Being the probability of introduction through legal imports of live pigs  higher than the risk of legally imported swine products . This could be caused due to the low probability of exposure associated with this type of commodity (products). The risk of feral pigs accessing to swine products discarded in landfills was slightly higher than the potential exposure of domestic pigs through swill feeding. The identification of the months at highest risk, the origin of the higher risk imports, and the location of the US states most vulnerable to those introductions , is valuable information that would help to design prevention, risk-mitigation and early-detection strategies that would help to minimize the catastrophic consequences of potential ASF/CSF introductions into the US.The US livestock safety strongly depends on its capacity to prevent the introduction of Transboundary Animal Diseases (TADs). Therefore, accurate and updated information on the location and origin of those potential TADs risks is essential, so preventive measures as market restrictions can be put on place. The objective of the present study was to evaluate the current risk of African swine fever (ASF) and Classical swine fever (CSF) introduction into the US through the legal importations of live pigs and swine products using a quantitative approach that could be later applied to other risks. Four quantitative stochastic risk assessment models were developed to estimate the monthly probabilities of ASF and CSF release into the US, and the exposure of susceptible populations  to these introductions at state level. The results suggest a low annual probability of either ASF or CSF introduction into the US, by any of the analyzed pathways (5.5*10 Asfarviridae family [Pestivirus, family Flaviviridae [Ornithodoros spp.) [O. moubata the main vector in Africa and O. erraticus in the Mediterranean [African swine fever (ASF) and Classical Swine Fever (CSF) are animal diseases notifiable to the World Organisation for Animal Health (OIE)  that have family , whereasiviridae . ASF andiviridae , 3. ASFVos spp.) , being Oerranean .ASF was first described in Africa in 1921, which remains endemic in most of Sub-Saharan countries . HoweverWhile ASF had been traditionally associated with the African continent (and Sardinia) until its re-introduction in Europe, CSF has been much wider distributed causing important problems around the world for decades . NotableThe United States of America (US) is the world's third-largest producer and consumer of pork, and the world’s largest pork exporter, with an average of 20% of pork annually produced in the US being exported . Therefohttp://bioportal.ucdavis.edu/) to facilitate their visualization and analysis, and the quantitative approach developed here will serve to develop a template for the assessment of the potential introduction of other TADs.ASF and CSF represent a risk for the global swine industry, and important economic consequences are expected if they were introduced in swine production countries as the US , 19. TheFour quantitative stochastic risk assessment models were developed to estimate the monthly probabilities of ASF and CSF introduction into the US by legal imports of live pigs and products during the high risk period . Specifically, the four models addressed the following event pathways: i) risk of ASF being introduced by legal imports of live pigs, ii) risk of ASF introduction by legal imports of swine products, iii) risk of CSF introduction by legal imports of live pigs and, iv) risk of CSF being introduced by legal imports of swine products.® and run 1,000 iterations using the Monte-Carlo sampling method.Finally, those four models were combined to estimate i) the probabilities of ASF introduction and ii) the probability of CSF introduction by any of the analyzed pathways. These two probabilities were finally combined to estimate the probability of either, ASF or CSF, introduction into the US by legal importations of pigs or products. All models were developed in @RISK 7.5  on Microsoft Excel 2007ASF∩PCSF).*Note: In order to simplify the text and avoid redundancies, the sentences, parameters and probabilities that apply for both diseases’ models (the model developed for ASF and the model for CSF), were referred as “ASF/CSF”. For example “the probability of ASF/ CSF introduction into the US by the legal imports of live pigs” is read as “the probability of ASF introduction into the US and the probability of CSF introduction into the US”. It is important to differentiate this notation with the probability of both events (ASF and CSF) occurring at the same time  of at least one ASF /CSF infected domestic pig or kilogram (kg) of swine product by legal imports from the country of origin (c) into a state of the US (s), was modeled following a binomial process of the form:The probability of release (Pncs refers to the number of pigs or kilograms (kg) of swine products (depending on the pathway assessed) legally imported from the origin country c to the destination states s; and pcs is the probability that an ASF/CSF infected animals or products  arrive from origin country c to destination states s.Where ncs) was obtained from the USDA Global Agricultural Trade System (GATS) [The information on imported live pigs and swine products (m (GATS)  and US Cm (GATS)  from 200pcs was calculated as the product of different conditional probabilities as follow:The probability csL) was calculated as the product of four conditional probabilities (1); the probability of selecting a pig infected with ASFV / CSFV in country c (P2L); the survival probability of ASF/CSF infection (P3); and the probability that infected pig survives transportation from country c to destination s (1-P4). Due to the lack of information related with carrier animals , only apparent clinical stages of the disease were considered in the models.Specifically, the probability of release for the legal importations of live pigs (pbilities . The procsP)  and P2P as the probability of selecting a kilogram of swine products infected with ASFV/CSFV in country c.The probability of release for the legal importation of products (pcsP)  was estic was parameterized differently depending on the status of the diseases in the country of interest. For those countries currently infected by ASF / CSF, an exponential function was used to estimate the probability of at least one outbreak in the considered time interval with the following form:t is the time interval (one month) and λ is the mean number of disease outbreaks per month obtained from historical outbreak information [a the total number of outbreaks in domestic swine and b the total number of months of the study period.The probability of infection in the origin country ormation  estimateigraph [sna [For those countries with no records of ASF/ CSF presence for the last 10 years, the probability of infection was estimated through a compartmental Susceptible (S)-Infected (I) model that considers the pig trade network and the disease status of trading countries. Specifically, the model used data on world pig trade from the latest year available 2013) , and disigraph  and sna aph [sna . Each sc013 , andimports of live pigs, the probability of selecting an infected live pig in origin country c was modeled using a beta distribution defined by the following parameters: α1L as the number of estimated infected pigs prior to ASF/CSF detection in the country c, and α2L as the total number of pigs in country c. The number of infected pigs in country c (NI) was estimated as the product of three independent parameters: i) undetected outbreaks (Ou) during the high risk period in country c [In the models assessing the risk associated with ountry c , ii) aveountry c , and iiiountry c   of potentially infected swine products in the country c, and α2P as the quantity (kg) of swine products produced (NP) in the country c. The quantity of infected products in the origin country c (QIM) was estimated by the product of three parameters: NI) estimated number of infected pigs in the country of origin c, Pm) probability of an ASF/CSF infected pig being transformed into meat, and MP) the average weight of products (kg) obtained per slaughtered pig (In the models developed to estimate the risk associated with ered pig .m), was estimated as follows: Pm = P3*(1-P4)*Psm*PusThe number of infected pigs (NI) was calculated identically to the live pig models, as the product of Ou* Hp *To. The probability of an ASF/CSF infected pig being transformed into meat  in the US getting in contact with the viruses strongly differ depending on the type of infected matrix (live pigs or swine products). Therefore, the exposure assessments for both pathways were analyzed differently and explained separately below.L) was estimated as following:The probability of a domestic swine in the US getting an effective contact with a legally imported infected pig , their R, WRTE