We show that these cells can cycle stably at a rate of 0.5 C  with 600 mAh g−1 reversible capacity and nearly 100% Coulombic efficiency. By means of spectroscopic and electrochemical analysis, we find that the particles form a sodium-ion conductive film on the anode, which stabilizes deposition of sodium. We also find that sulfur remains interred in the carbon pores and undergo solid-state electrochemical reactions with sodium ions.High-energy rechargeable batteries based on earth-abundant materials are important for mobile and stationary storage technologies. Rechargeable sodium–sulfur batteries able to operate stably at room temperature are among the most sought-after platforms because such cells take advantage of a two-electron-redox process to achieve high storage capacity from inexpensive electrode materials. Here we report a room-temperature sodium–sulfur battery that uses a microporous carbon–sulfur composite cathode, and a liquid carbonate electrolyte containing the ionic liquid 1-methyl-3-propylimidazolium-chlorate tethered to SiO Rechargeable sodium-sulfur batteries able to operate stably at room temperature are sought-after platforms as they can achieve high storage capacity from inexpensive electrode materials. Here, the authors use rationally selected cathode and electrolyte materials to design a room temperature Na-S battery. To maintain stable cell performance, the substrate must also be able to prevent loss of the intermediate sodium polysulfides (NaPS) speciesdp<1.8 nm) and high surface areas (SA≥843 m2 g−1) have been reported previously1924252627+ to form solid sulfide product species in the cathode—that is, without forming soluble LiPS25et al.192–4) species in microporous carbon substrates that on reduction with Li+ cannot form soluble high-order LiPS. Although this argument is a reasonable interpretation of the electrochemistry data, support from thermodynamic analysis of the electrode has been lacking so far. On the anode side, fundamentally based strategies for preventing dendrite formation in lithium metal batteries should necessarily be applicable for the sodium anode. Among the most fundamental of these approaches, are the efforts reported by Schaefer et al.29et al.3132333520To construct a room-temperature rechargeable Na–S battery, a conductive cathode substrate able to overcome the electronically insulating nature of both the fully charged and discharged products  additives as an agent for stabilizing electrodeposition. Na–S cells with this design are shown to achieve excellent cycling performance with nearly 100 % Coulombic efficiency at higher current density and with relatively high sulfur loadings in the cathode. Reversible storage capacities of over 860 mAh g−1 at 0.1 C  and 600 mAh g−1 at 0.5 C based on active sulfur mass are reported. Even at the higher current density (0.5 C) the batteries are able to cycle stably for over 100 cycles with 0.31% capacity decay per cycle. The fundamental origins of the superior performance of the constructed Na–S cells are studied using spectroscopic tools and analysis to understand the electrochemistry at the cathode on sodiation and desodiation processes. Notably, we find that no soluble NaPS species are formed and that the diffusivity of Na+ into the composite cathode is consistent with expectations for solid-state transport. Altogether, these results indicate that the Na–S cells follow a different electrochemical reaction mechanism compared to traditional metal–sulfur batteries, which likely contributes to the stability and high capacity retention on cycling.We herein report a stable room-temperature rechargeable Na–S battery  that ove2–IL–ClO4 particle additives in the electrolyte play a significant role in ensuring stable cell performance during the recharge cycle. To understand the role played by the particle additive, an electrochemical floating test was performed in the potential range from 3.0 to 5.0 V. As shown in 2–IL–ClO4 particle additives exhibit much lower leakage current and are stable at least up to 4.5 V. We believe that these effects stem from the same source—immobilization of a fraction of anions near the anode as a supporting electrolyte during cell recharge—as reported in lithium electrodeposition studies, where similar SiO2–IL–TFSI (bis(trifluoromethane)sulfonimide) particles were shown to provide orders of magnitude enhancements in the stability of lithium deposition in a PC-1M LiTFSI electrolyte314 electrolytes containing different concentrations of SiO2–IL–ClO4 particles. The measurements were performed using coin cells with a Na metal electrode. It can be seen that the particles stabilize the ionic conductivities of the electrolytes, particularly at intermediate temperatures. Cells with 10 vol% particles in the electrolyte exhibit stable bulk ion transport until just below the melting point of Na metal (97.72 °C). In contrast, control cells with no particles present in the electrolyte exhibit irregular changes in conductivity with temperature, suggesting less stability. A scanning electron micrograph is provided in 2–IL–ClO4 particles were present in the electrolyte. It is obvious from the figure that the particles form a dense monolayer on the Na-metal surface, which we hypothesize is the fundamental source of the enhanced electrochemical and thermal stability. On the basis of these observations, we propose that SiO2–IL–ClO4 additive could play two roles in stabilizing the cell: (i) the tethered ionic liquid forms a mechanically robust and chemically stable SEI layer on the surface of sodium metal, which limits contact and parasitic thermal and electrochemical side reactions with the electrolyte4− anions, which function as supporting electrolyte and reduce the electric field through the tethered anion effect discussed previously35The SiOIn metal–sulfur batteries, cathode design is now understood to play an important role in improving cycling performance. Due to the highly reactive nature of sodium, reaction between sodium and dissolved NaPS species is anticipated to be more vigorous, compared to lithium and LiPS. Cathode configurations that allow stronger anchoring of PS species are therefore required for Na–S cells192 g−1 calculated from the N2 adsorption–desorption isotherm (2 g−1) micropore surface area and pore size distribution ranging from 0.6 to 1.8 nm. To create cathodes, different amounts of sulfur were infused into the MCP and the resultant composites denoted as MCPS1 and MCPS2 with 47% and 65% sulfur loading, respectively  discharge plateau associated with formation of soluble polysulfide species in Li–S cells. This suggests that S8 can probably show different electrochemistry when under confinement in porous carbon.The state of sulfur inside micropores can play a significant role in the electrochemical stability of the cathode, but remains unclear species . Combini−1 at a current density of 0.1 C . The dimple and the lower-voltage plateau at the beginning of discharge, compared with the following cycles, indicates that Na+ ions need to go through a barrier, which is probably related to desolvation or solvation shell distortion462S/Na2S2, without creation of intermediate soluble NaPS species. A reversible discharge capacity of 800 mAh g−1 is stably achieved for 50 cycles  because of the phase limitation2S, and sulfur intensities concentrated inside the micropores and suggesting the solid-state reaction 432S were observed in the whole process for the cell utilizing EC/PC carbonate electrolyte and in the first discharge process for the cell based on the TEGDME electrolyte. In contrast, high-order BzSx are clearly observed in recharged cathodes in TEGDME. Ultraviolet–visible (ultraviolet–vis) spectra of a dilute Na2S6 solution, battery cathodes after 10 cycles in TEGDME, and carbonate electrolyte soaked in TEGDME are shown in 22− or S2− formed, while the high-order soluble PS are formed when the cells are cycled in TEGDMETo investigate the discharge reaction mechanism, X-ray photoelectron spectroscopy (XPS) was applied on the cathode side after galvanostatic cycling at different stages of discharge/charge to study the species formed at each stage . The prireaction . To furt+ into the cathode and the electrochemical reaction with sulfur in the cathode progress as solid-state processes. To verify this hypothesis, we first extract an approximate value for the Na+ diffusivity in the cathode based on the electrochemical data. Measurements utilizing a galvanostatic intermittent titration technique (GITT) was performed by discharging the cell for 30 min at 0.1 C followed by a 10-h relaxation . The reason why the solid-state reaction occurs only in microporous carbons with small pore sizes, but not in other carbon materials is open to argument and will likely be a subject for many future studies. On the basis of the empirical evidence in the present study, we attribute the difference to the stronger interactions between sulfur/sulfur species in the carbon relative to the strength of sulfur/sulfur species solvation due to their poor solubility in carbonate electrolytesOn the basis of the spectroscopic study above, one may more firmly hypothesize that the electrochemistry in the cathode reaction occurs completely inside the MCPS, which means that both the transport of Nalaxation . The diflaxation  at diffe4 electrolyte. The combination of cathode substrate and electrolyte are shown to provide sufficiently strong association of sulfur in the cathode to confine the electrochemical reactions in the cathode to an all-solid-state process in which Na2S appears to be the only product. We use both spectroscopic and analytical tools to show that for the carbons used in the present work, sulfur remains as S8 and that the cathode reaction occurs inside the microporous carbon composite. An additional problem associated with instability during the recharge process of Na–S cells operated at moderate and high current density was identified and resolved using SiO2–IL–ClO4 particles as additives in the electrolytes. Electron microscopy and electrochemical analysis indicate that the particles form a dense protective coating on the Na anode and stabilize deposition of sodium by at least two mechanisms. First, they form a particle-rich, mechanically strong SEI layer that protects sodium metal from parasitic side reactions with the liquid carbonate electrolyte. Second, they appear to utilize a previously reported tethered anion effect to stabilize deposition of Na. Our finding underscores the benefits of micorporous carbon–sulfur composite and nanoparticles for guiding new material designs for inexpensive rechargeable metal–sulfur batteries. Further investigations are needed to fully understand the interaction of microporous carbon and sulfur species as well as the specific role SiO2–IL–ClO4 plays on metal anode protection.We report an example of a room-temperature, rechargeable Na–S battery that can be cycled stably with high Coulombic efficiency at low and moderate current densities. The battery utilizes a micoporous carbon/sulfur composite in the cathode and an EC/PC-1M NaClO2–IL–ClO4 electrolyte were synthesized according to the previous methods with modifications27−1 was used for both heating and cooling. The final mass fraction of sulfur in the composites was determined by TGA . The synthesis of SiO2–IL–ClO4 was the same as well except the anion exchange step. In this work, NaClO4 was used to as anion exchange source.MCPS and SiOλ=488 nm). 