The Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) glycolytic pathways are largely conserved across glycolytic species in nature. Is this a coincidence, convergent evolution or there exists a driving force towards either of the two pathway designs? We addressed this question by first employing a variant of the optStoic algorithm to exhaustively identify over 11,916 possible routes between glucose and pyruvate at different pre-determined stoichiometric yields of ATP. Subsequently, we analyzed the thermodynamic feasibility of all the pathways at physiological metabolite concentrations and quantified the protein cost of the feasible solutions. Pareto optimality analysis between energy efficiency and protein cost reveals that the naturally evolved ED and EMP pathways are indeed among the most protein cost-efficient pathways in their respective ATP yield categories and remain thermodynamically feasible across a wide range of ATP/ADP ratios and pathway intermediate metabolite concentration ranges. In contrast, pathways with higher ATP yield (>2) while feasible, are bound within stringent and often extreme operability ranges of cofactor and intermediate metabolite concentrations. The preponderance of EMP and ED is thus consistent with not only optimally balancing energy yield vs. enzyme cost but also with ensuring operability for wide metabolite concentration ranges and ATP/ADP ratios. Uniquely among them, the canonical Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) pathways are by far the most prevalent in nature1. These two pathways differ in the first few but share six of the remaining enzymatic steps. They both generate two moles of reduced redox cofactor NAD(P)H, which can be used for generating additional ATP through oxidative phosphorylation, but differ in the overall ATP yield. The ED pathway, often found in organisms living in carbon/energy/oxygen-rich environments , sacrifices energy yield for a pathway with a larger driving force1. In addition, the canonical ED pathway confers higher tolerance to oxidative stress as it generates NADPH as opposed to NADH in the EMP pathway3. In contrast, the EMP pathway is common in prokaryotes and eukaryotes with higher energy demands or those living in anoxic or low-energy environments1. The presence of the key EMP enzyme 6-phosphofructokinase (PFK) or key ED enzymes 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase and 6-phosphogluconate dehydratase is often used to identify whether a strain is capable of using either pathways4. The two pathways, however, are not mutually exclusive and often co-exist in many organisms5. In particular, enteric bacteria such as Escherichia coli can switch between them in response to the availability of different substrates6.Billions of years of evolution led to highly genetically and phenotypically diverse organisms, yet most of them retain largely identical routes for sugar catabolism despite the presence of a myriad of ways in nature’s enzymatic repertoire for converting glucose to pyruvate7. Semi-phosphorylative and non-phosphorylative ED pathways were reported in anaerobic Clostridia and archaea, wherein the first ATP phosphorylation step is catalyzed by 2-dehydro-3-deoxy-D-gluconate (KDG) kinase or glycerate kinase, yielding one or zero ATP per glucose, respectively9. Modified EMP pathways are found in (hyper)thermophilic archaea employing variants of glycolytic enzymes utilizing alternative cofactors such as ADP-dependent glucokinase and PFK (in euryarchaeota), pyrophosphate (PPi)-dependent PFK (in Thermoproteus tenax)10, non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (in crenarchaeon Aeropyrum pernix and T. tenax) and GAP ferredoxin oxidoreductase (in microaerobe Pyrobaculum aerophilum)12. A recent study confirmed that Clostridium thermocellum operates GTP and PPi-dependent glycolysis and predominantly employs a malate shunt to convert phosphoenolpyruvate (PEP) to pyruvate in the absence of pyruvate kinase13. An additional ATP per glucose by using a PPi-dependent PFK9 could potentially be gained, however, the source of PPi in C. thermocellum remains elusive13. Another recently found variant of EMP relies on an NADP-dependent GAP dehydrogenase (in Kluyveromyces lactis)14. Notably, the NADP-dependent C. acetobutylicum gapC was expressed heterologously in E. coli replacing the native NAD-dependent GAP dehydrogenase to improve NADPH availability15. Alternate glucose processing pathways not conforming to the ED or EMP structure include coupling phosphoketolase (PK) pathway with the EMP pathway as a shunt for glycolysis by the heterofermentative lactic acid bacterium Lactobacillus reuteri ATCC 5573016. Bifidobacteria also utilize the bifid shunt for conversion of glucose to acetate: lactate: ATP in a 1.5:1:2.5 ratio17. In a groundbreaking work18, a non-oxidative glycolysis (NOG) was designed and constructed which operates cyclically to convert glucose to acetyl-coA in a redox-independent and carbon-neutral manner. The NOG pathway generates two ATPs and three acetate moieties per glucose molecule.Variants of the canonical ED and EMP pathways have also been discovered especially in extremophileset al. suggested that the canonical EMP pathway is an optimal series of chemical reactions that maximize ATP yield at high kinetic efficiency19. By designing a series of shortest pathways connecting different pairs of central carbon metabolites using a set of 30 reaction rules (that acts on carbohydrates), Noor et al. proposed that the canonical glycolytic pathway is the shortest pathway (in E. coli) that ensures the production of essential precursors of cellular biomass20. A recent biochemical analysis suggested that the glycolytic pathway uniquely manages to avoid toxic intermediates  and phosphorylates intermediates thus reducing metabolite leakage21. Furthermore, a recent analysis put forth the hypothesis that the lower glycolytic pathway shared by both ED and EMP pathways is able to sustain the highest flux when compared to other alternatives22. Absolute quantitative measurement of intracellular metabolite concentrations and fluxes by Park et al. revealed that the lower glycolysis has a higher overall driving force in terms of change in free energy (roughly six-fold higher) than currently stated in biochemistry textbooks23.Previous studies have already tried to shed light on why the canonical glycolytic pathways are so uniquely prevalent despite the presence of many alternative routes with higher carbon and energy yields. A theoretical study by Melendez-Hevia 25. For example, fast-growing cells have to invest more resources for the synthesis of growth-related proteins 25. Instead of simply making proportionally more protein to accommodate higher growth requirements, they often shift metabolism towards pathways with more modest catalytic resources per unit of growth at the expense of energy efficiency26. Basan et al. further verified that E. coli switches from respiration to the more proteome-efficient fermentation under high growth rates27. Cellular metabolism has been shaped by evolution to ensure that carbon catabolic pathways are carefully selected to be in tune with both growth rate requirements and resource availability. Optimal glycolytic pathways must thus be able to balance high ATP production capacity while generating important intermediates and redox molecules at minimal proteome cost. These requirements are in direct conflict with one another requiring the establishment of Pareto optimal curves to decipher the relative “weights” between objectives that nature responds to when selecting different pathway designs.Concomitant to energy production and precursor synthesis hypotheses, recent studies have reaffirmed minimization of enzymes production as a key driver of optimizing resource allocation28 database using a modified implementation of the optStoic protocol29 . Driving thermodynamics closer to the limit  lowers the overall thermodynamic driving force thereby demanding a higher protein cost to drive the same amount of flux through the pathway. High (>2) ATP yielding pathways are also less tolerant to the changes in metabolite concentration ranges and ATP/ADP ratios, whereas the two canonical glycolytic pathways remain feasible under highly varying redox and metabolite states.In this study, we aim to systematically assess the relative importance between various objectives driving pathway selection by exhaustively generating over 11,916 routes from glucose to pyruvate with varying energy production efficiency per mole of converted glucose and quantifying the corresponding total protein investment. The pathways were designed by combining annotated reactions from the entire chemical repertoire of organisms accessed from the KEGGWe first exhaustively traced pathways from glucose to pyruvate that conform to a general glycolysis stoichiometry while generating 1 to 5 ATP per glucose. We then filtered the pathways based on their thermodynamic feasibility and subsequently predicted the minimal protein cost of each pathway. We identified the Pareto optimal for the tradeoff between protein cost and ATP yield of glycolytic pathways and further determined the main factor(s) that affect the protein cost for a pathway. Several novel pathway designs with higher ATP yields are discussed and their lack of robustness to wide ATP/ADP fluctuations is demonstrated.(i)+ = 2 Pyruvate + 2 NAD(P)H + 4 H+ (Glucose + 2 NAD(P)(ii)+ = ATP + H2O (ADP + Phosphate + HA glycolytic pathway is defined here as the conversion of glucose into pyruvate accompanied by the generation of energy cofactor ATP and redox cofactor NAD(P)H. This conversion can be described by splitting the overall reaction into two balanced sub-reactions:n) of ATP produced is given by:The overall reaction allowing for a varying amount . Alternate pathways were identified by iteratively appending integer cuts (see SI for details).The maximum number of ATP that can be generated while maintaining 19. Using directly optStoic29 led to a significant number of pathways containing disjoint subnetworks such as ATP generating cycles  matrix of the reaction database resulting in the Sint matrix which contains only the internal reactions. The rows involving cofactors  of the Sred matrix is generated with each row indicating a closed loop of reactions that result in no net non-cofactor metabolite production. A disjoint subnetwork  that exchanges only cofactors with the main glycolytic chain is now a closed loop on its own. The constraints  derived from the loop law33 prevent net flux traversing any such a loop in a cyclic manner and essentially prohibit the selection of the disjoint subnetwork. Therefore, ATP production can occur only on the main carbon transfer pathway  ). When the concentration range of CO2 was also restricted based on experimental measurements35, twenty-four more 1 ATP-yielding pathways and one more 2 ATP-yielding pathways became infeasible (condition (iii)). In addition, when the cofactor ratios  were allowed to vary only within experimentally observed ranges (condition (iv)), 77% of the pathways designed were rejected including all of the 5 ATP yielding pathways. Finally, when the metabolite bounds were further restricted to between 1 µM to 10 mM (condition (v)), all of the 3 to 5 ATP-yielding pathways were found to be thermodynamically infeasible, whereas both canonical glycolytic pathways  remained thermodynamically feasible. This strongly suggests that EMP and ED not only lie on the optimal tradeoff curve for yield vs. protein cost but do so while maintaining robustness to cofactor and metabolite concentration changes.As expected the fraction of pathways deemed thermodynamically feasible strongly depends on the imposed metabolite concentration bounds. Tighter concentration bounds reduced the number of feasible pathways 37 to ensure the production of many intermediate metabolites and redox equivalents. We computationally explored whether it is possible to identify a glycolytic pathway variant with a lower cost than the canonical glycolysis (ED and EMP). The absence of a lower cost pathway would bolster the cost-benefit hypothesis that natural evolution converges toward parsimonious enzyme expression38. Often the sum of absolute fluxes through a pathway is used as a proxy to total enzyme requirement39, however, actual enzyme demand depends both on the enzyme catalytic efficiency and the metabolite concentrations. The lack of experimentally measured kinetic parameters, intracellular metabolite concentrations, and enzyme mechanisms hampers the development of a detailed mechanistic model for each pathway across different organisms.A significant fraction (from 10% to 20%) of the total proteome is allocated to glycolytic pathways 30. We employed this computationally tractable approach to evaluate the minimal protein cost for operating any glycolytic pathway variants in a host cell-agnostic manner by assuming that all enzymes are equally efficient (see Methods) and metabolite concentrations are allowed to vary between 1 µM and 100 mM. Note that this analysis provides a lower bound to the actual enzyme cost40. Due to a large number of glycolytic variants, an automated pipeline was developed for the generation of the kinetic models of each thermodynamically feasible pathway (under the same condition) and the subsequent analysis of the minimal enzyme cost.To this end, we used the scalable convex optimization-based enzyme cost minimization (ECM) algorithmThe minimal protein cost of glycolytic pathway variants for each ATP yield spans a wide range regardless of the redox cofactor(s) generated  see Fig. . By plotNotably, the canonical ED and EMP pathways lie close to the Pareto front suggesting that they are among the most protein cost-efficient pathways in their respective ATP yield category. The distance between the canonical ED pathway and the Pareto front is 0.129 mg Protein/mmol Glc/h, whereas the distance between the EMP pathway and the Pareto front is slightly larger at 0.429 mg Protein/mmol Glc/h. Out of the many possible ways  that nature can construct a glycolytic pathway, EMP and ED are consistent with a near-optimal protein resource allocation.M) for their respective substrates. Therefore, it is expected that the thermodynamic driving force for the thermodynamic bottleneck  of a pathway is the main factor that affects protein cost 46. ATP is first invested in the phosphorylation of glyceraldehyde that is generated from the cleavage of KDG by KDG aldolase. Three ATPs are then produced downstream through the conversions of 1,3-BPG to 3-PG, 2,3-BPG to 2-PG and PEP to pyruvate. This is made possible by the investment of inorganic phosphate to phosphorylate 3-PG to form the higher energy intermediate 2,3-BPG. The lack of phosphorylation of glucose to minimize the escape  of neutral charged glucose from the cell in the first step possibly made this pathway less favorable for most organisms. Nevertheless, the second step converts glucono-1,5-lactone into gluconic acid which is a polar compound with reduced membrane permeability19.The EMP glycolysis generates two ATP per glucose. It incurs 55% more protein cost than the ED pathway under condition i Fig. . The mos+-dependent phosphorylating GAP dehydrogenase and ATP-generating phosphoglycerate kinase, the other bypasses the ATP-forming phosphoglycerate kinase step by using the non-phosphorylating GAP dehydrogenase (GAPN). Both NAD+ and NADP+-dependent GAPN have been previously identified in hyperthermophilic archaea T. tenax as well as in photosynthetic higher eukaryotes47. The resulting 3-PG is routed through the modified Rapoport-Luebering shunt (described in the previous section) to form 2-PG, thereby generating two extra ATPs. Five ATPs are produced in lower glycolysis which compensates for the two ATPs invested in the upper glycolysis for a net of three ATPs.In this section, we will discuss a few identified pathways generating more than two ATPs per glucose molecule. To derive an extra ATP from a pathway resembling EMP, the carbon flux from GAP is first split into two branches Fig. . While oC. cellulolyticum48 glycolysis by utilizing a PPi-dependent PFK thereby bypassing an ATP investment upstream requirement  per glucose molecule through its glycolytic pathway. This is achieved by (i) using a reversible PPi-dependent PFK; (ii) generating 2 GTPs from the reactions catalyzed by phosphoglycerate kinase  and a GTP-dependent PEP carboxykinase ; and (iii) generating 2 ATPs from an ATP-dependent pyruvate carboxylase . The PPi consumed by PFK is suggested by Rabinowitz et al. to be regenerated through the conversion of sedoheptulose-1,7-bisphosphate (SBP) to sedoheptulose 7-phosphate (S7P)47. Accounting for PPi usage, this C. cellulolyticum pathway generates an equivalent of 4 ATPs. In the pathway designed using the modified optStoic procedure  and E4P, which is catalyzed by fructose-bisphosphate (FBP) aldolase. The latter returns to the previous steps, while DHAP is channeled into the lower glycolysis. The lower glycolysis is similar to the 4 ATP pathway in Fig. Finally, a 5 ATP yielding pathway bypasses an ATP investment in the upper glycolysis by combining transaldolase from the non-oxidative pentose phosphate pathway with the reverse of the Calvin cycle Fig. . It firsM). A reaction close to equilibrium is often followed by a reaction that is substrate significantly sub-saturated. Even though the overall pathway C. cellulolyticum, may become infeasible in a different organism with a higher growth rate and/or different intracellular metabolite pool. Overall, a combination of higher protein cost, lower pathway driving force and sensitivity to metabolite pools may explain why these higher ATP yielding pathways have not become dominant in glycolytic organisms. In addition, instead of optimizing ATP yield, organisms may choose to maximize the rate of ATP generation by increasing substrate uptake rate through lower yield pathways50.Comparison of the protein cost distribution between the low ATP Fig.  and highays Fig. , the oveΔrG′Fig. . Near-eqE. coli are all above unity52. We repeated the MDF test when the ATP/ADP ratio is constrained to be above unity  growth phase, where cofactors and intracellular metabolites in organisms undergo high fluctuations 55. We find that the robustness of a pathway to different intracellular concentrations may ultimately determine whether a pathway is universally adopted. We have demonstrated that at least for glycolytic pathways, the Pareto optimal surface for protein cost vs. energetic yield must be extended to include the dimension of robustness. It is plausible that robustness imperatives that apply to naturally selected pathways may also be important for synthetic ones. This may explain why it is important to a posteriori subject engineered cells to directed evolution56, conditioning57 or adaptive evolution58 to identify variants with robust phenotypes. Interestingly, evolved strains often alleviate these bottlenecks with mutations that confer robustness to the engineered pathway  and subsequently growth rate58. Alternatively, one may impose feasibility of operation of a pathway over a wide range of concentrations and relevant energetic and redox cofactor ratios H/NAD(P)) as a design imperative. Our procedure can be used in this case to facilitate the design, analysis, and down-selection of (synthetic) bioconversion pathways.While optimizing protein resource is an important factor in pathway selection, robustness to extracellular changes  also plays a major role in shaping metabolic pathwaysin silico experiments using the widest possible range of metabolite concentrations.In this study, we assume that the metabolite concentrations are homogeneous within a cell. Compartmentation of metabolites and proteins may affect the optimal metabolite concentration for MDF  as well as the protein cost required to operate the pathway. For example, cells may need specific proteins/energetic demand to transport the metabolites between compartments. However, it is possible that with compartmentations, multiple local concentrations of a metabolite are allowed in different compartments and a pathway that was infeasible may become feasible. In addition, the concentration of metabolites varies significantly across species. This would ultimately affect the feasibility of various glycolytic alternatives. Due to the difficulty of obtaining high quality absolute quantitative metabolomics data for a large number of species, we have decided to perform these C. cellulolyticum and C. thermocellum utilize59. Recent efforts have started to leverage these organisms for the biosynthesis of various biofuels and bioproducts61.Beyond the current focus of this study, this work provides templates for designing alternate glycolytic pathways for potential metabolic engineering applications , especially for metabolites near central carbon metabolism that require additional ATP for biosynthesis. While most of the designed glycolytic pathways generating >2 ATP become thermodynamically infeasible, we did identify several alternatives that can be coupled with other engineered pathways for improving overall yield, albeit with higher protein cost and lower flux rates. This introduces a separate Pareto optimality problem between yield and productivity and designing pathways to systematically optimize between them. For example, we identified multiple such high ATP yielding glycolytic pathways see Fig.  that res29 was curated to ensure that all reactions are elementally  balanced and updated with new reactions from the KEGG database28. The updated optStoic reaction database contains a total of 7,164 reactions and 5,969 metabolites  was retrieved from KEGG and converted into InChI using Open Babel62 or ChemAxon ). The Gibbs free energy of formation of these compounds was then calculated using the same method and added to the database. Note that the same standard Gibbs free energy change of a reaction ) were removed. The standard transformed Gibbs free energy of all reactions (Step 1:Step 2:Step 3: A reaction is deemed (i) irreversible in the forward direction if both 