration, the Clinical Pharmacy Department and the Nosocomial Infections Control Committee is a key to the success of this reorganization. In recent weeks, the imagery team of Ibn Sina University Hospital in Rabat (Morocco) has been confronted with a new activity, the COVID-19 activity . The graOrganizational component: Ibn Sina University Hospital in Rabat normally has 800 hospital beds. The non-urgent activity  was suspended and the hospital was geographically divided into two activities with separate circuits: a dedicated “Covid” activity located in the basement and on the ground floor which includes the emergency reception service, the emergency radiology service, a second so-called central radiology service and several hospital units; a non-Covid activity located on the floor level and reserved for urgent non-Covid activities. The imaging activity, like all hospital activity, has been divided into: a Covid activity which is taken care of by the emergency radiology service which performs computed tomography (CT) and chest x-rays for suspected or confirmed patients infected with Covid-19 arriving from the emergency reception service or other hospital units. Any respiratory symptomatology that is not clinically proven is considered suspicious; a non-Covid activity carried out in the central radiology department, which is responsible for imaging other hospital patients. A clinical information collection form has been developed to facilitate the referral of patients receiving Covid imaging to chest x-ray or CT rooms. The indications for CT have been widened due to its high sensitivity, although its specificity is discussed .Bio-cleaning, disinfection and Protection of personnel: bio-cleaning and protection of caregivers  working in radiology were not formalized in our hospital before the health crisis. In the urgency of the situation, the clinical pharmacy department, the nosocomial infection control committee and the radiology department set to work. In addition to a review of the recent literature on the contamination and contagiousness of SARS-COV 2, the effectiveness of the implementation of the protocols was conditioned by three essential factors: 1) The involvement of the personnel; 2) The effective application of the protocols:it was possible either by simulations or by its implementation when a Covid patient arrived in radiology; The operational hygiene team of the hospital, reporting to Nosocomial Infection Control Committee (NICC) and the Nursing Service, were of undeniable contribution; 3) The total acceptance of the administrative staff and the hospital pharmacy to all our requests in terms of organization, supplies of hygiene products and protective equipment. The available data in the literature prompted us to opt for draconian disinfection measures in an imaging environment considered to be a high-risk environment, among other things because of the confined nature of the radiology rooms. Studies conclude that SARS-Cov-2 persists on different surfaces and reveals that plastic and stainless steel offer greater stability to the virus ,6. As foSARS-COV-2 being an enveloped virus, its inactivation can be effectively carried out by surface disinfection procedures with solutions containing 62-71% ethanol, 0.5% hydrogen peroxide or 0.1% sodium hypochlorite with minimum contact time of 15 minutes . The EN It should be noted that after more than a month of Covid activity, 260 scanners and 180 chest x-rays, no one of the emergency radiology department staff was contaminated. The protection of the radiology staff in contact with the patients is achieved by the installation of a specific kit including  and by a short training on its use, especially on dressing and undressing. Ultrasound in suspected or confirmed COVID-19 patients is conceived in two distinct situations: 1) Pulmonary ultrasound as part of screening or monitoring of lung lesions. It is currently in the research field and it is recommended that CT replace it whenever possible. Nevertheless, in the eventual absence of the scanner under our skies and in case of availability of expertise in pulmonary ultrasound, the latter is probably an interesting alternative. 2) Ultrasound other than pulmonary in the context for example of digestive, renal, vascular or other symptomatology associated with the classic symptomatology of Covid-19 infection or in the context of a complication. Rigorous protective measures must be implemented during an ultrasound due to an extended contact time -16. The We wanted to bring our experience in the reorganization of hospitals in general and of the Radiology department in particular while facing the Covid-19 pandemic. The aim is to facilitate and inspire our colleagues, brothers and sisters from the rest of our continent, for the organization of their own units if it has not already been set up. To each thing woe and good as they say. This exceptional situation forced us to improve hygiene practices in our hospitals and to implement protocols that normally should have existed before.