1H NMR spectra were taken by Inova-400 Spectrometer. Ultraviolet–vis spectra were collected by Shimadzu UV–Vis–NIR Spectrometer. XPS measurements were performed with a Surface Science SSX-100 spectrometer using a monochromatic Al Kα source (1486.6 eV). Non-linear least squares curve fitting was applied to high-resolution spectra, using CasaXPS software.The morphology and elemental mappings of the materials were studied using a FEI Tecnai F20 Transmission Electron Microscope and A LEO 1550 high-resolution SEM. The nitrogen adsorption–desorption isotherms of the MCP and MCPS were obtained with a Brunauer–Emmett–Teller (Micromeritics ASAP2020). AccuTOF DART was used to get mass spectra for sulfur and MCPS composites. Raman spectra were collected using a Renishaw InVia Confocal Raman Microscope (4) in a mixture ethylene carbonate (EC) and diethyl carbonate  or in tetraethylene glycol dimethyl ether (TEGDME) or in a mixture of EC and propylene carbonate  with different amount of SiO2–IL–ClO4 were used as electrolyte for the cells. Cell assembly was carried out in an argon-filled glove-box (MBraun Labmaster) by using coin cell 2032 type. The room-temperature cycling characteristics of the cells were evaluated under galvanostatic conditions using Neware CT-3008 battery testers and electrochemical processes in the cells were studied by cyclic voltammetry using a CHI600D potentiostat. Electrochemical impedance and floating tests were conducted by using a Solartron Cell Test System model 1470E potentiostat/galvanostat. Ionic conductivities were measured using a Novocontrol N40 broadband dielectric spectrometer.The cathodes were prepared with MCPS1 or MCPS2, carbon black , and polymer binder (poly(vinylidene difluoride), PVDF, Aldrich) in a weight ratio of 8:1:1. A carbon-coated aluminium foil  was used as the current collector. The typical thickness of the active material film is ∼20 μm and sulfur loading is around 0.73 to 1 mg. Sodium foil (Alfa Aesar) was used as the counter and reference electrode. A glass fibre filtre paper (Watchman 934-AH) was used as separator. 80 μl 1 M sodium perchlorate .Supplementary Figures 1-10, Supplementary Table 1, Supplementary Note 1 and Supplementary References


Previous studies have not addressed rheumatoid arthritis (RA) patients’ help-seeking behaviours for RA flares, and only one small qualitative study has addressed how patients experience daily life on current treatment regimes. Thus, this study aims to identify clusters of opinion related to RA patients’ experiences of daily life on current treatments, and their help-seeking behaviours for RA flares.Using Q-methodology , two separate studies were conducted with the same sample of RA patients . Thirty participants sorted 39 statements about daily life (Q-study 1) and 29 participants separately sorted 23 statements about flare help-seeking (Q-study 2). Data were examined using Q-factor analysis.Daily life with RA (Q-study 1): Three factors relating to the experience of living with RA were extracted and explained. Patients belonging to Factor A  use effective self-management techniques to control the daily impact of RA. Those in Factor B  struggle to self-manage and cope. Whilst patients in Factor C  prioritise life responsibilities over their RA, reporting less impact.Flare help-seeking (Q-study 2): Two factors explaining the experience of flare help-seeking (unrelated to the factors from Q-study 1) were extracted and explained. Factor X (68.8% on biologics) reported seeking help quickly, believing the medical team is there to help. Factor Y (0% on biologics) delay help-seeking, concerned about wasting the rheumatologist’s time, believing they should manage alone. All participants agreed they sought help due to intense pain and persistent, unmanageable symptoms.Patients with different characteristics appear to manage RA life in different ways and men may struggle more than women. Whilst all patients are prompted to seek help by persistent, unmanageable symptoms, some delay help-seeking. Further research is needed to quantify the severity of daily symptoms, the level of symptoms needed for patients to define themselves as in flare and to understand the support needs of RA men.The online version of this article (doi:10.1186/1471-2474-15-364) contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease, characterised by daily fluctuation of symptoms  and unpredictable disease flares , 2. EarlThere is a scarcity of research that addresses how patients specifically manage the symptoms and consequences of their RA flares. One qualitative study  within aIt is not only when patients experience a flare that they need to learn to manage their condition. Daily life with RA has been identified as full of ‘uncertainty’ relating to patients being uncertain about their own interpretations of their symptoms and whether they would be able to receive adequate help to master their disease and manage their everyday lives . UncertaIt has been widely reported that living with RA impacts on hobbies, pastimes and with sexual activities –14. The A wide variety of self-management methods are recommended for people with RA and patients use technical aids, rest, exercise, heat packs, and joint protection on a daily basis . Thus, iHowever, these studies were all conducted prior to the use of current, more aggressive treatment techniques, and despite these advances in clinical management , 21, onlIt is likely that individual patients experience life with RA and help-seeking for RA flares in different ways. Thus, producing overall consensus on these experiences creates the potential problem of providing a ‘bland generalisation’ of the issues  and maskThus, the current study aimed to identify clusters of consensus  on patients’ experiences of daily life with RA; and to identify clusters of consensus on RA patients’ barriers and prompts for help-seeking for flares .Q-methodology involves participants sorting a set of statements into the order of their agreement across a normal distribution grid. Q-methodology evolved from factor analytic theory to provide a way to reveal the subjectivity involved in any situation . Q-methoParticipants are asked to rank-order statements of opinion (agree to disagree), which is known as ‘Q-sorting’. The statements are opinion only, not fact; Q-methodology assumes that opinions are subjective and can be shared, measured and compared , 30. TheTo produce statements regarding RA patients’ experiences of daily life and their flare help-seeking behaviours, data from previous interviews with an earlier sample of 15 RA patients  were exaOne grid for each Q-study was designed, one containing 39 spaces for the daily life statements to be sorted onto Figure a, the otPatients attending outpatient clinics at one of three NHS Trusts, in different socio-economic locations and with different methods for accessing care, were invited to participate. Patients with confirmed RA  for >2 yParticipants gave informed consent and ethics approval was granted . Thirty two of 72 patients (44%) agreed to participate but data were excluded from one who could not understand the task. In Q-study 1 (daily life), a second participant’s data were excluded as his wife completed the task on his behalf (n = 30). In Q-study 2 (flare help-seeking) this participant was included as he completed the task unaided, but two participants declined to complete Q-study 2  (n = 29).relative to the other statements. Each numbered statement was placed in a single box on the Q-sort grid  in participants’ homes or non-clinical outpatient rooms (participants’ choice). Each participant was asked to divide the statements into three piles . They were then asked to use these piles to help them arrange each numbered statement in approximate rank order of the degree to which they agreed with that statement Q-methodology combines qualitative and quantitative methods to produce a rounded interpretation of a single dataset (in contrast to a mixed-methods approach) . Q-methoData were analysed using the PCQ software package . In Q-meFor each factor, the open-ended  comments from the factor exemplars (significantly loading participants) are combined with the factor array  to provide one Gestalt explanation of each factor , 38. StaTable p < 0.01, indicating that each loading participant closely exemplifies the factor they load onto , they take responsibility for managing their disease :“It’s [taking medication exactly as prescribed] important and part of my routine” (P5)“You can’t just do this [what you like regardless of the consequences], people that do this make me angry. You can’t expect the doctors to help you if you don’t help yourself” (P4)“I don’t find they make any difference”: P2).Factor A exemplars know when to accept help from tools and devices or other people  and have learned which self-management techniques do not work for them  “Just a fact of life, they’re sorted lower as they’re not as interesting as the other statements” (P2)that’s a negative way of thinking” (P7).Thus they do not report low mood due to RA  and will not say that their body has let them down [D29: -2] as “Nine participants load significantly on this factor. Eight load negatively and one loads positively. Thus, this is a ‘bipolar’ factor, meaning that two opposed viewpoints are expressed, which each have a factor array that is the ‘mirror-image’ of the other, thus only the negative loading (Factor B-) will be presented. Negatively loading participants are predominantly male (71%), in comparison to the other two factors and overall study population 26%) “It [RA] gets me down every single day” (P15) [D13: -3]“don’t know what to avoid” (P12) nor try alternative medicines [D16: -3] because “they’re a waste of time and money” (P14). They feel unable to exercise [D20: -5], be spontaneous [D2: -3], or prioritise pleasurable activities over chores [D12: -2]. They struggle to do certain things [D3: +2] and find it takes longer to do things than they think it should [D6: +2], which they find frustrating [D27: +3]:Factor B- exemplars seem to struggle to find a way of managing their RA. They will not avoid certain foods [D18: -4] as they “Very frustrating for me as I used to do things quickly” (P11)“I always did things quickly, I didn’t ever sit around. I find the less I do, the less I want to do and I don’t want that. I find it very frustrating” (P14)The positive loading participant (P8) holds the polar opposite viewpoint to that presented above.Seven participants load significantly onto this factor; all female, younger, diagnosed for less time and report less disability in comparison to the other factors Table . Five haFactor C exemplars prioritise their responsibilities above their RA Table , D33: -4“I’ve lived with it for a long time now, I’m not going to allow it to ruin my life, it’s not fair on the children. I’ll do what I can for the children, even if I suffer for it” (P20)“Not going to happen [rest time]. I run my own business and have two small children” (P20)These patients experience very little impact of RA on their daily activities [D6: -2]. They are also able to forget about their RA [D30: +3], including when choosing food [D18: -3] or clothes [D18: -3]:“This works. I am able to forget about it a lot of the time. My Consultant said to me ‘This is you [draws circle] and this is your RA [draws much smaller circle]’. It’s a very small part of you, and I believe that” (P22)“I never think of my RA when I’m shopping for clothes, I just wear what I like” (P18)These patients do not get angry [D34: -2] nor allow RA to get them down [D13: +4]. They feel particularly lucky in comparison to other people [D24: +5]:“Definitely – most important [statement]. RA runs in the family, so I’ve seen relatives in wheelchairs and very unwell with it and you see other people in clinic. I look and feel so well with it.” (P17)“don’t hold others back” (P23) [D28: -4] and “don’t ask for help” (P19) from family and friends [D14: -3] nor use tools to aid them [D9: -5].They are self-sufficient; they “a constant worry” (P19) and therefore try to exercise [D20: +2].However, they are concerned about gaining weight and putting extra stress on their joints [D19: +3]; it’s p <0.01.Twenty nine patients 21 women) participated in the flare help-seeking Q-study (Table  women pabold in Table Of the statements scored similarly by both factors (highlighted “When I just don’t know what to do anymore” (P25)Sixteen participants significantly loaded onto this factor. They are predominantly female (11/16) Table  and takiFactor X exemplars will contact the medical team as soon as possible when in flare [F2: +1] and won’t wait for their next scheduled appointment [F17: -2]. They believe the medical team will be able to help them [F5: -3] and doubt their flare will go away without help [F6: -1]:“I’ll contact the next day and I get seen very quickly” (P11)“Minor aches and pains go away on their own, flare-ups don’t” (P8)“that’s what they’re there for” (P17).They don’t worry about wasting their own [F18: -2] or the rheumatology team’s time [F03: -3]: Tipping points for help-seeking specific to Factor X exemplars are worries about long term joint damage [F15: +1], believing they need a change in medication [F19: +2] and flare beginning to affect their quality of life [F8: +4].Six participants significantly loaded onto this factor. They are predominantly female (4/6) Table  and noneFactor Y exemplars appear both cautious and indecisive in seeking help and will not contact the medical team urgently when they are in flare [F2: -3]. They are reluctant to seek help and hope the flare will go away on its own . They worry more than Factor X exemplars about wasting the rheumatology team’s time [F3: 0], which appears to be due to beliefs they should manage alone. A statement scored neutrally can indicate cautious agreement, and these participants’ comments support this:“I was brought up to be self sufficient and not run for help, I know that I should know better, but it’s ingrained” (P27)“I do worry about this [wasting the rheumatology team’s time], even though I shouldn’t. I went to the Doctor and had to keep going back for help and it wasn’t getting any better – but it wasn’t getting any worse. So I thought, they know how bad it is, they don’t need to see me unless it gets worse, so I left it” (P9)These patients will try and manage their symptoms until the flare stops [F14: +1], and will wait until they are prompted by a loved one to seek help [F11: +2].This study indicates that in daily life some RA patients are able to keep a balance through effective self-management strategies, some (predominantly men) struggle to cope and others (predominantly younger women) put their life and/or work responsibilities before their RA self-management. Patients will seek help for flares due to pain intensity, flare longevity and uncontrollable symptoms. However, whilst some patients will quickly define their symptoms as a flare and make a ‘definite decision’ to seek help, others are indecisive as to whether their symptoms constitute a flare and are cautious of seeking help due to beliefs they should manage alone.This study suggests disease severity, importance and self-management affect the daily personal impact of RA and these can mediate each other. These may be the first data to support the proposal of such an ‘impact triad’ . FurtherThe flare help-seeking Q-study consensus supports previous research , 7 that The majority of patients who reported rapid help-seeking were taking biologic therapies, whereas those who reported waiting were not. It is possible that patients on biologics have a different experience of care due to regular contact with the team for repeated medication reviews. Patients not prescribed biologics may consider their symptoms invalid, due to not being offered this treatment, which they worry about failing to ‘qualify’ for .This study asked patients’ perceptions of whether they were in flare at baseline rather than using an objective measure, therefore it is unknown whether patients were in an inflammatory flare or not. Patients were not asked to provide their definition of flare, but this was a pragmatic study, and the current lack of consensus of flare definition may already pose a clinical problem , 44. A lClinicians should also be aware that there are at least four ways in which patients experience life with RA: ‘Feeling Good’, ‘Taking Active Control’, ‘Keeping RA in its Place’ and ‘Constant Struggle’ and these patients would require different levels and types of support. Some patients manage well due to low symptoms or expert self-management and therefore appear to need little intervention from the medical team. One group of patients of particular note are male RA patients, who appear to have a more negative experience of RA than female patients. These male patients focus on their symptoms, experience negative thoughts and feelings and do not seem able to identify effective self-management techniques. This has an important clinical implication for the way in which men with RA are supported. These male patients may benefit from a tailored intervention, but their specific support needs require further research before an effective intervention can be designed.The findings from this study also indicate that some patients would benefit from patient education programmes to ensure they are aware of when and how to seek help for their RA flares. Cognitive Behavioural Therapies (CBT)  are growDaily life with RA has less impact on some patients than others, which can be due to less disease severity, expert self-management or attaching less importance to their RA. However, RA has a larger impact on other patients, the majority of whom seem to be male, due to concentrating on physical symptoms and rejecting self-management techniques.In an RA flare some patients will seek help quickly, whilst others will wait due to beliefs that they should manage alone. Patients will seek help when their pain is too intense, their flare has gone on longer than expected and they are no longer able to control their flare. Patients may therefore see medical help-seeking as a last resort, when they are no longer able to cope alone. Further research is needed firstly to quantify the nature and level of symptoms still experienced in daily life while on current treatments; and secondly in understanding men’s experiences of RA and their support needs.


This shift in thinking offers a revolutionary approach to PD treatment, potentially enabling a transition from purely symptomatic therapy to direct targeting of the pathology that drives disease progression. In this short review, we appraise current experimental support for PD as a prion-like disease, whilst highlighting areas of controversy or inconsistency which must be resolved. We also offer a brief discussion of the therapeutic implications of these discoveries.Parkinson's disease (PD) is the world's second most common neurodegenerative disease and most common movement disorder. Characterised by a loss of dopaminergic neurons and the development of intraneuronal inclusions known as Lewy bodies, it has classically been thought of as a cell-autonomous disease. However, in 2008, two groups reported the startling observation of Lewy bodies within embryonic neuronal grafts transplanted into PD patients little more than a decade previously, suggesting that PD pathology can be propagated to neighbouring cells and calling basic assumptions of our understanding of the disease into question. Subsequent research has largely served to confirm this interpretation, pointing towards a prion-like intercellular transfer of misfolded  In the The traditional understanding of PD as a movement disorder centres on dopaminergic neuronal loss in the substantia nigra pars compacta (SNPC) , as typiαsyn, a presynaptic protein suggested to regulate neurotransmission, intensely labelled Lewy bodies [αsyn gene cause an autosomal dominant form of PD [αsyn in PD pathogenesis. The αsyn present in Lewy pathology exhibits a conformational change from the native soluble protein to an insoluble, fibrillar form. These fibrils are rich in β-pleated sheets, shown clearly by intense staining with thioflavin S. Over 90% of this αsyn, compared to 5% normally, is phosphorylated at serine-129 (pser-129), which promotes fibril formation [αsyn shows ubiquitin immunoreactivity [αsyn, most notably the autophagy-lysosomal pathway [Intraneuronal inclusions, known as Lewy bodies or Lewy neurites, are also present and are considered the hallmark of microscopic PD pathology. The main component of these abnormal aggregates was an enigma until it was discovered that monoclonal antibodies against y bodies . Both murm of PD , stronglactivity , indicatactivity , which m pathway .The science of infectious disease was revolutionised in 1982 when Prusiner postulated that proteinaceous infectious particles (prions) devoid of nucleic acids cause the disease scrapie in animals . This on a priori be a true prion disease according to this definition [Currently, there is no evidence of interorganismal infectivity in PD; therefore it can notfinition . DespiteDopamine replacement therapy, the mainstay of current treatment for PD, can demonstrate limited efficacy and may be further complicated by drug-induced dyskinesias. Furthermore, PD leads to many nonmotor symptoms, such as autonomic dysfunction and cognitive and mood disturbances, all of which respond very poorly to dopamine replacement. These problems emphasise the need to target the cause of the progressive neuronal loss . Consequαsyn-positive Lewy-like inclusions were observed within the mesencephalic grafts of 4 patients [αsyn-positive dopaminergic neurons in a 16-year-old graft compared to a 12-year-old graft from the same person [In a series of open-label trials in the 1990s , fetal mpatients , a complIn light of these observations, a hypothesis that PD was a transmissible disease with prion-like features was proposed . Howeverαsyn knockout tissues and animals, endogenous αsyn has been confirmed as similarly necessary for the development of αsyn pathology both in vitro [ in vivo [αsyn and PrP are structurally similar, being rich in β-pleated sheets [An important characteristic of prion disease is that a pool of endogenous, natively folded PrP (PrPc) is required as a substrate for conversion by, and to, the pathological, misfolded form (PrPsc) . Via thein vitro  and in v in vivo . Furtherd sheets , prone tαsyn seeding in Lewy body formation, which has best been demonstrated in vitro [There are two mainstream theories regarding the mechanism of conversion of PrP (PrPc) to the misfolded “scrapie” form (PrPsc): the refolding hypothesis and the currently more favoured seeding hypothesis  Figure . There iin vitro .αsyn preformed fibrils (PFFs) to cells overexpressing αsyn, leading to the formation of Lewy-body-like inclusions. Anti-myc antibodies only labeled the centres of inclusions; endogenous αsyn had been recruited and converted to form the periphery [αsyn were cocultured, generating inclusions with a central core of green-labeled αsyn surrounded by endogenous red-labeled αsyn [αsyn amyloid fibrils; a single exposure to these was sufficient to induce aggregation of endogenous αsyn in human neuroblastoma SH-SY5Y cells [One study added myc-tagged recombinant eriphery . Whilst led αsyn . A more 5Y cells .These similarities between prionopathies and PD on a molecular and cellular level would seem to create a stable foundation for the prion-like hypothesis.