and WH for the retail, restaurants and house-holds, respectively). The probability of pork being discarded at house-hold level (WH) was estimated as the product of the proportion of food being wasted at consumer level (WC) by the proportion of pork consumed at houses instead of restaurants (1-CRTE). The opposite applies for the waste at restaurant level WRTE = WC* CRTE.The detailed information about the inputs and calculations used to estimate the probability of exposure to imported swine products into the US can be found in SF, the probability of domestic pig getting in contact with infected products through swill feeding, and PLF, the probability of feral pigs getting in contact with infected products having access to landfills.Once discarded, food waste could have different destinations, including recovery processes , feeding animals (mostly swine) or disposed in landfills. The first option does not involve contact with pigs, and consequently no further studies were performed on it. The model assessed two potential pathways of contact with susceptible swine populations: Pswill feeding practices and the regulations applied, strongly differs depending on the origin of the food waste (i.e. retail and restaurants vs. household food), therefore the household waste was considered separately from the others. For the probability of swill feeding originated from retail (SFR) and restaurants (SFRTE) we considered the proportion of food waste later used as swill feeding from the retail sector (SF|WR) [RTE) [il), by a beta distribution with a 95% of confidence that the swill feeding illegal practices are less common than 10% and most likely under 5%. These assumptions were used to compute beta distributions by the use of BetaBuster [R = WR* SF|WR*(GF*((1- Ef) + Pil)). For the swill feeding from restaurants (SFRTE) the equation is exactly the same but applying the values related with restaurants waste (i.e. WRTE and SF|WRTE).The frequency of  (SF|WR)  and restR) [RTE) , 42, theR) [RTE) . Based otaBuster . The proH) we employed a similar approach than the one used by [H) [HP) was estimated considering the number of household with pigs vs the total number of occupied houses in the US [HP). Finally, the risk associated with swill feeding practices with waste food from households was estimated as: SFH = WC*(1-CRTE)* SF|WH* PHP*(SF|PHP).To estimate the probability of swill feeding practices in the households (SF used by . Firstlyd by [H) . Then, tn the US . Previoun the US . Therefon the US  and the n the US , a prorathe disposal in landfills and the potential access of feral pigs to the landfills (PLF). As the proportion of free-ranging domestic pigs (without barriers) in the US is very low, the possibility of domestic pig population having access to landfills was not considered in the assessment. To evaluate that risk, firstly we obtained the proportion of food waste discarded in landfills from the retail sector (LFR), restaurants (LFRTE) [FP). For doing that, the maps of feral pigs distribution [FP). No data was found related with the probability of feral pigs having access to the landfills content. Therefore, we assumed a conservative approach, with a minimum probability of feral pigs having access to landfills of 0.05, a most likely probability of 0.1 and maximum of 0.2 (FPA). The probability of feral pigs getting in contact with food waste disposed in landfills coming from any of the three sectors was estimated as following:The second potential destination of the food waste that could lead into ASF/ CSF outbreak is  (LFRTE) , 42 and  (LFRTE) . After tribution  and inforibution  were oveSFi) plus the probability of feral pigs getting in contact with infected imported swine products disposed in landfills (PLF), all of them calculated at state level.Assuming that both exposure routes are mutually exclusive, meaning that they can’t not occur simultaneously , the final exposure was calculated as the sum of all three probabilities of domestic pigs being exposed by swill feeding . The annual mean (sum of monthly probabilities) probabilities of ASF /CSF introduction were mapped in ArcGis 10.0 (ESRI®) using Natural Breaks (5 classes) as classification methods calculated by Jenks algorithm  and the ASF is the combined probability of ASF being introduced into the US by legal imports, PASFL is the probability of ASF being introduced into the US by legal imports of live pigs and PASFP the probability of ASF being introduced into the US by legal imports of pig products. The same calculations applied for the combined probability of CSF (PCSF).In order to provide a global picture of the risk for the introduction of these diseases in the US, we combined the four analyzed pathways. Firstly, we calculated the annual probabilities of introduction per country for each disease by any of the two pathways analyzed. Considering both pathways  as independent events not mutually exclusive, the combined probability per disease was estimated as follows:C) was estimated as follows:Finally, assuming the introduction of each disease being independent and not mutually exclusive from each other, the final combined probability of introduction of ASF or CSF into the US by legal imports (Pi) between each input and the annual probability of ASF/CSF introduction in the US. Afterwards, the inputs that were most likely to influence the final results (βi ≥ 0.1) were analyzed in detail using the advanced sensitivity analysis tool of @RISK 5.5 running 500 iterations for each scenario. A total of 10 scenarios were assessed for each selected parameter, by changing the base values in ten consecutive steps, from a minimum of 50% reduction to a maximum of 50% increase.Sensitivity analyses were performed for all the models previously described in two steps. Firstly, the most influential parameters in each of the models were identified by calculating the regression coefficients , while the probability of CSF by the same pathway was 2.5*10−3 . These values approximately correspond with one outbreak of ASF in 276 years, and one introduction of CSF in 201 years, if conditions remain constant. The overall mean annual probabilities of ASFV being released by imported pigs was 8.4*10−2 , whereas for CSFV this probability reached 1.2*10−1 . The mean exposure of US domestic pig population to imported pigs  pigs was 4.4*10−2 .Considering the current situation of both diseases (as of November 2016), the probability of ASF introduction into the US by legal imports of live pigs was estimated as 3.6*10−3 for ASF, and 4.9*10−3 for CSF. The monthly disaggregation of the risk revealed that January and March were the months at higher risk in both models, including also February for CSF. However, no important seasonal differences were observed for none of the diseases (data not shown).The distribution of the risk of introductions through imports of live pigs into the US was very similar for both diseases analyzed. Although small differences were found between states for ASF and CSF, when the values were categorized for producing the risk maps, the result was the same, so only one map was presented . The hig−2 , and 6.9*10−2 , respectively. The location of the risks of ASFV /CSFV being released into the US through import of swine products varies between both viruses and from the risk associated with live pigs imports. Specifically, the risk of ASFV release was highest in the states of New Jersey (2.6*10−2), Virginia (1.2*10−2), California (9.5*10−3) and Florida (9.4*10−3). However, the risk of release of CSF potentially infected products was concentrated in Florida (2.6*10−2), Illinois (8.1*10−3) and California (6.9*10−3) (The probabilities of both viruses ASFV/CSFV being released into the US by infected swine products were estimated as 7.8*10.9*10−3) . As it c−2) and Poland (1.4*10−2). However, for the introduction of CSF the origins of the risk were wider distributed, being Finland (4.8*10−2), Canada (7.1*10−3), Cayman Islands (7.1*10−3) and Denmark (1.7*10−3) the origins of the highest risk.A total of 91% of the risk of ASFV being released into the US was originated from Denmark , existing important differences between states. This risk of exposure was concentrated in the southern states, including California, mainly due to the presence of feral pigs with potential access to landfills, as well as to the smaller size of swine premises, which present higher risk of using swill feeding (−1) than the risk associated with swill feeding activities to domestic pigs (2.0*10−2). The waste originated from households contributed with 64% of the total risk of exposure, mainly due to the disposal of 94.5% of the generated waste in landfills. This risk was followed by the waste from the risk associated with retail sector (20%) and finally the restaurants waste (16%).The average probability of US swine populations of being exposed to imported products per state was 4.4*10 feeding . The ris−4 , and 8.3*10−4  