29. Consequently, the updated database contains a total of 5,014 reversible reactions  per glucose mole is given by:29 to identify the set of reactions that conform to the above glycolytic stoichiometry. The minFlux formulation is given byI and J represent metabolites and reactions, respectively. i and each column representing reaction j, vj is the flux of reaction j, ite i. The set of all exchange reactions vj and qi is the stoichiometric coefficient of metabolite i in the design reaction. j. The objective function  formulation from the optStoic procedure2O) exchanges. This often results in pathway designs where the entire driving force of the conversion is accomplished by futile cycles disconnected from the main metabolism  with only energy , redox H) or other cofactors  was constructed by first removing all exchange reactions (columns). Subsequently, rows containing the selected cofactors  ATP hydrolysis, (ii) redox generation or (iii) water-splitting, can be represented by linear combination of the null basis Nred.Step 3: The following constraints are then appended to the optStoic formulation:L is the set of all loops, l and reaction j, M is a large positive number , and the variable Gj is a pseudo free energy parameter33, which does not reflect the actual thermodynamic driving force equivalent of reaction j  modeling system and Gurobi Optimizer v6.5.1 using Python 2.7. The codes for the optStoic procedure are provided as a part of the 34. The MDF formulation in essence attempts to identify a set of metabolite concentrations that ensure the lowest free energy changes for all the reactions in a pathway. If the objective value of MDF is positive, then the pathway is thermodynamically infeasible. See details in SI supplementary text.The thermodynamic feasibility of each pathway under physiological concentration ranges are assessed using the max-min driving force (MDF) formulation40. See details in SI supplementary text.The minimal enzyme demand in units of mg protein/mmol glucose/h for each one of the thermodynamically feasible pathways is then estimated based on the enzyme cost minimization (ECM) methodoptstoic-python package described in the previous section.To assist in the analysis of a large number of pathways designed using the modified optStoic approach, each pathway and reaction are represented as a Pathway Class object and Reaction Class object, respectively. A directed bipartite graph for each pathway is generated and rendered as SVG, PNG or JPEG format using the Graphviz software accessed through the Graphviz Python Package. The Python codes for pathway visualization are included in the Supplementary InformationSupplementary Dataset


Routine magnetic resonance imaging evaluation of the spine is often limited by low spatial resolution and artifacts resulting from cerebrospinal fluid pulsation. Balanced steady-state free precession sequences can supplement routine spin echo sequences and provide exquisite anatomic detail and high cerebrospinal fluid-to-soft tissue contrast, adding significant diagnostic value to the evaluation of a wide variety of spine disorders. In order to cover a large tissue volume, routine axial and sagittal spin echo (SE) sequences are typically 3–4 mm in thickness which can result in partial volume averaging and low spatial resolution. With significant improvements in imaging technology over the past few decades, MRI sequences utilizing steady-state free precession (SSFP) have become the staple of many clinical applications in neuroimaging. Such sequences have been shown to be particularly valuable for the evaluation of the cranial nerves , the cis2In a gradient echo sequence, when the repetition time (TR) is kept shorter than the T2 relaxation of the tissue, residual transverse magnetization (TM) is left over after each radiofrequency (RF) pulse. In classic steady-state sequences, this residual transverse magnetization is not wasted (as in spoiled GRE sequences) but is instead fed back into longitudinal magnetization (LM) with the next RF pulse. After a train of RF pulses, the repeated flipping of longitudinal magnetization into transverse magnetization and vice versa results in a nonzero steady state for both LM and TM  6]..6].For SSFP image formation, the two types of signals generated after each RF pulse include the free induction decay (FID) signal which has mixed T1 and T2* weighting and a spin echo from the prior RF pulse which has T2 weighting . In a b-CISS (Siemens) and FIESTA-C (GE) are equivalent sequences which represent a further modification of the b-SSFP paradigm. These modifications help reduce phase shift errors that cause dark banding artifacts which may obscure the image. In these implementations, two successive three-dimensional (3D) b-SSFP sequences are acquired, one performed with RF pulses which have alternating +α and -α flip angles and the other with a constant +α angle. These two datasets will contain banding artifacts which are mutually shifted ,6. A maxOur patients were imaged on a 1.5 or 3.0 T GE scanner  using a spine array surface coil. Multiplanar reformats were created on an independent PACS workstation. Scanning parameters for FIESTA-C are listed in 3CISS MRI provides fine spatial resolution and high contrast between cerebrospinal fluid/fat and solid structures, allowing the delineation of fine anatomic details such as the ventral and dorsal rootlets of the spinal nerves and the denticulate ligaments . The hig4MRI with conventional spin echo sequences is considered to be the modality of choice in the evaluation of degenerative spine disease with neurologic symptoms. However, MRI findings sometimes do not correlate with clinical symptoms which leads to diagnostic ambiguity. In particular, lateral recess nerve root compression has been reported to be underestimated by MRI in 28–29% of surgically confirmed cases, compared to 5–7% by conventional myelogram  noted im5Traumatic pseudomeningocele are abnormal collections of CSF outside of the thecal sac formed secondary to nerve root avulsions. Its formation is suggestive of a preganglionic injury which portends a poor prognosis for recovery and may necessitate nerve transfer for partial functional recovery . Small pPseudomeningocele formation is a possible complication of intradural spinal surgery. Symptomatic pseudomeningoceles can lead to wound breakdown, nerve root and cord herniation, and intracranial hypotension . Post-op6Spinal dural arteriovenous fistula (dAVF) is an underdiagnosed entity which can lead to progressive myelopathy and morbidity if untreated . CISS co7Arachnoiditis refers to inflammation of the spinal roots, meninges and subarachnoid space. Post-inflammatory fibrinous exudates coat the arachnoid membrane and nerve roots, which adhere to each other and the dura, causing clumping and neurologic impairment. This may result from prior spinal hemorrhage, operation, trauma, infection, inflammation, and lumbar puncture. The high CSF-to-soft tissue contrast of CISS allows resolution of fine meningeal adhesions, nerve root displacement and clumping, and subarachnoid scarring. CISS delineates the level and extent of arachnoiditis and demarcates the site of adhesion which is important in planning surgical release ,19. CISSSpinal cord compression secondary to arachnoid cysts is a rarely reported condition . Intradu8Syringohydromyelia is defined as intramedullary cystic dilatation of the spinal cord with or without communication with the central canal. The underlying etiology may be related to CSF flow disturbance from injury (e.g. trauma or inflammation) or structural malformation (e.g. Chiari I or tethered cord syndromes) . In many9Contrast enhanced MRI is the modality of choice in the imaging workup of spinal leptomeningeal metastases . In the 10Pediatric spinal dysraphism encompasses a wide spectrum of congenital anomalies resulting from abnormal neural tube closure and are divided into two main categories of open and closed defects . MRI is 11While CISS utilizes consecutive runs of 3D b-SSFP to minimize banding artifacts, these can still occur in areas of field inhomogeneity such as air-bone and soft tissue interfaces. These artifacts can be more pronounced at higher magnetic field strength (3.0 T compared to 1.5 T) secondary to increased sensitivity to susceptibility . DespiteWhile CISS accentuates the contrast between CSF and solid tissues, the intrinsic contrast resolution of neural structures is poor compared to spin-echo images. Furthermore, in cases such as collapsed myelomeningocele or severe spinal stenosis where neural structures are no longer surrounded by high signal CSF, CISS is unable to provide any more useful information than conventional spin-echo sequences. Therefore, CISS must be interpreted in combination with conventional T1- and T2-weighted images and is best used as a supplemental problem-solving sequence. This added acquisition time can be problematic for patients who are unable to tolerate the increased scan time, increasing the likelihood of motion artifact and incomplete exams. However, acquisition time can be improved by scanning in the sagittal plane along the short axis of the spine and reformatting in the remaining planes.12CISS is a useful troubleshooting sequence which provides the fine spatial resolution lacking in conventional spin echo sequences and accentuated contrast between CSF and neural structures. When used in concert with routine sequences, CISS can provide significant added value in a wide variety of spine applications.


These viruses have been circulating in the Americas for ≈20 years and show recombination with other genotypes. Clues to norovirus natural history can guide development of treatment and prevention plans.We report multiple nontypeable genotype II noroviruses circulating in South America; nucleotides differed by  Norovirus is a leading cause of acute gastroenteritis .Advances in genome sequencing approaches enabled us to detect a novel (nontypeable) GII norovirus circulating in Peru in 2008  Table. TThe maximum-likelihood phylogenetic tree of the RdRp-encoding nucleotide sequences showed that the newly obtained strains from Argentina and Peru were closely related to each other and clustered with a group of nontypeable strains (recently classified as GII.P23–GII.P27) . This geSimilar to the RdRp phylogenetic tree, the analysis of the VP1-encoding nucleotide sequences showed a distinct group of nontypeable strains (recently classified as GII.23–GII.27) . This geThe analysis of the VP2-encoding nucleotide sequences also showed a distinct group of those GII.23–27 genotypes; however, clustering of genotypes (particularly GII.27) seen in VP1 was disrupted in the VP2 tree . These gAll strains in this group were detected in the Americas   Figure 2Although only a few genotypes  are most prevalent among humans, those with limited circulation can emerge, causing large outbreaks  (Supplemental data for study of recombinant nontypeable genotype GII human noroviruses in the Americas.Table showing mismatches of common quantitative PCR primer sequences.