αsyn. There are numerous mechanisms by which this could occur, from cellular release and uptake of αsyn to direct transmission via nanotubes [The spread of Lewy pathology in graft patients is likely to be due to intercellular transfer of anotubes .αsyn is present in the cerebrospinal fluid of individuals with or without PD [αsyn release in vitro, shown to be independent of cell death or membrane leakage. This secretion is reduced by low temperature, which also slows vesicular exocytosis, but is unaffected by Brefeldin A, which inhibits conventional exocytosis. These data suggest that αsyn is secreted via an unconventional vesicular pathway [αsyn can be found within large dense-core vesicles [αsyn is more likely to be internalised than free αsyn [αsyn secretory pathway.Despite a lack of secretory signal peptide sequence, thout PD . There i pathway , althougvesicles . Alternaree αsyn . Thus, iαsyn internalization [ in vitro and in vivo has been demonstrated by Desplats and colleagues [αsyn under the control of the brain-specific Thy-1 promoter. Four weeks later, 15% of grafted cells displayed human-αsyn immunoreactivity. Some cells developed αsyn-positive inclusions although, unexpectedly, fibrillar αsyn was not detected. This pathology was not observed in grafts inserted into nontransgenic mice: αsyn is therefore essential for host-to-graft transmission. However, the hippocampus is not a site relevant to grafts in clinical PD treatment, and MCNSCs are proliferative, unlike most neurons. Accordingly, Hansen and colleagues transplanted wild-type mouse embryonic mesencephalic neurons into the striatum of transgenic mice overexpressing human-αsyn under the control of the native mouse αsyn promoter. Six months later, 5% of transplanted neurons were human-αsyn-positive [The need for lipofection agents in many experiments has highlighted the difficulty in demonstrating meaningful lization , althouglleagues . Fluorespositive .αsyn uptake via the use of inhibitors [αsyn but that monomeric αsyn can be directly translocated across the plasma membrane, suggesting that at least two internalisation pathways may exist which are αsyn state-dependent [αsyn immunoreactivity (15% in 4 weeks versus 5% in 6 months). This is likely due to differences in experimental design, which include different promoters driving αsyn expression, the types of transplanted cell utilized, and differing brain areas studied. Furthermore, Hansen and colleagues did not observe seeding after uptake in vivo, and a similar study in rats also reported little aggregation [αsyn secretion and uptake.Both Desplats and Hansen give evidence for an endocytic mechanism of hibitors , 35. Othependent . Howeverregation . Despiteαsyn has been found within TNTs between glioblastoma cells [αsyn transmission in PD.TNTs are a recently discovered form of direct cell-to-cell communication. They are F-actin containing tubes with diameters of less than 200 nm that connect the cytoplasm of two cells. Intercellular transfer of PrPsc  has beenma cells . This prαsyn-positive subjects, it suggests that, in PD, Lewy pathology affects regions in the brain in a stereotypic, topographical manner. Lewy pathology severity is also suggested to correlate with the clinical progression of PD symptoms [αsyn aggregation form a positive feedback loop [αsyn, which have been shown to be capable of replication by self-propagation in vitro [The Braak hypothesis is perhaps the currently best-accepted model of PD progression and would be highly consistent with a prion-like mechanism of spread. Derived from the postmortem analyses of 100 symptoms . Braak sack loop , coupledack loop . Dopaminin vitro .αsyn pathology begins, due to environmental insult, within enteric epithelium. From here, it travels retrogradely via the axons of enteric nervous system (ENS) neurons to their somata within the intermediolateral column of the spinal cord and then to the dorsal motor nucleus of the vagus (DMNV) in the medulla. Thereafter, it is able to reach the pons and substantia nigra and, beyond that, vulnerable regions of the cortex [αsyn pathology [αsyn spread, as well as for interneuronal spread more generally. These include several studies that find distributions of αsyn pathology in PD that would not be predicted by the stereotyped Braak pattern of disease spread and the observation that αsyn pathology is also the predominant histopathological characteristic of other diseases, most notably multiple system atrophy (MSA), which feature a completely different pattern of CNS involvement and spread than PD [The Braak hypothesis proposes that e cortex  , with negligible cell loss. However, by 100 dpi, the total number of cells and striatal dopamine concentration had decreased, suggesting that αsyn Lewy pathology is cytotoxic [ In vitro, αsyn has been shown to lead to caspase-3 activation [αsyn overexpression in the right substantia nigra of rats, along with bilateral striatal transplant of embryonic dopaminergic neurons. This led to αsyn immunoreactivity in the right striatal transplant but not the left [αsyn-containing material elicited different and connectivity-dependent patterns of αsyn spread, although it must be noted that nonphysiologically high concentrations of αsyn were used to elicit widespread transfer in these experiments [In prionopathies, the protein itself is toxic and leads to rapid neurodegeneration . The priytotoxic . This cetivation . Cell-cethe left . Furthereriments .αsyn, but controversies remain; in particular, one of the most prominent models for PD propagation, the Braak hypothesis, which would be entirely consistent with prion-like spread, has not been clearly proved. To properly address this question experimentally, a technique to longitudinally monitor αsyn spread will need to be developed, which will also allow prion-like transfer to be examined over time.Overall, the evidence provides substantial support for prion-like transfer of αsyn spread is very young; however, the therapeutic implications could be huge. Inhibition of αsyn transmission could offer a disease-modifying approach with the potential to prevent both motor and nonmotor decline in patients.The field of research into prion-like αsyn transmission is represented in  in vitro for PD or other protein misfolding diseases. One possibility is to increase the resistance of αsyn to seeding. In transthyretin amyloidosis, where transthyretin misfolds to cause disease, compounds have been developed to stabilise the protein as a functional tetramer [αsyn can exist as a folded tetramer that is resistant to aggregation [αsyn available for misfolding, which may cause only few side effects since the protein shows a high degree of cellular redundancy in mammals. One study reported that interfering RNA against αsyn conferred resistance to MPP+ exposure, which usually causes PD-like degeneration [A summary of possible treatment strategies targeting tetramer . As αsynregation , this stneration . Such stneration .αsyn transfer, progress is hampered by many of the limitations of current experimental animal models, which are an imperfect replication of human PD. Of course, such problems are far from unique to this field. While many improvements would be desirable to ensure greater applicability to human disease, perhaps the most significant shortcomings are the chronicity of the studies, which take place in fairly short-lived organisms, and the nonphysiological levels of αsyn protein which have been used to elicit spread, both of which are clear confounds when considering a process which takes place over many years and with native levels of αsyn in PD. With regard to the latter problem, it is noteworthy that while the majority of studies use cells or animals overexpressing αsyn at high levels, this is not always necessary for PD pathology to develop if neurons are exposed to PFFs [ In vitro models also have an important role to play in allowing high throughput approaches and a level of access that in vivo models often cannot support. To this end, the use of human induced pluripotent stem cells is an exciting development which promises all the advantages of the in vitro approach coupled with the unprecedented ability to study dynamic disease processes in real time in living human neurons differentiated along the lineages of relevant populations such as SNPC dopaminergic neurons [While many studies have provided much good evidence for prion-like  to PFFs . In vitr neurons .αsyn spread via a prion-like infectious process is central to PD. The therapeutic implications of this conclusion are powerful indeed and have the potential to revolutionise treatment of both motor and nonmotor manifestations of this devastating disease.In summary, the role of prion-like spread in PD progression remains an exciting area of research. Whilst there are still unanswered questions, many studies corroborate the idea that


We selected Atlantic cod as a model animal due to the affluent amount of literature on this species, but also undertook additional dedicated experimental works. We found that specific gravity of yolk plus embryo is central in influencing ρegg and thereby the buoyancy. However, our established framework documents the effect on ρegg of the initial deposition of the heavy chorion material in the gonad prior to spawning. Thereafter, we describe the temporal changes in ρegg during incubation: Generally, the eggs showed a slight rise in ρegg from fertilization to mid-gastrulation followed by a gradual decrease until full development of main embryonic organs just before hatching. Ontogenetic changes in ρegg were significantly associated with volume and mass changes of yolk plus embryo. The initial ρegg at fertilization appeared significantly influenced by the chorion volume fraction which is determined by the combination of the final chorion volume of the oocyte and of the degree of swelling (hydrolyzation) prior to spawning. The outlined principles and algorithms are universal in nature and should therefore be applicable to fish eggs in general.It is generally accepted that the high buoyancy of pelagic marine eggs is due to substantial influx of water across the cell membrane just before ovulation. Here we further develop the theoretical basis by applying laboratory observations of the various components of the fertilized egg in first-principle equations for egg specific gravity (ρ Gadus morhua) as an object of study.Buoyancy of fish eggs has been studied in a wealth of articles over the last decades et al. The introduction of the density gradient column by Coombs egg model, first-principle equations should be applied and these algorithms should reflect the fact that a fertilized fish egg typically consists of three parts: chorion, perivitelline space (PVS) and yolk plus embryo (Appendix: Eq. 1). Thus, the total egg mass is the sum of masses of these components, which can be expressed by their separate volumes (V’s) and specific gravities (ρ’s):In the establishment of a true ρcho) may differ both among cho) is roughly constant, even between different species, but appears to be exceedingly high: e.g. 1.20 g cm−3 for Atlantic cod −3 for Atlantic halibut (Hippoglossus hippoglossus) water–ρegg) in pelagic fish eggs is less than 0.003 g cm−3water ranges from about 1.010 (brackish) to 