for CSF. Those probabilities were approximately eight times (ASF) and 6 times (CSF) lower than the estimated probabilities of introduction through imports of live pigs. The final risk maps for both diseases were quite similar, being in both cases Florida and California the states at highest risk . For CSF, the combined probability resulted in 3.3*10−3. The final combined probability, which estimates the risk of ASF or CSF being introduced into the US by legal importations of animals or products was estimated as 5.5*10−3, which is approximately equivalent to one outbreak every 181 years.The combined probability of ASF introduction into the US through legal importations (of pigs or products) was estimated as 2.2*10i ≥ 0.1), the following inputs were selected for the advanced sensitivity analysis from the pigs imports model: the probability of ASF/CSF infection in Canada (P1 CAN), the probability of selecting an infected pig from Canada (P2 CAN), the probability of survival to ASF/CSF infection (P3), the probability of pigs being undetected during quarantine (Pu) and the probability of imported pigs going through quarantine (Pq). The advanced sensitivity analysis reveals that both models were only noticeable influenced by one parameter: Pq or the probability of imported pigs going through quarantine (Based on the correlation coefficient assessment (βarantine . The resA) and the food losses at consumer level (WC) were selected for both models. In addition, from the ASF model of swine products the probability of selecting infected meat from Denmark (P2 ASF DEN), Canada (P2 ASF CAN) and The Netherlands (P2 ASF NETH) were included. Whereas for the CSF model, the probability of selecting infected meat from Finland (P2 CSF FIN), Cayman Island (P2 CSF CAY) and United Kingdom (P2 CSF UK) were selected (βi ≥ 0.1). Based on the advanced sensitivity analysis, both models were highly influenced by the likelihood of feral pigs accessing to landfills and the food losses at consumer level . However, the lack of detailed data and controversy associated with the estimation of those other pathways make these estimations very difficult and require to be supported by a series of risk assessments of each of the source countries, which was out of the scope and possibilities of this study. Therefore, although is not the perfect solution, it was considered that this approach provides more realistic information than traditional estimates, and could be easily applied in other risk assessment models.Another strength of this study was the integration of the release and exposure assessments in one single stochastic model for the assessment of the risk associated with imported products. The integration of both steps in a common model is not common, as in most of the cases, the quality of information are not comparable and each step is assessed separately (i.e. release and exposure in ). In addIf done in controlled manner , our model suggests that it implies less risks than disposing the food waste in landfills.In our model, the risk associated with swill feeding practices was mostly related with the food waste generated in households (64% of the risk of swill feeding was associated with waste from households vs retail and restaurants). Opposite to EU where swill feeding is totally banned, many states in the US allow swill feeding to swine under certain conditions (properly heat-treated and fed by a licensed facility) Therefore−2), posing a higher risk compared to other exposure pathways analyzed here as the swill feeding activities in domestic pigs. In addition, although not included in this study, feral pig could potentially have access to the food waste in bins and containers, before it arrives to the landfills, as well as contacting domestic pigs. The contacts and potential transmission of pathogens between domestic pigs and feral pigs has been documented in certain areas of the US [Most of the food waste generated in our houses ends in landfills (94.5%) , and manf the US  and posef the US  with des−3). However, it is important to remember that other pathways should be analyzed such as the importation of other biological products , animal feed and, importantly, illegal pathways, including the waste from planes and other international transports, in order to have a more complete picture of the risk of ASF and CSF introduction into the US. Among the four pathways analyzed, the imports of pigs posed the highest risk for the introduction of CSF into the US. This could be caused, firstly, due to the fact that CSF is more widely distributed in the world, and the probabilities of CSF infection in origin derived from the SI model were higher comparing with ASF. Secondly, the risk associated with the legal import of swine products was lower in both diseases (4.5*10−4 for ASF and 3.8*10−4 for CSF) compared to the live swine imports (3.6*10−3 for ASF and 4.9*10−3 for CSF), potentially due to the lower risk of exposure to the imported products, as they usually go directly to human consumption. However, the risk of ASF and CSF released by imports of animal products was considerable higher (0.1), which reflects the importance of controlling the exposure to these products to avoid potential outbreaks of these diseases in the US.Despite the global burden situation of ASF and CSF, and the assumptions used in the model, the risk of introduction of either ASF or CSF into the US by legal import of animal and products is considerably low , Minnesota (12%) and Wisconsin (10%). The models presented here estimated the existing risk associated with exporting animals before the detection in the country of origin (high risk period), assuming that US will not accept importation of swine from ASF or CSF infected countries. Canada is an ASF/CSF free country but the risk rises with increasing the volume of commodity imported. According to FAOSTAT , in the −2. However, the results of the sensitivity analysis identified the probability of imported pigs going through quarantine (Pq) as the essential parameter in the model. Therefore, the correct application of quarantine procedures is an essential component for maintaining the free status and reduce the risks associated with animal importations.The probability of domestic pigs in the US being exposed to a potentially infected (either with ASFV or CSFV) imported swine was estimated as 4.35*10For the legal importation of live pigs, no differences were found on the location of the risk between the analyzed diseases (ASF or CSF). However, for the risk associated with the legal imports of swine products, the location of the risk substantially varies between ASF and CSF, due to the trade differences between the states. Whereas for ASF the probability of release was highest in New Jersey (33%), Florida (14%), California (13%) and Virginia (11%); for CSF the risk of release was concentrated in Florida (38%), Illinois (12%), California (10%), and Texas (8%) . The proAlthough the consequences of the introduction of ASF and CSF into the US were not considered in this work, the example of previous outbreaks in other free territories suggest a potential huge economic impact. Presumable, the impact would be more serious in the case of ASFV introduction, as no vaccines are available for its control. However, in the case of CSF, there are highly efficacious and safe vaccines available, including a bait format for oral immunization which could be used in wild boar and even in domestic pigs in backyard conditions .http://bioportal.ucdavis.edu/) for the easy update and visualization of the risk estimates. Other pathways of introduction such as the illegal importation of pork and other swine products into the US by the different ports of entry  are currently being addressed and will be also incorporated in the platform.Quantitative risk assessments present certain benefits and advantages versus qualitative models, as they incorporate the variability of data, uncertainty of the estimations and evaluate the influence of the parameters in the final outcome through sensitivity analysis. However, this type of analysis results in very time intensive process due to the substantial efforts for collecting all the data required plus the computational requirements. For example, in this study, more than 15,000 inputs were used in the models for the legal importations of pig products. On the other hand, once the models’ structures are defined, data sources identified and available, the models can be easily updated. The studies developed here not only served to estimate the probabilities of ASF and CSF introduction through legal imports, but will constitute the basis for the documentation and quantitative analysis of the risk of other FADs into the US, that would help to prevent the negative consequences associated with these types of diseases. The epidemiological information obtained in the present study could be used to develop risk-based surveillance, prevention and early detection strategies that would help to prevent ASF/CSF introduction and protect US swine livestock and consumers as well as allocate resources effectively and efficiently. In addition, the model parameters and calculations of the present study will be integrated in an online user-friendly risk assessment platform , with most of the live pigs coming from Canada. In contrast, the final risk of ASF/CSF introduction for the products model was concentrated in states of California, Florida and Texas. However, the risk of entrance of potentially infected swine products into the US clearly differs between CSF and ASF. The epidemiological information obtained in the present study could be useful to develop prevention and early detection strategies that would help to prevent ASF/CSF introduction as well as allocate resources effectively and efficiently.