Cellular Dissociation Grade (CDG) composed of tumour budding and cell nest size has been shown to independently predict prognosis in pre-therapeutic biopsies and primary resections of oesophageal squamous cell carcinoma (ESCC). Here, we aimed to evaluate the prognostic impact of CDG in ESCC after neoadjuvant therapy.We evaluated cell nest size and tumour budding activity in 122 post-neoadjuvant ESCC resections, correlated the results with tumour regression groups and patient survival and compared the results with data from primary resected cases as well as pre-therapeutic biopsies.p = 0.016).CDG remained stable when results from pre-therapeutic biopsies and post-therapeutic resections from the same patient were compared. CDG was associated with therapy response and a strong predictor of overall, disease-specific (DSS) and disease-free (DFS) survival in univariate analysis and—besides metastasis—remained the only significant survival predictor for DSS and DFS in multivariate analysis. Multivariate DFS hazard ratios reached 3.3 for CDG-G2 and 4.9 for CDG-G3 neoplasms compared with CDG-G1 carcinomas (CDG is the only morphology-based grading algorithm published to date, which in concert with regression grading, is able to contribute relevant prognostic information in the post-neoadjuvant setting of ESCC. ESCC is often associated with an aggressive disease course, and a substantial percentage of patients are in locally advanced clinical stages at the time of initial diagnosis.14 Classical WHO-based histopathologic grading of ESCC, which is based on the morphological factors keratinization, nuclear pleomorphism and mitotic activity,19 is not applied after neoadjuvant therapy, not only because chemoradiation is known to profoundly influence critical factors of this grading system such as nuclear size21 but also because its prognostic significance is highly controversial even for primary resected ESCC and de facto non-existent after neoadjuvant therapy.18In the neoadjuvant therapy setting, patient prognosis and consequently the selection of postoperative treatment modalities are mainly determined by postoperative histopathologic staging and (to a lesser extent) histopathologic tumour regression grading.24 In ESCC, Cellular Dissociation Grading has been shown to be a stage-independent predictor of the disease course when measured in pre-therapeutic biopsies25 as well as in primary resection specimens.26 However, the feasibility and the prognostic significance of the application of the Cellular Dissociation Grade in ESCC after neoadjuvant treatment has not been investigated so far.Recently, a novel grading approach termed Cellular Dissociation Grade (CDG) based on the evaluation of tumour budding and cell nest size, which are histologic factors that measure the extent of cellular dissociation either from a quantitative (tumour budding) or a qualitative (cell nest size) angle, was proposed for squamous cell carcinomas from various anatomic sites.25 and with data from a cohort of primary resected ESCC.26In order to probe for the transferability of the Cellular Dissociation Grade to the post-neoadjuvant treatment setting in ESCC, we investigated tumour budding and cell nest size in a cohort of 122 ESCC resection specimens after neoadjuvant chemoradiation and correlated the results with postoperative pathological staging , histopathologic tumour regression scoring and overall-, disease-free- as well as disease-specific survival. Furthermore, we compared the Cellular Dissociation Grade derived from resection specimen after neoadjuvant treatment with data from pre-therapeutic biopsies of the same patientsn = 53), the remaining patients  received 5-fluorouracil (5-FU) monotherapy. Of the patients with Platin-based chemotherapy, Cisplatin monotherapy  was the most common therapy approach followed by Oxaliplatin combined with 5-FU  and Carboplatin combined with Paclitaxel . The time span from the date of the initial preoperative diagnosis (as a surrogate time point for the date of initiation of chemoradiation) to surgical resection was available for 113 of the 122 patients (93%) and ranged from 51 to 168 days (mean: 95.7 days). The general surgical procedure applied for all patients with ESCC was abdomino-thoracal resection after Ivor-Lewis,27 which is the standard surgical procedure for oesophageal cancer at our hospital.Our cohort comprised resection specimens from 122 patients suffering from ESCC, who underwent resection after neoadjuvant chemoradiation. Patients were surgically treated between 1991 and 2016 at the University Hospital Rechts der Isar of the Technical University Munich. Mean patient age was 57 years (range: 30–80); male patients were more common than female ones . Neoadjuvant chemotherapy combined with radiation therapy was administered in a synchronous fashion according to the guidelines in place during the time of treatment. The exact chemoradiation scheme was available for 66 of the 122 patients (54%). Radiation dose ranged from 28.8 to 60 Grey . Sixty-three of the 66 patients received a dose of 40 Gy or more. The chemotherapy scheme was Platin-based in 80% of these patients  patients, additional data on the Cellular Dissociation Grade from pre-therapeutic biopsy specimen of the same case were available.n = 6, cT3: n = 107 and cT4: n = 7; cN0: n = 23, cN1: n = 97). Post-neoadjuvant ypTNM data were available for all patients. Histopathological assessment after chemoradiation of the respective resection specimen resulted in 47 patients with carcinomas in a rather early local stage  and 75 patients with locally advanced tumours . Vital lymph node metastases were detected in 47 patients . In total, 95 patients (77.9%) died during follow-up, 86 deaths were tumour specific. A relapse of the disease was noted in 92 (75.4%) patients during follow-up , who was blinded for clinicopathological data and the disease course. Interobserver reproducibility was probed by randomly selecting 45 cases from the cohort, that were evaluated by a second experienced pathologist , fully blinded to the results of the initial evaluation by observer 1.14 subclassifying neoadjuvantly treated ESCCs in complete responders , subtotal responders , partial responders  and nonresponders . Tumours showing complete regression could not be included in the cohort for the obvious reason that in these cases no gradeable tumour was present anymore.Furthermore, all carcinomas were divided into keratinizing, non-keratinizing or basaloid neoplasms. Tumour regression was scored in analogy to the regression score proposed by Becker et al.,The dissociation of small tumour clusters consisting of <5 neoplastic cells into the peritumoural stroma in an invasive fashion was defined as “tumour budding”. Tumour budding activity was investigated throughout the whole slide and scored within the high- power field showing the highest budding activity. The visible area in ×40 magnification was defined as a high-power field (HPF). The field diameter for one HPF (0.55 mm) was determined through division of the field number (22 ×) by the objective magnification (× 40). Low tumour budding activity was stated if 1–4 and high tumour budding activity was stated if ≥5 budding foci were identified , all carcinomas were evaluated for tumour budding within one HPF, reporting the HPF that showed the highest budding activity. In addition, the whole tumour was evaluated for the smallest cell nest size. This approach is in complete analogy to the evaluation algorithm applied in the ESCC biopsy setting.25 A score of 1 received tumours without budding activity, tumours with low budding activity (<5 buds per HPF) were assigned a score of 2 and tumours with high budding frequency (≥5 buds per HPF) received a score of 3. Regarding cell nest size, tumours with large cell nests (>15 cells) received 1 point, while tumours with intermediate (5–15 cells) and small (2–4 cells) nests were scored with 2 and 3 points, respectively. ESCCs with single-cell invasion were scored with 4 points. The sum of both variables resulted in a final grading score ranging from 2 to 7. By using this score, we composed a Cellular Dissociation Grade algorithm for ESCC after neoadjuvant treatment, which is generally identical to the grading algorithm proposed for primary resected ESCC and pre-therapeutic biopsy specimens.26 CDG-G1 (well-differentiated) ESCC had a score ranging from 2 to 3, CDG-G2 (moderately differentiated) neoplasms had a score ranging from 4 to 5 and CDG-G3 (poorly differentiated) carcinomas had a score from 6 to 7. The algorithm for Cellular Dissociation Grading of ESCC after neoadjuvant treatment is summarised in Table We established a grading system that we termed “Cellular Dissociation Grade” (CDG) by using a combined score incorporating both parameters, tumour budding and cell nest size. The modified grade is in analogy to our previous work of primary resected ESCC, that evaluated the extent of both factors in 10 HPFs.2 (chi-square) test as well as χ2 test for trends. Survival analyses were performed by using the Kaplan–Meier method; a log-rank test was used to investigate the significance of differences in survival probabilities. Multivariate survival analysis was performed by using the Cox proportional hazard model. P-values ≤ 0.05 were considered significant. As only planned hypothesis testing was performed, no corrections for multiple testing were necessary in this study.29Statistical analysis was performed by using the Statistic Package for Social Sciences 25.0 . Comparisons of tumour budding, cell nest size and Cellular Dissociation Grade between primary resections, resections after neoadjuvant therapy and pre-therapeutic biopsies were calculated with Mann–Whitney U and Wilcoxon tests. Associations of histopathological characteristics with clinicopathological parameters were calculated with χ14 were grade 3  in 49 out of 122 cases (40.2%), grade 2  in 22/122 cases (18%) and grade 1B  in 51/122 cases (41.8%).In our neoadjuvantly treated cohort, keratinizing tumours  were more frequent than non-keratinizing cancers , 19 ESCCs showed a basaloid morphology . Regression grades according to Beckerr = 0.717, p < 0.001), as seen previously in a primary resected26 and a biopsy cohort.25Tumour budding activity per 1 HPF) was distributed as follows: 15 cases (12.3%) had no budding activity, 49 tumours showed low budding (2–4 buds per one HPF) activity (40.2%) and 58 cancers had high budding (5 or more buds per one HPF) activity (47.5%). The analysis of the minimal cell nest size revealed large cell nests (>15 tumour cells) in 11 cases (9%), intermediate nests (5–15 tumour cells) were present in 4 tumours (3.2%) and 19 cases (15.6%) showed small nests (2–4 tumour cells). In the vast majority of cases , infiltrative clusters of singular tumour cells were observed and these cases were therefore classified as having single-cell invasion  and cell nest size (1–4 points) for each specimen resulted in 15 (12.3%) CDG-G1 (2–3 points) neoplasms, 19 (15.6%) CDG-G2 (4–5 points) tumours and 88 (72.1%) CDG-G3 (6–7 points) carcinomas. Figure –3 pointsn = 135) of almost equal size.26 Tumour budding was significantly higher in the neoadjuvantly treated cohort than in the primary resected population (p = 0.004). The same was true for cell nest size (p < 0.001); specifically the number of cases that showed single-cell invasion was substantially increased. Consequently, overall the group of neoadjuvantly treated cases showed significantly higher Cellular Dissociation Grades than the group of primary resected cancers (p < 0.001). This difference was likely due to the fact that full responders (ypT0)—who are not present in our neoadjuvantly treated cohort since no evaluable tumour is left—often fall into the CDG-G1/G2 category.We compared the frequency of tumour budding, cell nest size and