1.028 g cm−3 (marine). As the chorion is permeable to the ambient seawater it is assumed that the specific gravity of the perivitelline space (ρpvs) is identical to ρwater. Evidently, ρyolk+emb varies intraspecifically; the reported values are 1.017 g cm−3 and 1.008 g cm−3, respectively, for marine and brackish cod eggs −3yolk+emb counteracts the high ρcho to create the necessary hydrostatic lift of the egg. Both egg diameter and thereby egg volume (Vegg) and chorion thickness and chorion volume (Vcho) are considered constant throughout embryonic development, i.e., after the aforementioned initial short period of swelling and eggshell hardening that settles the volume of the egg. Therefore, also the sum of the volumes of the perivitelline space and the yolk plus embryo must be constant, i.e. Vpvs+Vyolk+emb = constant, and hence changes in Vyolk+emb during development must result in compensating changes in Vpvs according to Eq. 3 (Appendix). Consequently, variability in ρegg during incubation, i.e. from fertilization to hatching, must be caused by changes in ρyolk+emb. In regards to egg metabolism and physiological development, these changes in ρyolk+emb can be caused by two factors: (1) changes in small molecules including ions  and (2) changes in macromolecules  Here the contribution of the chorion requires some special attention. This structure consists of proteins de facto determines ρegg, both at fertilization and during incubation, by including associated measurements of a series of relevant variables (cf. Appendix). These formulations show that Vyolk+emb, ρyolk+emb and Vcho play important roles in defining ρegg. We hypothesize that (1) initial variation in ρegg among individual newly spawned eggs from a single female, is due to differences in Vcho, and that (2) trends in ρegg during embryonic development are caused by changes in ρyolk+emb and Vyolk+emb. The value of ρyolk+emb (Appendix: Eq. 4) was achieved by consulting the new measurements. Hence, the present series of theoretical and practical approaches confirmed the role of ρyolk+emb in defining ρegg.During the work we encountered two hurdles: i) lack of sufficiently detailed algorithms of basic relationships and ii) missing or incomplete observations throughout incubation on key input variables for these equations. Hence we develop both the theoretical side (cf. Appendix) and a data collation programme undertaken under controlled conditions in the laboratory to provide values for the equations. The experimental study was performed to explore what http://conventions.coe.int/treaty/en/treaties/html/123.htm). Key experimenters were properly certified in laboratory animal science (FELASA category C researchers) (http://www.felasa.eu/accreditation-boards/accreditation-board-for-education-and-training1/). Permissions for collecting eggs were granted by the commercial hatchery of the Marine Harvest Cod (MHC) and the governmental hatchery at Austevoll Research Station, the Institute of Marine Research, Norway. Both hatcheries were properly approved and registered by Norwegian authorities  and followed strict veterinary and husbandry guidelines. Our study was restricted to the use of fertilized eggs only. An initial document search clarified that such early life stages are not classified as ‘animals’ . Hence, in the present case no specific permission was needed from the Norwegian Animal Research Authority.All experiments followed the ‘European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purpose’ (ETS123: th Jan.–16th Feb. 2009) and spring season (6th–31st May 2009) and by the hatchery at the Austevoll Research Station, Institute of Marine Research (IMR), for one fall season (7th–31st Oct. 2009). Female and male cod were stocked together in single spawning tanks  and induced to spawn naturally by artificial day light rhythms. MHC broodstock cod were originally captured near the entrance of Sognefjord (50 km north-east from Bergen), and female cod age and body weight were on average 9 years and 8–15 kg for the winter experiment and 12–13 years and 15–25 kg for the spring experiment. The cod at IMR Austevoll were born and raised in captivity from wild specimens captured in Øygarden (20 km west from Bergen), and were 5 years in the present fall experiment. Data on the female body weight was not available. Sea water of spawning tanks in the two hatcheries was maintained at temperatures of 6–7°C and salinities of 34–35.Experiments were performed on eggs provided by the commercial hatchery Marine Harvest Cod (MHC) for one winter  was determined using a density-gradient column  set up in the refrigerated room (6°C). All preparation of density gradient, calibration, accuracy, stability and operation have in general been described by Coombs et al. −3 at the surface of the column to 1.0325 g cm−3 on the bottom. At each experimental trial, five glass floats of known and calibrated densities (ρfloat) were first introduced into the column from the top. The densities of the floats were originally reported at 23°C (or 18°C) with certified instruments in the Martin Instrument Co. . A density correction at 7°C was obtained according to the equation based on thermal expansion: ρfloat at 7°C = ρfloat at 23°C+(23–7)×ρfloat at 23°C×0.000028. After 1 h the glass floats had positioned at their levels of neutral buoyancy in the column and could be used as reference levels of specific gravity. Prepared eggs were gently inserted from the top of the column and located at their neutral buoyancies. The vertical centre position of the glass floats were read with a precision of 1 mm and formed the basis of linear regressions between column position and specific gravity . Due to the large number of eggs in the column and because the density gradient was set up to observe very small differences in egg specific gravity, we found it sufficiently accurate to count the eggs at every 10 mm vertical intervals. The eggs were first counted 30 min after the introduction into the column. The egg specific gravity, ρegg, was derived using the regression equation .Egg specific gravity  The purpose of the former measurements was to track daily changes in specific gravity of the same eggs. About 50 eggs at early developmental stages of 2–3 dpf were submerged in a single column and continuously monitored for two weeks until hatching. ρegg was noted on a daily basis. Moribund and dead eggs in the column were counted every day for calculations of egg mortality rate and hatching rate from the initial number of eggs on the first day of the experiments. Moribund and dead eggs were apparent in that they were not transparent and with no sign of onward development from the previous day, or continuously sinking to the column bottom. Egg mortality rate was about 2–10% of the initial number of eggs. Hatching rate was about 2–8% at 12–13 dpf. (2) The latter measurements were aimed at studying co-variation of egg traits (see below) during development. About 100–300 eggs were introduced in another single column every second day. Sub-sets of these were recollected using a glass tube at assigned buoyancy layers (see below) for further scrutiny at different egg ages. Since the density gradient columns were newly made at each time point measurement occasion, the relatively large number of eggs did not affect the local oxygen conditions in the column. Due to the communal conditions of females and males in the same spawning tank, several egg batches were collected from different females on the same day. Generally, the individual differences in ρegg are considerably larger than the observed temporal changes of one single egg throughout incubation. Therefore, in measuring the temporal changes in ρegg we minimized the noise from the individual variations by selecting eggs at 2 dpf from a very narrow distribution (standard deviation: 0.0002 g cm−3) in the columns. Thus, egg specific gravity layers of different egg batches at 2–3 dpf were carefully selected based on the narrow distributions in the column  equipped on a stereomicroscope at 10×. For the measurements of egg wet weight, about 50 eggs were sub-sampled from the photographed eggs, rinsed carefully in distilled water for 60 sec to remove sea water ions from the perivitelline space cho).Three eggs representing each specific gravity layer were treated individually for this procedure. First, the diameter of an egg was measured under a fluorescent microscope  at 25×, and placed between two microscope slides on a soft cutting-off plate. The two glasses were placed side by side with a distance of an egg diameter, preventing the egg from moving during cutting process. The egg was sectioned into two halves under a stereomicroscope by a scalpel, and placed in a water-filled petri dish. Each half was observed under a fluorescent microscope at 400× during which the background light intensity was set at a grey level of 150–160. Chorion thickness (t) and egg diameter (2r) were measured in ImageJ and used for the calculation of chorion volume  the form of the embryo is starting to deviate from the ellipsoid shape. Hence, Vyolk+emb was not measured after ∼4 dpf. Perivitelline space volume (Vpvs) was calculated as: Vpvs = Vegg−Vcho−Vyolk+emb.With the captured egg images in ImageJ, individual yolk and embryo volume, V−4 g cm−3, while the certified specific gravity precisions of the calibrated floats are ±2×10−4 g cm−3 implying that recording of vertical level of eggs within 10-mm intervals was near the possible specified precision of the method.Statistical analyses were carried out using R version 2.10.0 and Statistica version 12 egg after the mid-gastrulation stage. Pearson correlation coefficient (r) was used to determine the strength of relationships between variables. In order to generate a predictive model of egg specific gravity we initially carried out a stepwise regression procedure with egg weight measures and different egg volume contributions  as independent variables to determine which of these variables significantly contributed to the determination of observed specific gravity. Only cases with no missing values were used. Subsequently a re-analysis was carried out with extra data left out at the initial analysis and a final multiple regression analysis was carried out with significant independent variables.Analysis of covariance (ANCOVA) was used to compare slopes of regressions between continuous and time point measurements for trends in ρegg) was observed among the three seasons (egg until mid-gastrulation (3–4 dpf). Thereafter, the specific gravity gradually decreased until full development of main organs (9–10 dpf). Subsequently, ρegg suddenly increased just prior to hatching (11–13 dpf). The egg total incubation period was 12 days for winter and spring and 13 days for fall at 7°C. The egg distribution in a column was relatively narrow at early stages, but widened at later stages. Within the single egg batches the standard deviations were not significantly different from those of egg volume, chorion volume, and yolk volume measured from a single batch. Also such variations tended to be a bit higher at later stages of 10 and 12 dpf, but it does not hamper understanding of the general trend during development.A similar pattern of temporal changes in egg specific gravity (ρ seasons –S3. In gegg was similar between the two types of measurements (continuous vs. time point) for winter and spring, but different for fall . The specific gravity of yolk plus embryo (ρyolk+emb) was estimated using Eq. 4 (Appendix) and appeared to be similar between 2 and 4 dpf. In terms of volume