Entanglement of states is one of the most surprising and counter-intuitive consequences of quantum mechanics, with potent applications in cryptography and computing. In organic materials, one particularly significant manifestation is the spin-entangled triplet-pair state, which mediates the spin-conserving fission of one spin-0 singlet exciton into two spin-1 triplet excitons. Despite long theoretical and experimental exploration, the nature of the triplet-pair state and inter-triplet interactions have proved elusive. Here we use a range of organic semiconductors that undergo singlet exciton fission to reveal the photophysical properties of entangled triplet-pair states. We find that the triplet pair is bound with respect to free triplets with an energy that is largely material independent (∼30 meV). During its lifetime, the component triplets behave cooperatively as a singlet and emit light through a Herzberg–Teller-type mechanism, resulting in vibronically structured photoluminescence. In photovoltaic blends, charge transfer can occur from the bound triplet pairs with >100% photon-to-charge conversion efficiency. et al. show that the immediate triplet pair is bound and emissive in a range of acene and heteroacene materials.Singlet fission in organic semiconductors can generate triplet exciton pairs that are crucial to the charge generation in a photovoltaic process, whilst their nature remains elusive. Here, Yong  Of those that discuss energetics, all consider 1(TT) to be higher in energy than T1+T1. Here we demonstrate this is incorrect and show that 1(TT) is bound with respect to free triplets.The entangled pair of triplet excitons ls1 ref. , the for1(TT) state experimentally and theoretically in a range of acene and heteroacene materials (1(TT) state as the immediate product of singlet fission. The processes of formation and decay of 1(TT) are shown in et al.131(TT) is temperature-independent in polycrystalline films and is thought to occur via a combination of intra- and intermolecular motion from the photoexcited singlet1213142134361(TT) can undergo one of three detectable decay processes as follows: (i) form free (unbound) triplets, a process aided by disorder at low temperatures and thermally activated above 50 K; (ii) emit (red arrows) by way of symmetry-breaking distortions enabling intensity borrowing from nearby bright states; and (iii) with sufficient energy, ‘back transfer’ to S1 leading to delayed fluorescence. We also infer that a significant proportion of 1(TT) states undergo non-radiative decay, presumably directly to the ground state. In photovoltaic blends, we find that charge transfer from this bound, entangled triplet pair is as efficient as that from free triplets giving photon-to-charge conversion quantum efficiencies of >100%.Here we probe the aterials . We demo2-TES ADT), rubrene and tetracene1538392-TES ADT are shown in 2-TES ADT and tetracene, we suggest a similar origin for the red-shifted emissive species. We propose in the following that this emission arises from a bound, entangled triplet pair, 1(TT), the immediate product of singlet fission.2-TES ADT solutions , see below. At higher temperatures, the increased intensity in the delayed component at 585 nm, co-incident with the singlet 0–0 emission, suggests thermally activated delayed fluorescence with the same lifetime as 1(TT) (process (iii) in In concentrated solution, the singlet converts into a red-shifted species maintaining vibronic structure. Likewise, in solid films decomposition reveals correlation between prompt and delayed emitter kinetics: the delayed species grows at the expense of the prompt. Although the ‘prompt’ emitter lifetime remains essentially unchanged over the temperature range, the ‘delayed’ emitter lifetime increases substantially with decreasing temperature. Conversely, the prompt emission spectrum changes dramatically with temperature. The enhancement of the 0–0 peak, a property common to materials such as tetracene, which show super-radiance at low temperatureS〉 is not an energy eigenstate of the system and |XX〉, |YY〉 and |ZZ〉 acquire relative phases, evolving in time with frequencies governed by their energy spacing. As the state periodically approaches its initial composition ), there is significant enhancement of the rate to re-form the original, fluorescent singlet through TTA if this is energetically allowed. The observed PL quantum beats thus reveal modulation of the radiative TTA probability due to spin conservation. The oscillation amplitude follows the extracted delayed PL kinetics in F2-TES ADT and tetracene, clearly linking the delayed emission to the 1(TT) state generated by singlet fission. The beats’ persistence over the 1(TT) lifetime indicates that spin coherence is maintained over at least that timescale, and that 1(TT) is thus an entangled triplet-pair state.Interestingly, following femtosecond excitation at 300 K we observed temporal oscillations of the PL, as shown in 4747asis see  and detaasis see ). |S〉 is1(TT)) to emit, especially with the well-defined vibronic progression seen in ab initio ) excited-state calculations on π-stacked dimers in the crystal geometry (details in the Methods section below and 1(TT) relative to the singlet and non-interacting triplet pairs. Importantly, the bright singlet contribution to the adiabatic 1(TT) state vanishes at the equilibrium geometry161(TT) instead mixes only with a dark singlet, with emission thus forbidden. Geometric distortion activates slight mixing with the bright state, resulting in finite but low radiative rate . Nonetheless, the 1(TT) emission can dominate the spectrum at intermediate temperatures due to its long lifetime. This mechanism is equivalent to Herzberg–Teller couplingIt is unexpected for this doubly excited state (2-TES ADT (341(TT) emission may be preferentially regenerated by annihilation from ‘trapped’ triplets due to its lower electronic energy341 and 1(TT) energies; even pentacene has been shown to demonstrate similarly shaped emission in films1(TT) spectrum to the 0–1 peak, akin to emission from the ‘triplet-pair’-like1 state in polyenes1(TT) 0–0 peak from the spacing between observed 0–1 and 0–2 peaks . This is shown in We propose that this mechanism explains the vibronically structured red-shifted PL seen in many acenes in the literature. As discussed above, tetracene demonstrates very similar temperature-dependent PL to F-TES ADT , 42, 43,ES ADT 34. Even on–2 peaks . We find2-TES ADT and similar materials, 1(TT) and S1 are roughly isoenergetic. Indeed, the delayed spectra in 1(TT) PL spectrum through the Herzberg–Teller model. The ability of 1(TT) to emit and the spin-conservation considerations above suggest that quantum beating could likewise be anticipated in the direct 1(TT) emission, suggesting a need for spectrally resolved quantum beating studies.This model has critical implications for singlet fission energetics in ‘slow’ tetracene-like materials. In F1(TT) we used TA spectroscopy. 