Cellular Dissociation Grade in our neoadjuvantly treated cohort with our own previously published distribution data in a primary resected cohort . The time span between the initial diagnosis and the surgical resection was also not correlated with post-neoadjuvant Cellular Dissociation Grade (p > 0.5).In the subset of patients where the exact chemoradiation scheme was available, neither the type of chemotherapy nor the radiation dose were statistically associated with the post-neoadjuvant Cellular Dissociation Grades . As a consequence, composite Cellular Dissociation Grade was also associated with regression, minimally regressive tumours were significantly less differentiated than tumours with strong regression .Both tumour budding as well as cell nest size were strongly associated with the extent of tumour regression induced by neoadjuvant therapy. The smallest cell nest sizes and the highest budding activity was observed in those tumours that showed minimal to no regression (more than 50% viable tumour) as response to neoadjuvant treatment. In contrast, those cases with strong regression (<10% viable tumour) usually did not show single-cell invasion and strong budding activity (p < 0.05) but not with age, sex, ypT or ypM (Supplementary Table p = 0.09), but not perineural invasion (Pn) or venous invasion (V) was associated with a poorer differentiation according to CDG.When we correlated cell nest size, tumour budding and Cellular Dissociation Grade with staging data, a higher budding activity, smaller cell nest size and poorer differentiation was significantly associated with higher ypN stage  than for cell nest size , disease-specific (DSS) and disease-free (DFS) survival Table . The assp = 0.016, Table p = 0.029, Supplementary Table p = 0.150, Supplementary Table When we performed a multivariate analysis under inclusion of ypT, ypN, ypM, regression grade and Cellular Dissociation Grade, the only prognostically relevant factors that emerged were ypM and Cellular Dissociation Grade. Patients whose tumours were poorly (moderately) differentiated had a hazard ratio of 4.99 3.35) for DFS, when compared with their well-differentiated counterparts (.35 for Dp = 0.006) and DSS (p = 0.016).Although there is a certain overlap with nodal positivity, we additionally performed an exploratory multivariate analysis under inclusion of lymphatic vessel invasion (L0/L1), this resulted in an even stronger p-value for CDG for DFS  and DFS (p = 0.047) compared with well/moderately differentiated tumours (grouped because of low numbers). A clear impact on OS, however, was not observed. Regression grade 3 tumours were almost exclusively poorly differentiated .Interestingly, when we subdivided our cohort according to regression grade and stratified the subgroups for Cellular Dissociation Grade,26 as well as in pre-therapeutic biopsies of oesophageal squamous cell carcinoma,25 to the neoadjuvant setting by evaluating these factors in a large cohort of 122 ESCC specimens resected following neoadjuvant treatment. We were not only able to confirm the prognostic power of the Cellular Dissociation Grade in post-neoadjuvant resection specimens, but were also able to demonstrate that the Cellular Dissociation Grade remained the only prognostic factor for DSS/DFS besides the presence of distant metastases in a multivariate analysis incorporating postoperative pathologic staging , and tumour regression.14In this study, we investigated the transferability of Cellular Dissociation Grading based on tumour budding and cell nest size, which has already been established in primary resection specimens25The proposed grading is reproducible in this setting, which is in line with previous observations not only in biopsy specimens of ESCC but also in other tumour entities with squamous morphology.9 with consecutive surgical resection reflects the common therapy approach for locally advanced but still operable ESCC in the absence of distant metastases.8 In the postoperative setting of neoadjuvantly treated ESCC, histopathologic stage and tumour regression are considered to be among the strongest factors that determine the further course of the disease.14 Nevertheless, a considerable amount of ESCC patients with a partial or even subtotal response to neoadjuvant treatment suffer from disease relapse and consecutively die of the disease,12 underlining the need for additional biomarkers in addition to staging and tumour regression grading that allow for an improved prognostic patient stratification in the postoperative setting.Combined platinum-based chemotherapy and radiation15 and relies on traditional histomorphologic parameters such as nuclear pleomorphism, mitotic activity and degree of keratinization. In neoadjuvantly treated ESCC, WHO-based grading is usually omitted, not only because chemoradiation is known to considerably disarray relevant factors to this algorithm such as nuclear size/pleomorphism21 but also because it is almost irrelevant for clinical decision-making even in primary resected ESCC, due to its at best-limited prognostic significance.19Classical histopathologic grading of ESCC is currently performed in accordance with the algorithm of the WHO classification of tumours of the digestive system36 and other squamous cell carcinoma entities.38 Our proposed grading algorithm that has been shown to nicely discriminate prognostic subgroups in primary resected ESCCs26 as well as in pre-therapeutic ESCC biopsies25 and several other squamous cell carcinoma entities,39 pairs up tumour budding with cell nest size, a second parameter measuring cellular dissociation from a more qualitative angle. Therefore, our novel grading approach has been termed “Cellular Dissociation Grade”. While we already demonstrated preoperative Cellular Dissociation Grading performed on biopsies to be a strong pre-treatment predictor for ESCC survival independent from clinical staging in a previous study,25 the prognostic implications of Cellular Dissociation Grade in the post-neoadjuvant setting of ESCC have not been investigated until now. In this study of 122 post-neoadjuvant ESCC resection specimens, the Cellular Dissociation Grade retained its prognostic power in all survival comparisons, making the Cellular Dissociation Grade—to our knowledge—the first fully and solely morphology-based prognostic system working on tissue after neoadjuvant therapy in ESCC. The prognostic relevance of the Cellular Dissociation Grade is further underlined by our multivariate analysis incorporating the only currently known prognostic factors in the postoperative setting  of ESCC after chemoradiation, where Cellular Dissociation Grade remained the only multivariate prognosticator for DSS/DFS besides distant metastases. Even in the subset of ESCC with high tumour regression , tumour remnants showing a poor differentiation according to the Cellular Dissociation Grade were predictive of shortened DSS/DFS, which means that the grading system is able to further prognostically stratify patients even when they showed a good initial response to neoadjuvant treatment.Tumour budding, a histologic parameter that measures the quantitative extent of cellular dissociation of a neoplasia, has been identified as a highly prognostic factor in ESCCBesides the retained impact of Cellular Dissociation Grade on survival even after neoadjuvant therapy, specifically the association of the grading system with different therapy responder groups is very interesting. When we correlated tumour differentiation according to the Cellular Dissociation Grade with tumour regression grades, almost all of the nonresponders showed a poor differentiation, while well-differentiated and moderately differentiated tumours were much more common in subtotal responders, indicating that poorly differentiated tumours according to the novel grading system likely do not respond well to current chemotherapeutic approaches. This finding is supported by the association of a poor differentiation according to the Cellular Dissociation Grade in pre-therapeutic biopsies with a poor response to the neoadjuvant treatment in the respective resection specimen, although this finding needs further validation in light of the rather small subset of patients where pre- and post-neoadjuvant grading data were available. Another supporting argument is the fact that high Cellular Dissociation Grade cases enrich in the population of neoadjuvantly treated when compared with primary resected tumours, which is likely due to the fact that a certain number of cases with optimal response (ypT0) are for obvious reasons “deselected” from the neoadjuvant resection cohort evaluated here.The Cellular Dissociation Grade itself and thus the capacity of Cellular Dissociation of a given carcinoma does not seem to be strongly influenced by neoadjuvant chemoradiation, which is nicely demonstrated by the largely stable post-therapy grade in the subgroup of patients where pre-therapeutic grading data were available. This finding strongly argues against a mere selection of more poorly differentiated tumour areas of a given neoplasm by neoadjuvant therapy.The lack of predicting factors allowing us to choose the best adjuvant therapy option not only in those patients with but also in those without neoadjuvant therapy is a continuing nuisance for us and dismal for our patients. Although it has not been prospectively tested (and thus proven) regression grade—as an indirect measure of chemotherapeutic sensitivity—might have some potential in this regard. Our data suggest that CDG might be a second factor that could add some information here, since obviously CDG high-grade cases do not respond well, thus arguing that in those cases post-surgery adjuvant treatment might also be less beneficial.In conclusion, our study demonstrates that Cellular Dissociation Grading for oesophageal squamous cell carcinoma provides relevant independent prognostic information in the post-neoadjuvant setting. This makes Cellular Dissociation Grading the only morphology-based grading algorithm for ESCC published to date, which is able to independently contribute prognostic information in the post-neoadjuvant setting in concert with regression grade and pathological/clinical staging. In addition, evidence is mounting that Cellular Dissociation Grade holds potential as a predictor of response to conventional neoadjuvant chemotherapy.Supplemental Material