changes, Vpvs increased and Vyolk+emb decreased from 2 to 4 dpf while Vcho apparently was constant . Water content was positively correlated with age in two samples  and Batch4-b . Given the needed high precision of the measurements of dry weight and water content it is however, not surprising that trends are different for these measurements. Vyolk/Vegg (where Vyolk in this figure is the yolk excluding the embryo volume) consistently showed a decreasing trend  , Batch4-b , Batch5-a  and Batch7-a   . Vcho/Ve= 0.002) . Howeveryolk+emb and Vcho contributed significantly to the specific gravity of the eggs , while the Vpvs and wet- and dry weights did not. Based on the initial analysis, a re-analysis was carried out with cases that had been excluded due to missing values in non-significant independent variables. The residuals of the extra cases from the original relationship was not significantly different from 0 , and the re-analysis with all available data provided the following relationship based on the multiple regression: ρegg = 1.02697–0.01253×Vyolk+emb+0.000278×Vcho  (Only the V = 0.91) .egg), chorion thickness (t), egg diameter (2r), and yolk plus embryo volume (Vyolk+emb) into Eq. 4, we calculated ρyolk+emb at 2–3 dpf. The relationship between ρegg and ρyolk+emb was highly significant. The linear regression was ρegg = 1.6326×ρyolk+emb–0.6376 (r2 = 0.94)  .egg) for marine pelagic fish eggs based on first principles. It shows the relative contribution from the various components of the egg to the resulting egg specific gravity. Chronologically, the critical events in determining the changes in egg specific gravity are as follows: The first determinant starts in the ovary with the deposition of the heavy chorion material (Mcho) associated with each oocyte egg prior to spawning as addressed in the work by Craik & Harvey pvs/Vegg) as it manifests just after fertilization This study presents the first coherent explanation of the developmental and individual variability of egg specific gravity (ρegg is attributable to the volume decrease in yolk plus embryo and the corresponding volume increase in PVS (yolk+emb) without expansion of the chorion was also detected by Davenport et al. yolk+emb, presumably resulting from osmotic water loss, may cause eggs to become slightly heavier at mid-gastrulation.As the early notable feature of ontogenetic changes, eggs increase their specific gravity up to 0.03% of the initial value from fertilization until mid-gastrulation (∼4 dpf). The slight increase in ρe in PVS . The shregg until 10–11 dpf, but with a rapid increase at 12–13 dpf just before hatching. The rate of decrease in ρegg is 0.0002 g cm−3 day−1  eggs, wet weight, dry weight and water content were approximately constant until hatching cho) do not properly explain temporal variability in ρegg during development.After 4 dpf, eggs generally show a gradual decline in ρ−3 day−1 , implyinyolk+emb) and specific gravity (ρyolk+emb) can cause the gradual decline in ρegg, since egg diameter (2r), chorion mass (Mcho), and chorion specific gravity (ρcho) are approximately constant throughout development. Although a slight decrease in chorion thickness (t) prior to hatching was observed, resultant changes in Mcho are too small to significantly affect the ρegg. The change in ρyolk+emb during development can, in turn, have two causes: (1) volume and mass changes in the yolk-sac linked to the water balance. Riis-Vestergaard egg of about 0.00142 g cm−3, i.e. very similar to our observation of 0.0014 g cm−3 calculated by the reduction rate. (2) The other is caused by volume and mass changes in embryo structural composition such as lipids, proteins, muscles and cartilage, as the embryo develops. Finn et al. yolk+emb×Vyolk+emb = K, where K is constant. This means that a decrease in ρyolk+emb must be compensated by a corresponding increase in Vyolk+emb. Under these assumptions, we can calculate the change in Vpvs (Appendix 5–8) derived from:Only changes in yolk plus embryo volume (Vegg (0.0014 g cm−3) throughout incubation into the equation, the calculated changes in Vpvs are only from 13.3 to 13.4% of the Vegg for an average egg . This is in contrast to the much larger variation in Vpvs which varies from 17 to 22% of total egg volume during development egg and Vpvs throughout the first part of the incubation period onto 11 dpf are caused significantly by the changes in yolk osmolarity rather than the changes in embryo composition of proteins, bones, and muscles. Accordingly, the increase in osmolarity of yolk plus embryo after spawning results in loss of water and, consequently, reduced Vyolk+emb, increased ρyolk+emb, and subsequently increased Vpvs from fertilization to 4 dpf. Thereafter, the ρyolk+emb decreases since the decreasing yolk and the increasing embryo begin to actively take up water resulting in the increase in Vyolk+emb and the corresponding decrease in Vpvs.By inserting the observed reduction of ρegg is consistent with egg osmoregulatory work egg have been reported for other species where continuous measurements of egg specific gravity have been conducted. These include anchovy (Engraulis encrasicolus) Merluccius capensis) Micromesistius poutassou) Our explanation for developmental variability in ρegg are also associated with catabolism of various energy substrates , and lipids). For Atlantic cod eggs, FAA, which is markedly denser than ambient seawater, contributes 75% to aerobic catabolism Moreover, developmental changes in ρcho) at early egg stages consists from 2 (thickness: 3.8 µm) to 3.6% (thickness: 7.8 µm) of total egg volume (Vegg) and significantly associates with ρegg ranging from 1.017 to 1.026 g cm−3. Similar positive relationship was observed for Baltic cod eggs in brackish water egg ranging from 1.010 to 1.014 g cm−3. Kjesbu et al.cho) would have different egg specific gravity depending on egg diameter, because the ratio Vcho/Vegg would change. Hence, a larger egg would have lower specific gravity than a smaller egg. Our results confirm the significant contribution of chorion volume to determining the initial egg specific gravity.Of egg structural materials, carbohydrates and proteins are denser than sea water and provide downwardly directed forces on the egg. In contrast, lipid and pure water are less dense and provide upward forces pvs has been earlier reported to be about 20% of total egg volume pvs largely varies from 9 to 18% at 2–3 dpf. Also the ρyolk+emb estimated in here ranges largely from 1.014 to 1.019 g cm−3, but mean ρyolk+emb is surprisingly similar to the previous measurement of Kjesbu et al.vs. our estimate of 1.0171 g cm−3. Measurements on specific gravity on the hardened chorion material, ρcho, from cod eggs Kjesbu et al.−3. Also independent measurement on chorions from six Atlantic halibut eggs showed similar average value, i.e. 1.1842 g cm−3egg to the presently observed ranges of egg size, Vcho, Vpvs, and ρyolk+emb. It appears from the table that the specific gravity of the cod eggs changes by 0.0063 g cm−3 across the range of egg size and Vcho, i.e. the smallest eggs with thickest chorions are 0.0063 g cm−3 heavier than the extreme large eggs with thin chorions. In comparison, the extreme changes in ρyolk+emb cause ρegg to change by 0.0046 g cm−3, which is about 70% of the variation caused by the chorion volume fraction. It should be emphasized that total range of ρyolk+emb among individual eggs (i.e. 0.0046 g cm−3) is 3.3 times larger than the individual change in ρegg throughout incubation (i.e. 0.0014 g cm−3). We have not measured the initial degree of hydrolyzation of the proteins in the yolk of the present study. However, noting that the large difference in ρegg between Baltic cod and Norwegian Atlantic cod can partly be ascribed the variation in hydrolyzation of the yolk yolk+emb among individual eggs in Norwegian Atlantic cod could be explained by moderate individual differences in yolk hydrolyzation and, hence, the degree of egg swelling prior to spawning. While the chorion volume (Vcho) is determined during the oocyte stage cho/Vegg) is determined by subsequent hydrolyzation prior to spawning. The present findings on the variation range of the Vpvs are larger than earlier findings in the literature; 9–18% compared to 18–22% in Kjesbu et al. egg: 0.0006 g cm−3, which is less than 10% of the variation in ρegg caused by the chorion volume fractions.The Vegg during development may also be related to differences in the proximal composition of the yolk and embryo component, resulting in slight changes in ρyolk+emb from fertilization to hatching. For example, relatively higher free amino acid (FAA) content is expected in the yolk compared to the embryo yolk versus ρemb based on unfed newly hatched cod larvae, suggest that these differ by approx. 0.014 g cm−3 (own unpubl. results). During the initial few days after fertilization this difference is not expected to be of importance in the overall ρyolk+emb due to the relatively high mass of yolk relative to mass of embryo. However, as the volume of the embryo increases rapidly towards the end of incubation it is expected to contribute more to increasing the overall ρegg.Some of the changes in ρegg is largely influenced by chorion volume fractions, and that the ontogenetic variability in ρegg during incubation is attributable to the volume and mass changes of yolk plus embryo. With the previous findings of water balance and metabolism during egg stages, we demonstrate that swelling of embryos by uptake of water and drop in yolk osmolarity are main causes of decreasing ρegg from mid-gastrulation to full development of main organs.In conclusion, based on our working hypotheses, the present study confirms that the initial ρFigure S1Time point egg specific gravity measured in winter every second day during development. Horizontal lines refer to initial specific gravity of the selected layers  at 2–3 dpf. Symbol of # indicates sampled levels for further analyses on egg composition shown in (TIF)Click here for additional data file.Figure S2Time point egg specific gravity measured in spring every second day during development. Horizontal lines refer to initial specific gravity of the selected layers  at 2 dpf. Symbol of # indicates pick-up levels for further analyses on egg composition shown in (TIF)Click here for additional data file.Figure S3Time point egg specific gravity measured in fall every second day during development. Horizontal lines refer to initial specific gravity of the selected layers  at 3 dpf. Symbol of # indicates pick-up levels for further analyses on egg composition shown in (TIF)Click here for additional data file.Figure S4Initial changes in egg specific gravity caused by perivitelline space and yolk volume dynamics. Left panel is changes in mean egg specific gravity and right panel is changes in mean volume fractions of yolk plus embryo, perivitelline space and chorion from 2 to 4 dpf, represented by spring time point measurements of (A) Batch4-b, (B) Batch5-a and (C) Batch6-a. Points refer to mean and one standard deviation.(TIF)Click here for additional data file.Table S1Egg characteristics at 2–3 days post-fertilization (dpf) from time point measurements.(DOC)Click here for additional data file.Table S2Comparison of two types of measurements (continuous vs. time point).(DOC)Click here for additional data file.Appendix S1Theoretical equations for egg specific gravity containing (1) list of symbols in equations and (2) algorithms of specific gravity at fertilization and during development.(DOC)Click here for additional data file.