2-TES ADT films collected at 300 K and we can immediately distinguish two primary species: the initially formed singlet exciton and a subsequent long-lived photo-induced absorption (PIA) band centred at 970 nm. To aid assignment of this feature, we examine F2-TES ADT solutions at several concentrations211+T1; sensitization1(TT) emission (pink dashed trace), enabling assignment to 1(TT). The last panels in 1(TT) acts as a distinct singlet fission intermediate even in the solid state. We expect the 1(TT) PIA absorption cross-section to be similar to that of two free triplets. Therefore, the reduction in signal from 3–100 ns in the raw TA kinetic (pink trace in For further insight into the formation and decay of 1(TT) intermediate in all three  decay was completely independent of intensity, indicating that any recombination is geminate. By contrast, free triplets exhibit fluence-dependent recombination. Interestingly, our results show that S1 decay and 1(TT) growth are largely temperature-independent  and free triplet population dynamics for F2-TES ADT films from 4–250 K. Data for other molecules can be found in 2 ADT single crystal), 1(TT) is stabilized with respect not only to the initial singlet but also to two free triplet excitons, confirming its bound natureg state in polyenes relative to two free triplets5456A temperature dependence does, however, appear in the subsequent process, in which the bound triplet pair separates into free triplet excitons . It is n1(TT) into free triplets based on Marcus–Levich–Jortner rate theory parameterized against ab initio calculations  or the ground-state geometry (G). Bound and separated triplet pairs then correspond to TTG and TGT configurations, respectively. The free energy stabilization going from TGT to TTG completely dominates the activation energy ΔE. The stabilization energies are overestimated by these calculations, probably due to the presence of polymorphism and the assumed single crystal geometry. For instance, we calculate that increasing the π-stack distance in TIPS-pentacene by 0.1 Å decreases the ΔH by ∼30%. Nonetheless, the predicted trends in the ΔE values match well with measurements.To better understand this binding and thermally activated decay, we have analysed conversion from H (50–100 meV) correlate well with the admixture of singlet and charge-transfer configurations (1(TT) stabilization, although orbital delocalization also contributes. The electronic coupling for intermolecular triplet transfer VT−T was estimated using π-stacked dimer models and agrees well with previous studiesMoreover, the magnitudes of Δurations , indicat1(TT) separation, in good agreement with experimental results given the simple model. Importantly, we reproduce the large rate difference between TIPS-pentacene and F2-TES ADT, and the smaller difference between F2-TES ADT and rubrene. These differences cannot be attributed to ΔH  and instead follow VT−T. Thus, the underlying rate for 1(TT) separation is governed by the intrinsic triplet mobility. At the same time, we note that the overall activation energy is dominated by ΔH rather than the inter- or intramolecular reorganization energies. We can thus conclude that the measured high-temperature activation energies reflect the energy stabilization of bound versus free triplet pairs, and that triplet-pair separation can be described in terms of triplet hopping from the bound state. The most significant discrepancy between theory and experiment is at low temperature: the model predicts vanishingly small 1(TT) separation, whereas we measure very little change in the rate from ∼50 K to lower temperatures. We propose that structural and/or energetic disorder, for instance as would be found at grain boundaries, accounts for the persistence of low-barrier free triplet formation.As shown in 2-ADT, 1(TT) emission, with clear temperature dependence of the 1(TT) lifetime above 200 K but little temperature dependence below. In TA, we found free triplet formation at ≥200 K but observed no free triplets at lower temperature. These results support our model for the role of disorder: in the absence of structural or energetic disorder, the 1(TT) state must be separated through thermal energy alone, leading to complete suppression of the process at low temperature.To verify this, we have performed similar detailed TA and PL measurements on single crystals of difluorinated anthradithiophene F2-ADT, . We obse1(TT) separation in F2-TES ADT, tetracene1(TT) lifetime is only ∼200 fs (ref. 1(TT) in photovoltaic blend films and devices, we have studied bulk-heterojunction films of F2-TES ADT and -phenyl C71 butyric acid methyl ester (PC71BM) in 4:1 molar ratio, in which we expect significant demixing. The device structure is shown schematically in 2-TES ADT should either be free or bound spin-entangled triplets, respectively  drops as the temperature is lowered  PL and free triplet TA lifetimes. There is thus a competition between two modes of 1(TT) decay: diffusion to a PC71BM interface with direct charge generation, and thermally activated separation into free triplets, which in turn diffuse to the interface to form charges.The 300 K extracted kinetics shown in 1(TT) to charge formation can be isolated, as free triplet formation is strongly suppressed. Indeed, we find that the relative yield of free triplets is >50% lower at 50 K than at 300 K (1(TT) lifetime in neat and blend films is strikingly similar at 50 K. We can only reconcile this by citing the same rate-determining process for both 1(TT) separation to free triplets and charge separation from 1(TT): exciton diffusion to the interface. In blend films this is to a PC71BM-rich phase; in neat films diffusion is to grain boundaries or other defect sites (see above).At 50 K, the contribution of at 300 K . In the 1(TT) and free triplets to the charge yield, we focus on 1(TT) should be the dominant pathway at low temperature.To determine qualitatively the contribution of 1(TT). An interesting property of 1(TT) is that it emits as a singlet but can transfer charge from each of its constituent triplets. We find that non-radiative decay of 1(TT) is a dominant loss mechanism limiting further exploitation. Similarly fast non-radiative 1(TT) decay has been observed in covalent acene dimers246256651(TT). Our work also has critical implications for TTA upconversion efficiency models1(TT)) converts to S1 with unity efficiency. Instead we find that 1(TT) →S1 conversion efficiency is determined by the competition between singlet formation, triplet-pair separation and non-radiative decay.We have demonstrated the general presence of the bound, entangled triplet-pair state More broadly, we consider that the generality of our findings across singlet fission materials affords a powerful platform to study the interactions between triplet excitons and the properties of bound multiexciton states and could lead to a new material set for solid-state quantum computing applications.2-TES ADT was synthesized as described previously71BM, tetracene, rubrene, TIPS-pentacene and pentacene were purchased from Sigma Aldrich. F2-TES ADT and TIPS-pentacene thin film samples were spin-cast  on polyimide precoated fused-silica substrates (the polyimide aids wetting of the organic semiconductor). For F2-TES ADT: PC71BM thin film samples, 4:1F2-TES ADT:PC71BM blend solutions  were spin-cast on polyimide precoated fused-silica substrates. Samples were dried on a hot-plate at 50 °C for 10 min. Tetracene, rubrene and pentacene thin-film samples were prepared via thermal evaporation at a base pressure of <6 × 10−6 mbar.F2-ADT were grown through a physical vapour growth method. Details of the relevant method can be seen elsewhereSingle crystals of F−2 to avoid bimolecular singlet–singlet annihilation effects and the pump and probe polarization were set to magic angle (54.7°) to avoid reorientation effects. For ns-TA measurements, pump pulses were generated using a frequency-doubled Q-switched ∼500 ps Nd:YVO4 laser (532 nm). Delay times from 1 ns to 1 ms were achieved using an electronic delay generator. For time-resolved PL measurements to generate spectral maps, the samples were excited by 40 ps pulses (excitation wavelength: 470 nm) operating at a repetition rate of 2.5 MHz. PL decay dynamics were resolved using electronic gating through time-correlated single-photon counting with a temporal resolution of 180 ps and detection based on a cooled microchannel plate photomultiplier tube coupled to a monochromator. PL quantum beating measurements were performed on a separate time-correlated single photon counting system (Becker-Hickl module) system, using 100 fs pulses at 500 nm with a repetition rate of 80 MHz. Here, the emission was detected with a silicon single-photon avalanche diode, yielding a temporal resolution of around 40 ps. For all solid-state time-resolved optical measurements, samples were kept at a fixed temperature in a cryostat with dynamic helium gas flow. For solution measurements, a fused silica 1 mm path length cuvette was used.For TA measurements, 90 fs pulses generated in a Ti:sapphire amplifier system (Spectra-Physics Solstice) operating at 1 kHz were used. The broadband probe beams were generated in separate home-built non-collinear optical parametric amplifiers for visible (500–800 nm) and near-infrared  ranges. For fs-ps TA measurements, we used either narrowband <200 fs pulses from a commercial travelling-wave optical parametric amplifier of superfluorescence (TOPAS) or compressed pulses  from a home-built non-collinear optical parametric amplifier to excite the samples. The time delay between pump and probe pulses was controlled by a mechanical delay stage. Unless otherwise noted, pump intensities were kept below 5 μJ cmN-methylfulleropyrolidine (NMFP) and F2-TES ADT in a molar ratio of 4:1 were prepared at a concentration of 1 mg ml−1 in chloroform. The NMFP was excited by the 355 nm frequency tripled output of a Q-switched sub-ns Nd:YVO4 laser. Following intersystem crossing on