Tg(HuC:GCaMP5G) zebrafish larvae swimming in response to whole-field visual motion. We combined large-scale population calcium imaging in the hindbrain with simultaneous high-speed recording of the moving tail in animals where specific markers label glycinergic inhibitory neurons. We identified cells whose activity preferentially correlates with the visual stimulus or motor activity and used brain registration to compare data across individual larvae. We then morphed calcium imaging data onto the zebrafish brain atlas to compare with known transgenic markers. We report cells localized in the cerebellum whose activity is shut off by the onset of the visual stimulus, suggesting these cells may be constitutively active and silenced during sensorimotor processing. Finally, we discover that the activity of a medial stripe of glycinergic neurons in the domain of expression of the transcription factor engrailed1b is highly correlated with the onset of locomotion. Our efforts provide a high-resolution, open-access dataset for the community by comparing our functional map of the hindbrain to existing open-access atlases and enabling further investigation of this population’s role in locomotion.Locomotion in vertebrates relies on motor circuits in the spinal cord receiving inputs from the hindbrain to execute motor commands while dynamically integrating proprioceptive sensory feedback. The spatial organization of the neuronal networks driving locomotion in the hindbrain and role of inhibition has not been extensively investigated. Here, we mapped neuronal activity with single-cell resolution in the hindbrain of restrained transgenic  A great challenge in the study of motor control has been the technical difficulty of recording activity from neurons responsible for motor actions, in the midst of active motor actions. The experimental solution to this has mostly been to work in reduced or paralyzed preparations, where proprioceptive signals are either missing or reporting a failure to move. In order to best capture the neural activity representing these processes, functional recordings can be performed during active motion.When exploring their environments, animals constantly perform updates based on their motor actions and available sensory information. In the larval zebrafish, which swim via discrete bouts of tail oscillation4. By taking advantage of these existing transgenic lines, and with open-access larval zebrafish brain atlases, we have an opportunity to look at the activity of large populations of neurons and place that functional activity in the context of known markers, without the painstaking process of fixing and staining.While large scale population imaging data can be extremely informative on their own, new tools are emerging to genetically target specific populations of neurons and to register them in brain atlases, with close to cellular resolutionet al., revealed an intricate organization of the hindbrain in alternating stripes of neurons based on glutamatergic and glycinergic neurotransmitter expression7. These stripes reflect an underlying organization of the hindbrain, and are stacked according to developmental age, input resistance, and anatomical properties6. Yet the functional recruitment of interneurons from these stripes during active swimming is not yet known. GABAergic neurons were not investigated in that work, although we know they are found in large numbers in the hindbrain and cerebellum, previously identified by transgenic lines and in situ staining9.Previous work can inform our expectations regarding excitatory (glutamate) and inhibitory (glycine and GABA) neurotransmitter phenotypes in the hindbrain. Kinkhabwala et al., has been shown to be positive for the expression of the transcription factor engrailed1b 14. The only functionally identified neuronal class in the engrailed1b+ glycinergic stripe are the feedback-inhibitory neurons (FINs), known to provide glycinergic inhibition to the Mauthner cells during a very specific fast type of movement, the escape response6. Thus, there is still much left unexplored.The medial-most glycinergic hindbrain stripe identified in Kinkhabwala 4.Here we ask which neurons responsible for motor commands and sensorimotor processing are located in or near the zebrafish hindbrain. We use population calcium imaging to record from the brains of actively swimming but restrained larval zebrafish in an attempt to incorporate multisensory signals into our dataset. Since such information processing may incorporate both excitatory and inhibitory cell types, we then ask if these active populations are inhibitory by mapping them onto known transgenic lines in the ZBB atlasTg double transgenic animals, expressing pan-neuronally the GCaMP5G calcium indicator15 with mCherry expressed in glycinergic neurons9, homozygous for the mifta mutation (mitfa−/−17)  Fig. , Methodscue Fig. , and taicue Fig.  without cue Fig. , the kinet al.22. First, we applied an automated calcium source extraction method to identify potentially interesting activity in the calcium imaging data23  expression as a bridge, we registered our dataset with the ZBB brain atlas4. Once this bridge was established, we were able to easily compare the pixels associated with the clusters identified above with the transgenic lines available in the ZBB atlas.We then used the identified cluster means as regressors to identify the brain regions in each recording showing activity correlated to any of the clusters. We therefore calculated the pixel-wise correlation of each cluster mean during each swim event (for swim cluster 1) and each stimulus presentation (for stimulus clusters 1–3). To compare activity related to each cluster between larvae and brain regions, we registered the resulting average correlation maps for all 10 larvae to a common reference brain. Using Tg(glyt2:mCherry) line from our own dataset, and also with the images of the Tg(glyt2:GFP) line in the ZBB atlas, indicating activation of glycinergic cells during swimming  we concluded the swim cluster resides in the engrailed1b+ glycinergic stripe in the hindbrain  transgenic line which co-localize   Fig. , which lion Fig. . GABAergOur study provides the community with an open-access, single cell-resolution population imaging dataset of hindbrain calcium activity during active locomotion induced by optomotor stimuli. Here we investigated which inhibitory neurons are recruited during sensorimotor integration and found interesting responses in diverse populations of glycinergic and GABAergic neurons whose activity correlates with motor generation and/or visual integration.32. These larval brain atlases have enabled the integration of functional datasets with anatomical markers, in both fixed and live brains. By using a combination of live transgenic markers and registering with the ZBB atlas, we found an anatomical stripe of glycinergic neurons recruited during locomotion. Previous work on glycinergic contributions to zebrafish locomotion has shown that mutants and pharmacological blocks of glycine receptors and transporters cause locomotor defects, although these global methods are not able to target specific regions of the brain and spinal cord37. Anatomical characterizations of glycinergic neurons in the brain across development have revealed both a striped pattern of organization and a heavy innervation of the Mauthner cells by glycinergic neurons, implicating them in the regulation of the escape response39.In order to identify the neurotransmitter phenotypes of functional clusters, we relied on the combination of transgenics as well as the use of the recent creation of open-access zebrafish larval brain atlases by several groups40. Capelli et al., revealed the distinct roles of glutamatergic and glycinergic interneurons within brainstem paragigantocellular nuclei, which could not be resolved by activating the entire anatomical nucleus. Only by separating these intermixed populations did neurotransmitter-specific activation produce locomotor responses: either high-speed initiation of locomotion or full stop. These observations highlight the need for studies deciphering the opposing functions of neighboring cells of differing neurotransmitter phenotype and might explain contradicting observations from deep brain electrical stimulation in humans and macaques42. Capelli et al., specifically found that optogenetic activation of glycinergic cells of the lateral paragigantocellular nucleus induced locomotor halt or speed reduction proportional to stimulation laser intensity. It is an enticing possibility that a portion of the zebrafish hindbrain glycinergic stripe may represent a basal homologous structure common to vertebrates, although further functional, molecular, and anatomical characterization will be necessary.A recent paper in mammals has brought the role of glycinergic neurons in the brainstem into renewed focusengrailed1b+. The engrailed transcription factor has long been studied across vertebrates for its role in development of particularly the midbrain-hindbrain boundary and the spinal cord45. In the spinal cord, where engrailed+ neurons have been best characterized, they perform a critical role providing ipsilateral ascending inhibition across vertebrate species46. In the mammalian midbrain, engrailed1 has been implicated in a role in Parkinson’s disease48. Apart from the previously identified FIN neurons6, also ipsilateral and ascending, the role of the vast majority of engrailed1b+ hindbrain neurons has not been explored in zebrafish. In this study, we find activation of these neurons concurrent with locomotion. Future work should attempt to disentangle the various types of locomotor actions, whether it be comparing swimming to turning, or various swimming speeds, in order to further clarify whether engrailed1b+ neurons participate in all forms or specific forms of motor output. Intersectional genetic approaches in the future will allow disambiguation of midbrain-hindbrain engrailed1b+ neuronal function from their spinal counterparts.The swim cluster 1 region we identify appears to co-localize with the medial-most glycinergic stripe, which is 51, faster imaging techniques such as light-sheet imaging56, combined with cell-targeted electrophysiology will most likely be instrumental to resolve the recruitment of engrailed1b+ cells relative to the start and end of movement. The combination of light-sheet imaging and electrophysiological techniques will give the community a great opportunity to further explore the role of feedback in active circuits.Further investigation of the neuronal populations we identified here with electrophysiology or systematic single-cell ablation methods, combined with whole brain imaging, will elucidate their precise contribution to movement coordination. The temporal kinetics of voltage indicatorsDanio rerio larvae of AB, TL, or mixed background of mitfa -/- genotype (nacre17). Triple transgenic animals were Tg referred to as Tg for simplicity throughout, by crossing Tg(elavl3:GCaMP5G)15 with Tg9 and screening for double-positive embryos. Additionally, transgenic Tg(vglut2a:loxP-DsRed-loxP-GFP)6 larvae referred to as Tg(vglut2a:DsRed) were used for comparing and bridging datasets.All procedures were approved by the Institut du Cerveau et de la Moelle épinière (ICM) and the National Ethics Committee on E.U. legislation  based on EU legislation or were in accordance with institutional and national guidelines. Larvae were raised in an incubator set to 28.5 °C under a 14/10 light/dark cycle. Experiments were performed at room temperature (22–26 °C) on 5–6 days post-fertilization (dpf) larvae. All experiments were performed on Tg larvae were mounted in glass-bottom dishes  filled with 1.5% low-melting point agarose, and the agarose surrounding the tail was removed. Calcium imaging was performed on a two-photon microscope  including a Mai Tai Deepsee laser (Spectra-Physics) tuned to 980 nm and a Zeiss Axio-Examiner Z1 with a Zeiss W Plan-APO 20X water-immersion objective. Fluorescent images were acquired using Slidebook software  at 5.81 Hz with 1.2 µs dwell time during scanning. Each trial collected in a single plane was 315 s in duration and the image size was 512 × 249 pixels. Single-plane acquisitions were spaced by 10 µm in z and collected from dorsal to ventral in the brain. Optomotor stimuli were presented using an MP160 pocket projector (3 M) filtered with a Wratten #29 deep red filter . The stimulus was created using Labview 2012  and consisted of a square-wave grating alternating between 10 s of motion in a caudal-to-rostral direction with 5 s static presentation, delivered to a screen positioned on one side of the animal. To record tail movement, a similar setup was used in Bӧhm, Prendergast et al., 201657 where an 890 nm LED was used to illuminate the larva and behavior was recorded at 250 Hz from below through a 5x microscope objective and imaged on a camera (Basler) with a 50 mm camera objective. Infrared light from the imaging laser was blocked with a filter located in front of the camera. Calcium imaging, behavior, and stimulus presentation were synchronized via a Digidata series 1322 A digitizer, and pClamp 8.2 software . After each experiment, the larva was anesthetized with MS-222 and a