APOE gene has three polymorphic alleles that influence the risk for various types of cancer and neurodegenerative diseases. A potential association between APOE allele and the risk for gastric cancer has been implicated, but the specific allele involved and potential associations with the subtype and the grade of cancer malignancy need further clarification. We screened the APOE genotype in 550 gastric cancer patients and 550 non-cancer control individuals and found that the presence of the APOE ε2 and lower serum total cholesterol are associated with an increased risk for gastric cancer . Interestingly, APOE ε2 is also correlated with increased risk for both intestinal and diffuse histotypes but not with TN classification or stage in gastric cancer patients, suggesting that APOE polymorphic alleles are associated with the risk of development but unlikely the progression of gastric cancer. Since ε2 carriers have lower levels of serum total cholesterol than non-ε2 carriers, our findings suggest that the increased risk for gastric cancer by APOE ε2 allele might be mediated through lowered serum total cholesterol levels.Apolipoprotein E (apoE) mediates lipid metabolism both in peripheral and in the brain. The human  Gastric cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide1APOE gene consists of four exons and three introns, and is polymorphic on two single nucleotides resulting in three different alleles  and six APOE genotypes 13APOE genotypes were shown to determine its protein levels in the brain, cerebrospinal fluid, and serum, with ε2/ε2 > ε3/ε3 > ε4/ε4ApoE is a 299-amino acid glycoprotein that plays a key role in lipid transport and lipoprotein metabolism by binding to members of the low-density lipoprotein receptor family568910APOE gene polymorphism leads to an alteration in lipid and lipoprotein metabolism16161819APOE gene polymorphism and high-density lipoprotein cholesterol (HDL-cholesterol) level was noted in some studies1620232425APOE ε2 allele is also associated with higher risk for both intestinal and diffuse types of gastric cancer. Since ε2 carriers have lower levels of serum total cholesterol than non-ε2 carriers, our findings suggested that the increased risk for gastric cancer by APOE ε2 allele might be mediated through lowered serum total cholesterol levels.In our current study, we also observed a strong inverse association between serum total cholesterol levels and the risk for gastric cancer. In addition, we observed significant lower levels of total cholesterol, LDL-cholesterol and apoB in the ε2 carriers which are consistent with previous reports. However, we only observed a trend of higher levels of total cholesterol, LDL-cholesterol in the ε4 carriers. Intriguingly, the presence of the APOE genotypes for both patients and controls are also summarized in A summary of the characteristics for the 550 gastric cancer patients and the 550 cancer-free controls is displayed in APOE ε4 and ε2 alleles in all subjects are displayed in Comparisons of demographic variables and serum lipid profile according to the presence or absence of the APOE ε2 allele; however, this did not survive correction for multiple testing (P = 0.032). There was no statistically significant evidence of an association with risk of gastric cancer for APOE ε4 (P = 0.87) or apoA1/B ratio (P = 0.087).An evaluation of risk factors for gastric cancer is provided in APOE ε2 and increased risk of gastric cancer strengthened and became significant after adjustment for multiple testing (P = 0.0004). Associations with gastric cancer for triglycerides (P = 0.061), LDL cholesterol (P = 0.35) and apoB (P = 0.48) weakened in multivariable analysis and were no longer even nominally significant, while the lack of association of gender and APOE ε4 with risk of gastric cancer remained unchanged (P ≥ 0.30). Intriguingly, when examining the association between APOE ε2 and gastric cancer subtype, an increased risk of gastric cancer was noted in ε2 carriers for both intestinal gastric cancer (P = 0.017) and diffuse gastric cancer (P = 0.0006)  .APOE ε4 and ε2 with T classification, N classification, and stage in gastric cancer patients are displayed in Associations of APOE gene polymorphism and serum cholesterol levels on the risk of gastric cancer among a Chinese Han population. Our results show that the presence of APOE ε2 allele and low serum levels of total cholesterol, HDL-cholesterol, apoA1 and apoA1/B ratio are associated with an increased risk of gastric cancer. Interestingly, APOE ε2 is also correlated with increased risk for both intestinal and diffuse histotypes. However, APOE genotypes do not appear to influence the invasion and metastasis of gastric cancer as reflected by TN classification and stage.Our case-control study of 550 gastric cancer patients and 550 cancer-free controls evaluated for the first time the effects of APOE genotypes, with individuals carrying the ε2 allele having lower and those carrying the ε4 displaying higher levels compared to the more common ε3 allele161819APOE ε2 allele might be mediated through lowered total cholesterol level.In gastric cancer, serum cholesterol levels have been reported to inversely correlate with the risk of disease2527APOE genotypes on the risk of numerous cancers has been previously investigated, including breast, prostate, ovarian, colorectal, head and neck cancer29303132333435APOE ε2 allele against gastric cancer in an Italian Caucasian populationAPOE ε2 and gastric cancer risk in both ethnic populations.The effect of APOE genotypes and T classification, N classification, and stage in gastric cancer patients. Our data showed no significant association between APOE genotypes and various classifications or stage of gastric cancer, suggesting that APOE polymorphic alleles are associated with the risk of development but unlikely the progression of gastric cancer.ApoE has recently been identified as a potential tumor-associated marker in gastric cancer from gene expression analysis438APOE ε2 as a risk allele for both intestinal and diffuse types of gastric cancer in our studied population, which might be partly attributed to the lower serum total cholesterol of ε2 carriers compared to those with ε3 or ε4 alleles.In summary, our study confirmed the associations of lower levels of serum cholesterol with the incidence of gastric cancer in a Chinese Han population. Importantly, our study reports for the first time that Approval for this study was obtained from the Ethics Committees of the Xiamen University and Fujian Medical University Union Hospital. All experiments were performed in accordance with the approved guidelines. All individuals who participated in this study gave written informed consent.rd English editionA total of 550 gastric cancer patients with histological confirmation and 550 cancer-free controls seen at the Fujian Medical University Union Hospital in China between 2011 and 2014 were included in this case–control study. Information was collected regarding age, gender, body mass index (BMI), and serum lipid parameters which include the levels of triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, apoA1, apoB, and apoA1/apoB ratio. None of the patients underwent pre-operative chemotherapy or radiation therapy. Histopathological evaluations were performed with reference to the Japanese Classification of Gastric Carcinoma, 3APOE SNPs  was carried out using the APOE SNP genotyping kits  and the Applied Biosystems® 7500 real-time PCR Systems . Data analysis was performed by measuring the allele-specific fluorescence. As a measure for quality control, three samples with known APOE genotypes were included in each assay. Additionally, the genotyping analysis was blinded to the subject’s case or control status. Finally, 10% of the total samples were randomly selected and retested with 100% concordance. There was no evidence of a departure from Hardy-Weinberg Equilibrium in study controls for either rs429358 (P = 0.79) or rs7412 (P = 0.71).Genomic DNA was extracted from peripheral blood using a DNA extraction kit . Genotyping of the two th percentile, 75th percentile, and maximum. Categorical variables were summarized with number and percentage of patients. Due to the small number of subjects with APOE ε4/ε4 and ε2/ε2 genotypes, we utilized a dominant model in analyzing both APOE ε4  and ε2  in all analysis. Comparisons of demographic variables and serum lipid parameters according to the presence of the APOE ε4 or ε2 allele were made using a Wilcoxon rank sum test or Fisher’s exact test. Risk factors for gastric cancer were evaluated using single variable (i.e. unadjusted) and multivariable logistic regression models. Multivariable models were adjusted for all variables that were associated with risk of gastric cancer in single variable analysis with a p-value of 0.10 or lower, although there were some exceptions to this due to the high degree of correlation between many of the variables of interest and the resulting potential for collinearity  and multivariable logistic regression models. Multivariable models were adjusted for all variables that were associated with risk of gastric cancer in single variable analysis with a p-value of 0.10 or lower .In order to adjust for multiple testing, we utilized a Bonferroni correction separately for each group of similar tests. Specifically, p-values ≤ 0.0050 were considered as significant when comparing demographic variables and serum lipid parameters according to presence of ε4 and ε2; p-values ≤ 0.0042 were considered as significant when evaluating risk factors for gastric cancer; p-values ≤ 0.0167 were considered as significant when evaluating associations of ε4 and ε2 with TN classification and stage in gastric cancer patients; and p-values ≤ 0.025 were considered as significant when evaluating the association between How to cite this article: Kang, R. et al. Apolipoprotein E epsilon 2 allele and low serum cholesterol as risk factors for gastric cancer in a Chinese Han population. Sci. Rep.6, 19930; doi: 10.1038/srep19930 (2016).