NMFP, triplet transfer to F2-TES ADT occurs and is monitored by TA spectroscopy. Results are shown in Blends of To obtain samples that gave phosphorescence, the molecule of interest was spin-coated into films doped with platinum octaethylporphyrin (purchased from Sigma Aldrich). The weight ratio of target molecules to platinum octaethylporphyrin in solution was varied from 95:5 to 98:2, to give the clearest phosphorescence signal. Mixtures were spin-coated on Spectrosil at 800–1,200 r.p.m. for 1–2 min in a nitrogen-filled glove box and the films were then annealed at 60 °C for 30 min. The final samples were encapsulated to prevent sample degradation and triplet quenching by oxygen. Phosphorescence was detected using a calibrated infrared InGaAs photodiode array (ANDOR iDus 490A) coupled to a spectrograph (ANDOR Shamrock), with CW excitation at 532 nm (∼0.5 mW).2-TES ADT. In these measurements, the change in PL is monitored under magnetic resonance. ODMR gives a direct way of identifying triplet excitons, as the dipole–dipole interaction between electron and hole spins within the triplet exciton gives rise to characteristically broad ODMR spectra, governed by the zero-field splitting Hamiltonian D* and E* are the zero-field splitting parametersOptically detected magnetic resonance (ODMR) experiments were performed to investigate the presence of free triplets in FB. Excitation was provided by a 532 nm laser with variable intensity I. The integrated PL was collected by a photodetector, after removing the laser line with a 550 nm long-pass filter. Microwaves were square-wave modulated at frequency fM, and the change in PL due to microwave transitions ΔPL was recorded by monitoring the photodetector response at this microwave chopping frequency using lock-in detection.Films were placed on a microwave stripline operated at a frequency of 6.2 GHz and mounted inside an optically accessible cryostat magnet, providing the static magnetic field λ=1.5418 Å, 40 kV, 40 mA) and an X’Celerate detector operating in a one-dimensional mode. A variable slit optics choosing an illuminated length of 10 mm was used in combination with 0.04° Soller slits at the primary as well as at the secondary side. Low temperatures down to 85 K were reached with the low temperature attachment TTK600 from Anton Paar Ltd using vacuum (2 × 10−5 bar) conditions. Starting from room temperature the samples were cooled down in steps of 25 K with a cooling rate of 5 K min−1. After reaching a temperature of 85 K, the sample was rapidly heated up to room temperature and cooled down again in steps to a temperature of 230 K, to confirm the measurements were reproducible after thermal cycling. Careful alignment of the sample height and sample tilt were made to obtain reliable results. Peak parameters were determined by a fit of a Gaussian curve to the experimentally observed Bragg peaks.Specular X-ray diffraction was performed with a PANalytical Empyrean system in Bragg–Brentano geometry using a sealed copper tube  using a wavelength of 1.5406 Å.2-TES ADT/PC71BM blend was dissolved in 1 ml of mesitylene and spin-coated on top of the PEDOT layer at 1,500 r.p.m. and annealed for 10 min at 80 °C. Finally, the LiF/Al cathode (50 nm) was vacuum-evaporated onto the annealed photoactive layer. All devices were encapsulated before testing.Solar cells were fabricated on 10 mm × 15 mm indium tin oxide-coated glass substrates that served as the anode. The substrates were ultrasonically cleaned in detergent, deionized water, acetone and isopropanol. A layer of 30 nm PEDOT:PSS :poly(styrene sulphonate) (Clevios PH 1000) was spin-coated onto the indium tin oxide substrate and dried in air at 120 °C for 10 min. Fifteen milligrams of 4:1 F2 using a set of lenses to illuminate individual pixels of size 0.08 cm2. The current–voltage (I–V) characteristics of the devices were measured under standard AM 1.5G conditions using an Abet Sun 2000 solar simulator, at an intensity equivalent to 100 mW cm−2. Spectral mismatch correction was performed before the measurements. The dark and bright I–V characteristics were measured using the Keithley 2635 source measure unit.A 100 W tungsten halogen lamp  and a 120 W Xenon lamp (350–500 nm) dispersed through a monochromator (Oriel Cornerstone 260) were used for external quantum efficiency measurements. For wavelengths between 375 and 900 nm, a set of silicon diodes (ThorLabs SM05PD1A) were used. A Keithley 2635 source measure unit was used to measure the short-circuit current as a function of wavelength. The incident light was focused to a spot size of ca. 1 mmλ)/A(λ), where A is the absorbance of the photoactive layer, which was computed with a transfer matrix approach, where A was modelled according to published literature proceduresn and the extinction coefficient k were determined via ellipsometry, see Internal quantum efficiency plots were calculated from external quantum efficiency  intermediate state kinetics in the TA measurements with that in the transient PL measurements. These two measurements are both conducted in the linear regime at similar excitation fluence to ensure the same photophysics are observed in both measurements.We assess the reliability of the results of the optimization in three ways: (1) by testing the reproducibility of the results from >10 runs of the genetic algorithm data, as shown in 1(TT) PL, we analyse the bright exciton component in the 1(TT) state based on ab initio excited-state calculations. The 1(TT) states of π-stacked dimers are calculated using the complete active space self-consistent field (CASSCF) method, where the active orbitals consist of the highest-occupied molecular-orbital and lowest-unoccupied molecular-orbital of two monomers, that is CASSCF. The doubly excited 1(TT) diabatic state is optically dark and thus the 1(TT) PL necessitates mixing with singly excited bright exciton states. Yet, as found for the acene crystals1(TT) states completely vanishes due to the relative phase factor of the wavefunctions in H-aggregates. In this case, the symmetric 1(TT) mixes only with the dark exciton and charge transfer states, which do not contribute to PL. However, the bright exciton can be mixed with 1(TT) via symmetry-breaking effects due to intra-molecular vibronic coupling. We model this in the simplest way possible by displacing the geometry of one of monomers from the triplet minimum to the ground state minimum, (see PL from the triplet pair state: to clarify the origin of 1(TT) to free triplets based on the Marcus–Levich–Jortner rate theory parameterized against ab initio calculations. The electronic coupling for the intermolecular triplet transfer, VT−T,  separation was obtained from CASSCF/ CASPT2 calculations on π-stacked trimer models, with the monomers adopting either the fully relaxed triplet geometry (T) or the ground-state geometry (G). Thus, in this notation, the bound and separated triplet pairs correspond to TTG and TGT configurations, respectively.Triplet pair separation rate: we analysed the transfer rate from the bound 1(TT) separation rate, KT−T, is evaluated by the Marcus–Levich–Jortner theory:The k, T and S are the Boltzmann constant, temperature and entropy change, respectively. The Franck–Condon factor for the intramolecular effective mode, 〈0|0〉intra, is evaluated from the reorganization energy, λintra, and the frequency of 0.18 eV corresponding to the usual breathing/stretching mode in aromatic/conjugated molecules. The low-frequency intermolecular reorganization energy, λinter, is small for covalent excitations such as triplets and its calculation cumbersome. Here we take a conservative value of 0.05 eV; modifying it in a reasonable range (up to a factor of 3) hardly affects the conclusions below. eW account for ΔS considering the numbers of spatial and spin configurations for the bound 1(TT) and separated triplets. The 1(TT) separation pathway for π-stacked TIPS-pentacene, F2-TES ADT and rubrene can be regarded as a one-dimensional chain; in this case, the spatial ΔS from the bound 1(TT) to nearest-neighbour T+T is unity. Considering the dissipation of spin correlation, the number of spin configurations of the separated T–T (3 × 3) is three times as many as the total-singlet 1(TT), that is exp(ΔS/k)=3.where H indicates that the 1(TT) separation is endergonic for the three molecular materials investigated (see 1(TT) is stabilized by orbital delocalization as well as the admixture of singly excited electronic configurations into the nearest-neighbour TT pairs (TTG), both contributions vanishing in the case of separated triplets (TGT). The free-energy stabilization going from TGT to TTG completely dominates the activation energy ΔE. Although these are overestimated by the calculations, the predicted trends in the ΔE values (namely larger in TIPS-pentacene compared to rubrene) match reasonably well with the measurements. It is interesting to point out that for rubrene in its equilibrium