high-resolution Z-stack was recorded with 1 µm z-step for both red (mCherry) and green (GCaMP5G) channels simultaneously. Parameters were: 1024 × 499–1024 pixels, 10 pixel averaging, 3–5 frame averaging, 4–8 µs dwell time per pixel.Double transgenic 58. From the tail-bend angle over time, we detected local maxima and minima from which bout and interbout durations were extracted. All bouts were visually inspected by experimenters and bouts were manually excluded from the dataset in cases of tracking failures, or when belonging to obvious escape or struggle behavior. Bouts were not further subdivided or categorized for this analysis. All custom MATLAB scripts are available upon request.Using custom MATLAB software , 8 segments of the tail were extracted between manually chosen points at the end of the swim bladder and the tip of the tail59. Initial ΔF/F fluorescence traces were extracted using the automated source extraction methods and code published in Pnevmatikakis et al., 201623 . After testing a range of number of components with our data, the algorithm always selected less than 150 meaningful components, thus we selected 150 to ensure none were missed. This method resulted in 150 traces per trial, without verifying that all of them contain significant activity.Image time series were motion corrected in 2D using fast non-rigid motion correction (NoRMCorre)To define the traces whose activity was most locked to either stimulus presentation or the occurrence of a swim bout, we used the following two-step approach. First, we extracted the parts of the trace around each swim bout (2 s before and 5 s after the start of a swim bout and >0.5 s after a previous bout) and around each stimulus presentation (5 s before and the full 10 s during which the stimulus is moving) in order to calculate the average swim-triggered and stimulus-triggered traces respectively. Second, we calculated the average correlation coefficient of each individual swim-triggered and stimulus-triggered trace to its swim- and stimulus-triggered average respectively.We used this measurement to evaluate how consistently the calcium signal changed during either swimming or stimulus presentation and to categorize each trace as swim-driven or stimulus-driven. Traces were defined as stimulus-driven when having an average correlation coefficient >0.6 with their stimulus-triggered average and swim-driven when having and average correlation coefficient >0.6 with their swim-triggered average. Traces with an average correlation coefficient >0.6 with both their swim-triggered and stimulus-triggered average were considered ambiguous and excluded from the dataset . To map the occurrence of each identified clusters back to the original anatomical stack, we extracted the swim-triggered and stimulus-triggered frames from the original calcium imaging time series. Each frame was filtered with a 2 pixel mean spatial filter before performing pixel time series correlation to the swim-triggered clusters and stimulus-triggered clusters respectively. The individual correlation maps for each cluster were then averaged over all swim bouts and stimulus presentations to obtain the final average correlation maps. For a schematic representation of this process please refer to Supplementary Fig. To identify different clusters of activity within each of these categories, the previously identified average swim-triggered traces and stimulus-triggered traces were then clustered for each category independently. This allowed us to define cluster centroids that correlate either with the occurrence of swim bouts or the presentation of the visual stimulus. Clustering was based on methods published in Bianco http://nitrc.org/projects/cmtk) and registration parameters were selected based on Marquart et al., 20153. High-resolution stacks of the GCaMP5 expression of each brain were registered to a common reference brain using affine registration  brain served as the reference. To reduce a low level of bleed-through of the DsRed fluorescence into the GCaMP5G channel, a bleed-through factor was estimated from the scatter plot of pixel values of the DsRed channel vs. the GCaMP5G channel. The pixel values in the GCaMP5G channel were then reduced by the value of the same pixel in the DsRed channel times the bleed-through factor.Brain registration was performed using the Computational Morphometry Toolkit , the recording of the DsRed in the Tg(vglut2a:DsRed) from our reference brain was used to perform affine registration to the DsRed in the Tg(vglut2a:DsRed) larvae from the ZBB reference brain . The resulting transformations were used to register all correlation maps emanating from a given larva to the reference brain to compare anatomical location across larvae. To bridge our functional and anatomical data to publicly available z-brain-browser data (Marquart Swim cluster 1 pixels (green) and HuC:GCaMP5G (magenta) moving dorsal to ventral.Stimulus cluster 1 pixels (green) and HuC:GCaMP5G (magenta) moving dorsal to ventral.Stimulus cluster 2 pixels (green) and HuC:GCaMP5G (magenta) moving dorsal to ventral.Stimulus cluster 3 pixels (green) and HuC:GCaMP5G (magenta) moving dorsal to ventral.Swim cluster 1 pixels (green) and engrailed1b (magenta) moving dorsal to ventral.HuC:GCaMP5G averaged over all larvae (green) registered to our reference brain HuC:GCaMP5G stack (magenta) moving dorsal to ventral.Reference brain with Vglut2a expression (green) registered to the ZBB Vglut2a brain (magenta) moving dorsal to ventral.The average brain across all larvae with glyt2:mCherry expression (green) registered to the ZBB glyt2:GFP brain (magenta) moving dorsal to ventral. Note there are two different transgenic lines used.The ZBB vglut2a:DsRed stack (magenta) compared with the ZBB inhibitory lines .Supplementary information


Glycine max leaves” [1]. Phosphorylation plays a critical role in the regulation of the biological activities of proteins. However, phosphorylation-mediated regulation of proteins and pathways involved in ethylene (ET) and abscisic acid (ABA) signaling is currently poorly understood. Therefore, we used a shotgun proteomics approach to identify the phosphopeptides and phosphoproteins in response to ET, ABA and combined ET+ABA treatments. Here, we present the Mass spectrometry, protein–protein interaction, Gene ontology and KEGG data associated with the ET and ABA signaling in soybean leaves [1].The data reported here are associated with the article “Comparative phosphoproteome analysis upon ethylene and abscisic acid treatment in  Daewon seeds were germinated in the soil and allowed to grow in a growth chamber at 25 °C  for one month 2.22 based phosphopeptide enrichment kit (Pierce Biotechnology) following manufacturer׳s protocol.Protein extraction and phosphopeptides enrichment were carried out as described previously 2.3QExactive™ Orbitrap High-Resolution Mass Spectrometer  was used for the identification of enriched phosphopeptides exactly using the same protocol as described earlier 2.4https://david.ncifcrf.gov/tools.jsp) with integrated PANTHER Gene Ontology (GO) and KEGG pathways was used for the functional annotation of the identified phosphoproteins. For the construction of protein–protein interaction (PPI) network, homologs of the identified phosphoproteins were searched in the Arabidopsis database obtained from Phytozome . The obtained Arabidopsis homologs were used for the PPI analysis by the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database (http://string-db.org/) and arranged using Cytoscape tool.DAVID functional annotation tool (


BTBD9 is associated with a higher risk of RLS. Hemodialysis patients also exhibit higher rates of RLS compared with the healthy population. However, little is known about the relationship of BTBD9 and end-stage renal disease to RLS pathophysiology. Here we evaluated sleep and leg muscle activity of Btbd9 mutant (MT) mice after administration of serum from patients with either idiopathic or RLS due to end-stage renal disease  and investigated the efficacy of treatment with the dopamine agonist rotigotine. At baseline, the amount of rapid eye movement (REM) sleep was decreased and leg muscle activity during non-REM (NREM) sleep was increased in MT mice compared to wild-type (WT) mice. Wake-promoting effects of rotigotine were attenuated by injection of serum from RLS patients in both WT and MT mice. Leg muscle activity during NREM sleep was increased only in MT mice injected with serum from RLS patients of ideiopatic and renal RLS. Subsequent treatment with rotigotine ameliorated this altered leg muscle activity. Together these results support previous reports showing a relationship between the Btbd9/dopamine system and RLS, and elucidate in part the pathophysiology of RLS.Idiopathic restless legs syndrome (RLS) has a genetic basis wherein  Although the detailed pathological mechanisms of RLS are unclear, RLS occurs in an idiopathic form of genetic origin. Variants in BTBD9, MEIS1, MAP2K5/SKOR1, and PTPRD are known to confer a significant risk for RLS5. The most common conditions associated with RLS include iron deficiency, renal failure, neuropathy, spinal cord pathology, pregnancy, and multiple sclerosis. The use of certain medications, including selective serotonin reuptake inhibitors, lithium, dopamine antagonists, and caffeine can also increase the rate of RLS8. Patients with end stage renal disease (ESRD) receiving hemodialysis also have a remarkably higher incidence of RLS relative to the healthy population10.Restless legs syndrome (RLS) is a common sensorimotor disorder, whose basic components include a sensory experience, akathisia, and a sleep-related motor sign, periodic leg movements during sleep (PLMS), both associated with an enhancement of the individual’s arousal state12. Rotigotine, a non-ergot dopamine agonist that stimulates all dopamine receptor subtype (D1-D5), is approved for the symptomatic treatment of moderate to severe idiopathic RLS13. In a rat model of RLS caused by lesioning of dopaminergic nuclei, exposure to the neurotoxin 6-hydroxydopamine (6-OHDA) prevented increases in the frequency of limb movement14, whereas treatment with the dopaminergic agonist pramipexole decreased limb movement frequency14, suggesting that the dopamine system is involved in pathological mechanisms associated with RLS.Non-medication therapies such as iron supplementation and life guidance and drug therapies involving dopamine agonists have been widely used to treat RLS. Dopamine therapies have also been reported to be effective in treating RLS symptoms associated with chronic kidney diseaseBTBD9 encodes a protein that has two highly conversed domains, the BTB/POZ domain and the BACK domain, which are both associated with transcriptional regulation and protein ubiquitination. In humans, a genome-wide association (GWAS) study indicated that individuals carrying BTBD9 variants have increased risk of familial RLS3. BTBD9 variants are also associated with RLS in ESRD15, although another study reported differing results9. In animal models of RLS, Btbd9 mutant mice exhibited an increased travel distance in an open field test and an increased amount of voluntary activity in a wheel running analysis during the rest phase; these changes were accompanied by altered serum iron levels16. In Drosophila, loss of the BTBD9 homolog CG1826 (dBTBD9) resulted in sleep fragmentation and increased motor activity17. The same report indicated that BTBD9 controls dopamine levels in fly brains and iron homeostasis in human cell lines17. However, little is known about the physiological function of BTBD9 and its relationship to RLS pathophysiology19. In particular, how BTBD9 is associated with RLS that frequently occurs in hemodialysis patients is unclear. We also used a mouse model to examine how genetic mutations such as those occurring