Detecting changes of spatially high-resolution functional connectivity patterns in the brain is crucial for improving the fundamental understanding of brain function in both health and disease, yet still poses one of the biggest challenges in computational neuroscience. Currently, classical multivariate Granger Causality analyses of directed interactions between single process components in coupled systems are commonly restricted to spatially low- dimensional data, which requires a pre-selection or aggregation of time series as a preprocessing step. In this paper we propose a new fully multivariate Granger Causality approach with embedded dimension reduction that makes it possible to obtain a representation of functional connectivity for spatially high-dimensional data. The resulting functional connectivity networks may consist of several thousand vertices and thus contain more detailed information compared to connectivity networks obtained from approaches based on particular regions of interest. Our large scale Granger Causality approach is applied to synthetic and resting state fMRI data with a focus on how well network community structure, which represents a functional segmentation of the network, is preserved. It is demonstrated that a number of different community detection algorithms, which utilize a variety of algorithmic strategies and exploit topological features differently, reveal meaningful information on the underlying network module structure. A comprehensive insight into brain processes requires an understanding of information flow between and within structures of the underlying neural system. Commonly, based on non-invasive measuring modalities such as EEG, MEG, and functional MRI (fMRI), this is accomplished by a quantification of directed interactions between recorded time series, where prominent approaches rely on various conceptual principles. Common to all of them is that functional connectivity characteristics are indirectly estimated on the basis of statistical dependencies between measured time series. Due to a limited availability of temporal samples, usually smaller sets of selected or aggregated time series are considered for inferring functional connectivity from estimated statistical characteristics ,2. Thus,s in diseased and healthy states, or in response to behavioral or pharmacological interventions.An alternative to time series subset pre-selection or aggregation is a reduction of spatial dimensionality by a suitable coordinate transformation such as Principal Component Analysis (PCA), or Independent Component Analysis ,5. TheirBesides approaches for dimension reduction it has also been proposed to circumvent the indeterminacy of parameter estimation equations while keeping the data dimensionality unchanged ,7. For iIn this work, we propose a novel methodological concept, where spatially high-dimensional data are incorporated into connectivity analysis. These data originate from a (possibly large) system of connected and interacting elements  and thusWe propose a general large scale Granger Causality (lsGC) approach, a purely data-driven procedure which involves incorporating a PCA data dimension reduction step into low-dimensional (LD) space, but ultimately attains connectivity patterns in the original high-dimensional (HD) space. This concept is comprised of an orthogonal back projection of LD MVAR model residuals to HD space and using these back-transferred residuals for proper definitions of vertex by vertex interactions.This preservation of high spatial data dimensionality may result in networks representing functional connectivity consisting of several thousand vertices connected by millions of edges. With methods from network science these large functional connectivity networks can be further processed, and essential information about their underlying connectivity structure can be revealed. In contrast to a segmentation based on vertex activation (voxel time series in the case of fMRI) we are particularly interested in obtaining a functional segmentation of groups of strongly interacting network vertices and in tracing changes in such functionally segmented regions. This functional segmentation can be analyzed naturally within the framework of network module (network community) detection ,14. NetwThe paper is organized as follows: Firstly, we describe the generation of a synthetic data set and the real data used in this study. Thereafter, we present the novel lsGCI approach with embedded dimension reduction in detail, followed by an analysis of the effects of dimension reduction on the topology of the resulting lsGCI network using a synthetic network ensemble with known ground truth. We particularly investigate the impact of the edge pattern information loss caused by dimension reduction on the quality and the recoverability of network modules in the resulting lsGCI networks. Then, we give an example for an application of the lsGC approach and use it to obtain high-dimensional networks from resting state fMRI data. Finally, we analyze their functional segmentation given by their module structure and demonstrate that the network modules detected in these functional brain networks are plausible from the physiological perspective.To investigate the reliability and suitability of the novel methodology with respect to its effects on the topology and module structure of the resulting networks, we devised a network model to generate synthetic networks with a pre-defined and possibly clear-cut module structure, whose degree of definiteness depends on the chosen parameterization and that should be identifiable by module detection algorithms. In the following, these networks are called ground truth networks.D ∈ {100,200,…,800} vertices and can be partitioned naturally into non-overlapping modules restricted to consist of ten to fifteen vertices each. The dimensions D were chosen to obtain networks that can be still processed with standard GCI methods for the purpose of quantitative comparisons. The number of modules was scaled with the network size, so that for every increase of 100 vertices eight additional modules are gained. The size of each module was chosen randomly, such that the sum of module sizes equals D. The edges connecting vertices were placed randomly  under constraints that define the module structure. Edge patterns were constrained by probabilities for intra-module edges and inter-module edges. All column sums in the resulting adjacency matrices were restricted to be less than or equal to fifteen. In detail, the probability for directed intra-module edges was pint = 0.5, whereas the probability for directed inter-module edges pext depends on the network size so that the constraint on the column sums holds true.Each ground truth network consists of pext = 3/(D − 15). Moreover, additional conditions on minimum internal and maximum external in- and out-degrees have to hold true, such that the simulated module structure is more clear-cut, while the modules remain connected. That means vertices are limited to maintaining a minimum number of 4 edges both, to and from member vertices of their own module, while their number of interactions with vertices of other modules must not exceed a maximum of 4 edges in each of both directions. The computational costs for the simulations increase strongly with network size D as the aforementioned constraints become increasingly harder to satisfy. Finally, we simulated networks up to dimension D = 800, for which the computational costs of network generation were still manageable. Based on these settings we generated 100 different instances of ground truth networks for each dimensionality D.To account for outlier vertices that have an above-average number of edges  that would violate the column sum constraint, we determined that on average each vertex has three edges to and from vertices of different modules. Consequently, we defined the probability of inter-module edges by p-th order D-variate autoregressive (AR) models formally given byD-dimensional state vectors D-dimensional random variables.The corresponding multivariate time series were realized on the basis of D-dimensional MVAR process of order p = 1. The number of available temporal samples was held constant with N = 1000. The effect of N was already demonstrated in . It is defined as the difference of the fraction of intra-module edges and the expected fraction of such edges in a suitable random network. Modularity for directed networks takes into account “surprising” edges given the degree information of their tail and head vertices, e.g. edges that fall between pairs of vertices where the tail-vertex has small out-degree and the head vertex has small in-degree. The following algorithms for network module identification were used: “leading eigenvector” (1), “Louvain” directed (2), “Walktrap” (3), “fast greedy” (4), “leading eigenvector” (5) “Potts spin glass” (6), “Louvain” undirected (7). (A) ground truth network; (B-D) lsGCI network with variance explanations from 70%-90%; (E) GCI network.(TIFF)Click here for additional data file.S2 FigThe reduction of uncertainty of vertex assignments in one partition due to knowledge about the other partition is given by the mutual information . The following algorithms for network module identification were used: “leading eigenvector” (1), “Louvain” directed (2), “Walktrap” (3), “fast greedy” (4), “leading eigenvector” (5), “Potts spin glass” (6), “Louvain” undirected (7). (A) ground truth network; (B-D) lsGCI network with variance explanations from 70%-90%; (E) GCI network.(TIFF)Click here for additional data file.S3 FigThis measure computes for each pair of corresponding modules in both partitions the Levenshtein edit distance  of intra-module edges, which is the cost for their optimal alignment. For it, the adjacency matrix for each module is vectorized and typecasted to a string in the alphabet {0,1}. The Levenshtein distance is the minimum number of insertions, deletions and substitutions to make both strings equal. Single edit distances for each pair of corresponding modules are added up to yield the partition edit distance. The following algorithms for network module identification were used: “leading eigenvector” (1), “Louvain” directed (2), “Walktrap” (3), “fast greedy” (4), “leading eigenvector” (5), “Potts spin glass” (6), “Louvain” undirected (7). (A) ground truth network; (B-D) lsGCI network with variance explanations from 70%-90%; (E) GCI network.(TIFF)Click here for additional data file.S4 FigFor a network partition uncovered by a module detection algorithm, the fraction of correctly “interpreted” vertex pairs with regard to the network adjacency information is called performance . It takes into account the vertex pairs that are assigned the same module and that interact via an edge and those vertex pairs where both vertices are classified to belong to different modules that are not connected by an edge. In other words, the performance measure penalizes edges that are ignored by a given network partition (when both end-vertices are assigned to different modules) and it penalizes edges implied by the network partition that are not present in the network . The following algorithms for network module identification were used: “leading eigenvector” (1), “Louvain” directed (2), “Walktrap” (3), “fast greedy” (4), “leading eigenvector” (5) “Potts spin glass” (6), “Louvain” undirected (7). (A) ground truth network; (B-D) lsGCI network with variance explanations from 70%-90%; (E) GCI network.(TIFF)Click here for additional data file.S5 FigIn practice the Rand index does not necessarily range over the entire  interval and instead often concentrates in a small interval close to 1. Therefore, it might be adjusted for chance assignment of modules . The following algorithms for network module identification were used: “leading eigenvector” (1), “Louvain” directed (2), “Walktrap” (3), “fast greedy” (4), “leading eigenvector” (5), “Potts spin glass” (6), “Louvain” undirected (7). (A) ground truth network; (B-D) lsGCI network with variance explanations from 70%-90%; (E) GCI network.(TIFF)Click here for additional data file.S6 FigThe split-join distance  measures the extent to which two partitions are subpartitions of each other by means of accounting for their module overlap. The following algorithms for network module identification were used: “leading eigenvector” (1), “Louvain” directed (2), “Walktrap” (3), “fast greedy” (4), “leading eigenvector” (5), “Potts spin glass” (6), “Louvain” undirected (7). (A) ground truth network; (B-D) lsGCI network with variance explanations from 70%-90%; (E) GCI network.(TIFF)Click here for additional data file.S7 FigThe variation of information  compares two network partitions by measuring the change in their information  when one partition is converted to the other one. It is a metric on the space of network partitions and can consequently be used to calculate the distance of two partitions of the same network data. This measure does not depend on topological information of the input network as it relies only on module affiliations of vertices. The following algorithms for network module identification were used: “leading eigenvector” (1) “Louvain” directed (2) “Walktrap” (3), “fast greedy” (4), “leading eigenvector” (5), “Potts spin glass” (6), “Louvain” undirected (7). (A) ground truth network; (B-D) lsGCI network with variance explanations from 70%-90%; (E) GCI network.(TIFF)Click here for additional data file.S1 File(PDF)Click here for additional data file.