crystal structure at 0 K, the singlet-triplet mixing is strictly null because of symmetryE value computed in that case reflects only the energy stabilization associated with direct wavefunction overlap between neighbouring triplets. The relatively large 1(TT) separation rate of TIPS-pentacene compared with F2-TES ADT and rubrene is rationalized by the difference in electronic coupling for triplet diffusion. Thus, based on these theoretical results, we believe the measured activation energies reported in The calculated Δhttps://doi.org/10.17863/CAM.9032.The data that support the findings shown in both the main figures and How to cite this article: Yong, C. K. et al. The entangled triplet pair state in acene and heteroacene materials. Nat. Commun.8, 15953 doi: 10.1038/ncomms15953 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In oral cancer, loss of function due to surgery can be unacceptable, designating the tumour as functionally inoperable. Other curative treatments can then be considered. Currently, predictions of these functional consequences are subjective and unreliable. We want to create patient-specific models to improve and objectify these predictions. A first step was taken by controlling a 3D lip model with volunteer-specific sEMG activities. We focus on the lips first, because they are essential for speech, oral food transport, and facial mimicry. Besides, they are more accessible to measurements than intraoral organs. 3D lip movement and corresponding sEMG activities are measured in five healthy volunteers, who performed 19 instructions repeatedly, to create a quantitative lip model by establishing the relationship between sEMG activities of eight facial muscles bilaterally on the input side and the corresponding 3D lip displacements on the output side. The relationship between 3D lip movement and sEMG activities was accommodated in a state-space model. A good relationship between sEMG activities and 3D lip movement was established with an average root mean square error of 2.43 mm for the first-order system and 2.46 mm for the second-order system. This information can be incorporated into biomechanical models to further personalise functional outcome assessment after treatment. Oral cancer, including that of the lips, is the sixth most common cancer worldwide . SurgeryAccurate prediction of the functional consequences of surgery is an urgent need to make the right choice of treatment . FunctioThis paper focuses on the lips, since these are essential for speech, oral food transport and facial mimicry. To create a predictive model, a continuum of 3D lip shapes is needed ultimately to perform virtual surgery on a model. Former research on lip modelling, utilising surface electromyography (sEMG) of facial muscles, is more phonetic in nature and is mainly focused on the categorisation of facial expressions , categorTo our knowledge, only two studies have described lip shape modelling in combination with quantitative lip pose estimation using facial electromyography (EMG). Honda et al.  recordedThe goal of this study is twofold. First we want to demonstrate that sEMG signals contain enough information for controlling 3D dynamic models of facial expressions, particularly lip movements. The second goal is to establish the optimal processing configuration to extract information from facial sEMG data. To avoid the complexity and pitfalls of detailed biomechanical models, we first focus on an empirical model. If the results of this empirical model are promising, the premise is justified that sEMG signals are very useful to solve the ambiguity problems in inverse dynamic modelling . In addi® Porti™ system . The micro-sEMG sintered disc-shaped surface electrodes  were placed above eight muscles on both sides of the face Data were obtained from five healthy volunteers mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimA study of van Son et al., showed that Dutch (experienced) lip readers were able to recognise five consonantal and five vowel visemes . Visemesentclass1pt{minimaThe sEMG signals Many different sEMG feature types have been proposed in the literature. Based on the results of Phinyomark et al., who examined 37 feature types, and our results of a preliminary experiment, we chose to investigate four feature types given in Table ® Porti™ system when the cameras started their recordings. Thereafter the sEMG signals were cut and resampled to 100 Hz, equivalent to the frame rate of the cameras.The videos were recorded concurrently with the sEMG. To synchronise the recorded sEMG signals with the video recordings a synchronisation pulse was fed to the TMSiThere is a small time delay between a sEMG activity and the corresponding muscle activation. It is difficult to define a default value for this delay. Honda et al.  used 70 The facial markers were tracked in the images of the three cameras, and the 2D coordinates were reconstructed to a set of 3D coordinates. The root mean square (RMS) error of the 3D localisation of markers, obtained via the leave-one-out method, was 0.73 mm. The resulting 3D positions of the ten markers on the lip, corrected for head movement, are denoted by State-space estimation requires the availability of a measurement model that links the sEMG features To establish the measurement model, first a principal component analysis (PCA) was applied. Suppose that a training set consisting of b, and subsequent decoding, occurs according to:The dimension of the vectors The mean of the coefficient vector To arrive at a (pseudo-) linear measurement model, we constructed the matrix Suppose that the residuals of This can be regarded as a linear measurement model of For dynamic modelling, two state-space models were implemented, a first-order and a second-order system. In the first-order system, a time series In the second-order system, the state vector was defined as:Preliminary experiments showed that the submatrices Equation  models \The process noise The estimation of the coefficients of the PCA was done with a discrete Kalman filter. The dimension of the state vector is in the first-order system The measurement noise covariance matrix The process noise covariance matrix The diagonal matrices The algorithm needs training data to find the PCA components This resulted into three design parameters, We performed cross-validation for training and testing. The procedure is depicted in Fig. p = 0.03). No significant difference was found between the two dynamic systems. Four subjects showed the best results when the WAMP feature was used. The optimal threshold The best results for the static, first-order and second-order state-space models are summarised in Table The RMS errors for the different features for the first-order and second-order system are presented in Table The influence of the different parameters and window length on the error in the second-order system can be seen in Fig. For the prediction of the functional and aesthetic consequences of treatment in oral cancer, dynamic models of the lips are required. Biomechanical modelling is physics based and as such the most direct method to predict these consequences. However, finding the patient-specific muscle activation signals needed for the biomechanical models is difficult . sEMG siThe approach used here, incorporated the dynamics of the system by means of a state-space model. To test whether dynamical modelling was superior to static modelling, we implemented both. As expected, incorporation of dynamics improved the model. In comparison with the static system RMS errors decreased in every volunteer, with an average of 0.27 mm for the first-order system and 0.24 mm for the second-order system. An advantage of a dynamic system is that bandwidth can be sacrificed to improve the signal-to-noise ratio. Apparently, in the current application such a sacrifice pays off, but not drastically.The difference between the first-order and second-order system is negligible. A higher-order system has more parameters which have to be estimated, making the filter more sensitive for deviations in those parameters to the optimum settings. An optimal equilibrium has to be found between modelling accurate dynamic behaviour for which higher-order systems are beneficial, and confining the impact of errors in the estimated parameters for which a lower-order system is preferred. In this study, the advantages of a second-order system over a first-order system did not outweigh the errors induced by the deviations in the estimated parameters.The fudging parameters were used to optimise the model per volunteer and hence make it volunteer-specific. Only the regularisation parameter The different features also had a noticeable influence on the RMS error. The WAMP features with k et al. .Because of different error assessments, the differences of our model compared to studies in the literature will be discussed qualitatively. The main differences are stated in Table