in BTBD9 contribute to the exacerbation of RLS symptoms and whether these symptoms are ameliorated by rotigotine. We also injected wild-type (WT) and Btbd9 mutant (MT) mice with serum from RLS patients with and without renal involvement who had and had not undergone hemodialysis to examine whether substances in RLS patient serum affect response of RLS symptoms to rotigotine.Btbd9 was detected by reverse transcription polymerase chain reaction (RT-PCR) assay based on total RNA extracted from mice prefrontal cortex. On the other hand, WT mRNA expression of Btbd9 was amplified obviously  sleep observed for ice Fig. , but theice Fig. . MT miceice Fig. .Figure 1To evaluate sleep homeostasis, mice were sleep-deprived (SD) for 6 hours. During the recovery period after SD, both WT and MT mice showed increased SWA in NREM sleep Fig. .In a comparison of representative waveforms of EEG/EMG for WT Fig.  and MT  mice, EMNext, we examined the effect of the dopamine agonist rotigotine  on sleep and muscle activity in mice injected with serum from five RLS patients  decreased the amount of REM sleep during the light phase in WT mice injected with placebo and serum from healthy subjects, and increased the amount of REM sleep during the dark phase Figs  and 5a,b3 receptor subtype have a higher efficacy on periodic leg movements and RLS than a drug that preferentially targets the D2 receptor subtype20. These results indicate that serum from an RLS patient diminished the wake-promoting effect of rotigotine treatment in mice. However, the influence of dopamine D2/D3 agonist to sleep structure, human studies will be necessary in the future.Dopamine agonists targeting the dopamine DLeg movement measured by EMG during NREM sleep was increased in MT mice injected with serum from RLS patients with or without hemodialysis Fig. . In mice21. Moreover, the amount of slow wave sleep for RLS patients was similar to that for healthy controls, but NREM sleep duration was also reduced21. Thus, our results were partially consistent with earlier studies in that we observed decreased amounts of REM sleep in the RLS model, yet saw similar basal amounts of NREM sleep for MT and WT mice. Meanwhile, basal EMG data for leg muscle showed that MT mice exhibited increased leg muscle activity during NREM sleep compared with WT mice. These results support published previously studies that reported a strong linkage between BTBD9 and increased PLMS22.To our knowledge, this is the first study to measure changes in sleep conditions and periodic limb movements in sleep (PLMS) in an animal model of RLS in a related genetic background after administration of serum from a patient with severe chronic kidney disease and to assess the effects of a dopamine-receptor agonist on PLMS in these animals. In baseline sleep recordings, the amounts of wakefulness and NREM sleep of MT and WT mice were similar, whereas the amount of REM sleep was decreased for MT mice relative to WT mice. Reportedly, RLS patients show a reduction in REM sleep duration and increase in REM sleep latency2/D3 receptor agonist, is frequently used to treat RLS and has a sleep disrupting effect in mice23. In rats, systemic injection of a D2 receptor agonist induces biphasic effects, wherein low and high doses reduce and increase wakefulness, respectively24. Here we found that a high-dose rotigotine treatment indeed increased the amount of wakefulness in both WT and MT mice injected with placebo and serum from healthy controls. However, this wake-promoting effect of rotigotine was not observed in mice injected with serum from RLS patients who did or did not undergo hemodialysis. Although the explanation for why a wake-promoting effect of rotigotine was induced only in mice injected with placebo and healthy control serum is unclear, our results suggest that serum from patients with idiopathic and renal RLS may contain substances that counteract the arousal effect of rotigotine. These substances may be a factor in the increased muscle activity of RLS patients, regardless of the presence of uremic substances. In addition, genotype also contributed to these differences in that WT mice tended to show more conspicuous effects of rotigotine compared to MT mice, suggesting that the presence or absence of Btbd9 gene mutations may also be involved in the wake-promoting effect of rotigotine. Such mutations could be associated with RLS pathophysiology, a possibility that requires further exploration.Pramipexole, a dopamine DBTBD9 polymorphisms22. Here we confirmed that the PLMS findings in the EMG of MT mice were similar to those of the putative rat RLS model14.We considered that an increase in muscle activity during sleep in MT mice corresponds with PLMS, a major RLS symptom. PLMS is also associated with other diseases, but PLMS with and without RLS was significantly related to In EMG taken during NREM sleep, we observed increased leg movement in MT mice injected with serum from RLS patients who either had or had not undergone hemodialysis. Rotigotine treatment ameliorated this effect, suggesting that the injected serum could contain substances that induced increased muscle activity and that the dopaminergic system mediates these effects. Future studies will be needed to detect and identify such substances, which could guide the development of novel therapeutics for RLS.BTBD9 and the dopaminergic system are linked to RLS, and partially clarify the pathophysiological mechanisms of RLS, which may inform new approaches for treating RLS.In conclusion, we found that administration of serum from RLS patients increased muscle activity during NREM sleep in MT mice with disrupted Btbd9, and this increase in muscle activity was suppressed by rotigotine treatment. We also observed that serum from RLS patients could counteract the arousal effect of rotigotine treatment in mice. Our data strongly support previously published reports showing that Btbd9 gene were obtained from the Mutant Mouse Resource & Research Center  and injected into blastocysts. These mice were identical to other mice previously described25. Chimeric mice were crossed to C57BL/6 mice. Heterozygous mice (Btbd9+/− mice) were further backcrossed to C57BL/6 mice for at least eight generations and the resulting heterozygous animals were crossed to produce homozygous mice. Btbd9 MT mice were then transferred from Gunma University to Tokushima University for experiments. PCR was used for identifying the wild-type heterozygous-type and homozygous-type alleles for genotyping. When we used BtbGenome primers 1 (Btb F: TTCCTGGTCCTCCTGCTACC) and 2 (Btb R: ACCCGTGGGAAAGCTTAGTG) for identifying the 900 bp wild-type allele. To confirm the presence of the 450 bp trap allele, we used Btb F and trap vector primers 3 (Trap R: CCACAACGGGTTCTTCTGTT). We used homozygous mice for all experiments.MT mice were prepared at Gunma University. Mice were kept under pathogen-free barrier conditions, and animal procedures were performed in accordance with institutional and national regulations following approval by The Gunma University Animal Care and Experimentation Committee. The commercial embryonic stem (ES) cell line  containing a gene trap vector in intron 6 of the The mRNA expression of Btbd9 was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was purified from mouse cerebrum using Trizol reagent , followed by DNase digestion and purification using the RNeasy kit . cDNA synthesis was performed on total RNA extracted from the mice prefrontal cortex using SuperScript™ VILO™ cDNA Synthesis Kit . Then PCR was performed with two primer sets, the forward primer located in the exon 4 (Exon5-6F – 5′- AGCTTCTGAACGTCGTGAGG-3′)and reverse primer in the exon 5 before the intron containing the gene-trap (Exon5-6R – 5′- TCATCATCAATGGGGTGCCG-3′), the others forward primer located in the junction of exon 6 before the intron containing the gene-trap and exon7 after this intron (Exon6-7F – 5′-GCCCGTGTCTGCAGGTATAT-3′) and the reverse primer located in the exon 10 (Exon10R – 5′-TTCGGATGAAGGATGCAGGC-3′).  was maintained throughout the study (lights on at zeitgeber time [ZT] = 0 at 08:00 am). Room temperature was maintained at 23 ± 1 °C throughout the experimental period. The study protocol received approval from the Gunma University Gene Recombination Experiment Safety Committee and the Animal Experimentation Committee of Tokushima University. All experiments involving mice were performed in accordance with Guidelines for the Care and Use of Animals approved by the Council of the Physiological Society of Japan.Seven-month-old male MT mice and their wild-type littermates were used in this study. Food and water were available Mice were anesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (25 mg/kg) for EEG/EMG implantation surgery. Stainless steel miniature screw electrodes were implanted in the skull of the mice to record EEGs, and Teflon-coated stainless steel wires were implanted in the gastrocnemius muscle of both legs to record EMGs. After a 2-week recovery period, the mice were transferred to plastic cages (20 × 24 × 30 cm) in a sound-proof recording room and allowed to habituate for two days. The mice were connected by flexible cables to a polygraph and computer-assisted data acquisition system. EEG/EMG signals were captured at 128/500 Hz using a sleep recording system . Mice were also monitored with a video recording system.Off-line sleep scoring was performed by visual assessment of EEG and EMG activities using the SleepSign analysis program . Vigilance states were classified for each 10-second period as wakefulness, REM sleep, or NREM sleep. The EEG and EMG power spectrum in the period that was scored as NREM sleep was calculated by Fast Fourier Transform using the SleepSign analysis program. The EEG delta and theta frequency bands were set at 0.5–4.0 Hz (SWA) and 4.5–10.5 Hz, respectively. The delta and theta power were normalized and described as a percentage of the total power (0.5–30 Hz), and the data were averaged at hourly intervals. The EMG total power (50–250 Hz) in each vigilance stage was integrated and normalized as a ratio to that of REM sleep. The EEG/EMG data in periods including artificial/electrical noise were excluded from analysis after checking the video recording and movement of the mice.To evaluate sleep homeostasis, mice were sleep-deprived (SD) for 6 hours, between ZT0 and ZT6, using a small, soft brush to touch the backs of mice that appeared to be somnolent. At ZT6, SD was terminated, and the EEG and EMG were recorded for an 18-hour period of uninterrupted recovery sleep.Serum from 5 healthy subjects and 5 patients with idiopathic RLS and 5 RLS patients with secondary to end-stage renal disease was filtered using a Minisart filter . After filtration, identical volumes of serum from each subject in the same group  were mixed and the mixtures were used for intraperitoneal (ip) injection into the mice. All 5 RLS patients with secondary to end-stage renal disease were received hemodialysis for 4 hours three times a week. The serum was obtained from the different patients before hemodialysis. The administration of dopamine agonist was withdrawn for seven days. This study was conducted in accordance with the Declaration of Helsinki. Informed consents for participating this study had obtained from all participants. Research protocol and informed consent were approved by Medical Research Ethics Committee of Tokyo Medical University. All experiments were performed in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan.Four hours after ip injection with human serum, rotigotine was subcutaneously (sc) administered to the mice. Human serum  or placebo  was administered to each animal at ZT0. Rotigotine  was administered to each animal at ZT4 (Supplementary Table Results are expressed as the mean ± SEM. The data in Figs Supplementary information