A limitation of the proposed set-up is the high number of required sEMG electrodes. This makes the current approach time-consuming which is inconvenient in future clinical practice. Monopolar derivations can be used to halve the number of electrodes; however, this configuration does not reduce unwanted noise from the recordings by using the differential amplifier design . AnotherAnother difficulty is the variability in muscle anatomy, and overlying soft tissue, which makes standardisation of the measurements difficult. Additionally, physiological orofacial functions usually require simultaneous contraction of various muscles. These muscles therefore lack training in isolated contractions, resulting in relatively high co-contraction of muscles. Also volunteers can use different muscle activation patterns to perform the same instruction. We saw similar results when facial expressions were performed as described by Schumann et al. . Most voThe two main pillars of our study were to demonstrate that sEMG signals contain sufficient information to control 3D dynamic models of lip movements and to determine the best sEMG processing configuration for this purpose. These two steps are necessary for our ultimate goal to enable inverse biomechanical modelling of the lips, oral cavity and tongue, in order to retrieve patient-specific muscle activation signals inducing oral functions. These activation signals are needed to enable prediction of functional consequences after surgery. Besides patient-specific activation signals, patient-specific anatomical information and tissue parameters are required for simulating treatment effects. The current model does not account for these aspects yet. However, a biomechanical model should incorporate this physical relationship and patient-specific parameters before mimicking performed treatments. The simulated activations controlling the biomechanical model should be similar to the actual muscular control of the patient. sEMG is an instrument to provide the information for these simulated activations. Unfortunately, sEMG is a rough estimate, because of a nonlinear relationship, crosstalk, misplacement of electrodes and other artefacts. We showed that the relationship between 3D lip motion and sEMG can be accurately described by a statistical model. So it can be expected that with our approach the ambiguity problem of inverse modelling can be solved. Our next studies will focus on the relationship between activation signals and sEMG in biomechanical models.This study presented a next step towards the personalisation of the functional outcome assessment after treatment of oral cancer. The two dynamic modelling methods proved that a continuum of 3D lip positions can be predicted based on volunteer-specific sEMG features. The discrete Kalman filter with a first-order state estimation performs slightly better than a second-order system, with a mean RMS error of 2.43 mm. The optimal sEMG processing configuration was found to be the WAMP feature with


Vascularization of atherosclerotic plaques has been linked to plaque vulnerability. The aim of this study was to test if the vascularization was increased in upstream regions of early atherosclerotic carotid plaques and also to test if the same pattern of vascularization was seen in complicated, symptomatic plaques.We enrolled 45 subjects with early atherosclerotic lesions for contrast enhanced ultrasound and evaluated the percentage of plaque area in a longitudinal ultrasound section which contained contrast agent. Contrast-agent uptake was evaluated in both the upstream and downstream regions of the plaque. We also collected carotid endarterectomy specimens from 56 subjects and upstream and downstream regions were localized using magnetic resonance angiography and analyzed using histopathology and immunohistochemistry.Vascularization was increased in the upstream regions of early carotid plaques compared with downstream regions . Vascularization was also increased in the upstream regions of advanced atherosclerotic lesions compared with downstream regions  and was associated with intra-plaque hemorrhage and inflammation.Vascularization is increased in the upstream regions of both early and advanced plaques and is in advanced lesions mainly driven by inflammation. Upstream and downstream vascularization has not yet been measured in vivo and whether there are differences along the longitudinal axis early in plaque development is not known.In the atherosclerotic plaque, the regions upstream and downstream of the maximum stenosis will be exposed to different hemodynamic forces. High wall shear stress characterizes the upstream region while the downstream region is associated with low wall shear stress and turbulent blood flow. DifferePlaque vascularization is thought to be driven by hypoxia , but also by inflammation itself. During in vivo and ex vivo using contrast enhanced ultrasound (CEUS) and histology/immunohistochemistry respectively. The upstream and downstream regions were carefully identified in relation the point of maximum stenosis using in vivo ultrasound or Magnetic Resonance Angiography (MRA). We also aimed to determine whether hypoxia or inflammation is the main driving force for vascularization in late stage atherosclerotic plaques and to describe the expression of key angiogenic proteins in the upstream and downstream regions.The aim of this study was to investigate the distribution of vessels between upstream and downstream regions of early and late stage human carotid plaques The selection of early stage plaque patients for CEUS and late stage plaque patients, for immunohistochemistry have been described in detailed elsewhere, 15. ForThe study sample for the immunohistochemistry consisted of symptomatic carotid atherosclerotic plaques obtained from the Gothenburg Atheroma Study Group (GASG) bio bank of patients who underwent carotid endarterectomy (CEA) at the Sahlgrenska University Hospital  between October 2003 and April 2008. Criteria qualifying for surgery were minor ischemic stroke, transient ischemic attack (TIA) or amaurosis fugax (AFX), and a high-grade carotid stenosis (≥ 70% according to the ECST). From tAll subjects were asked to complete a questionnaire to provide clinical and lifestyle data and venous blood samples were drawn as previously described. This stThe early stage plaque subjects were imaged using a Siemens S2000, update VA16D, ultrasound platform  equipped with a 9L4 probe. We used 9MHz for B-mode ultrasound imaging and 4 MHz for cadence imaging. The carotids were scanned and the largest plaque was selected for contrast enhanced ultrasound imaging using a standardized protocol. In shorAll CEUS images were handled and analyzed using the Contrast Quantification Program (CQP). We usedIn the group with advanced plaque disease, MRA examinations were performed in a Philips Gyroscan Intera 1.5, Release 9 unit with a head and neck coil . In the in vivo MRA images, the distance from the flow divider to the site of maximum stenosis was measured.After surgical removal, endarterectomies were immediately fixed in formalin. From along the whole longitudinal axis of the plaque, 3-mm thick tissue blocks were prepared and for each block the distance from the flow divider in the bifurcation was measured. To determine the distance from the maximum stenosis for each tissue block the distance from the bifurcation was related to the MRA measurement. As we have reported previously, there is a good agreement between the point of maximum stenosis evaluated by MRA and the narrowest lumen found by histopathological assessment. From eaHistological staining and histopathological classification, including the American Heart Association (AHA) classification, were peIn addition to AHA classification, classification regarding presence of the different components of the AHA classification was also performed . The histopathological definitions used were adopted from Lovett et al. Immunohi+ vessel density was analyzed at high magnification (x40) and was assessed on the entire tissue sections and expressed as number of vessels per total section area excluding the lipid core.Extent of immunohistochemical staining is expressed as percentage stained area of the total section area (lipid core and lumen were excluded). CD34IBM SPSS Statistics 20.0 for Windows  was used for analyses. Results are given as means (SD) for parametric or medians (quartiles) for non-parametric variables. Categorical variables are presented as counts (%). Upstream and downstream sections where treated as related samples when compared. Parametric and non-parametric methods were used as appropriate. Correlations were tested using Spearman’s rho. Two-sided p-values <0.05 were considered significant.Fifty-two patients were recruited to the CEUS examination. Seven patients were later excluded because they had more than one visible plaque .During the study period, plaques from 265 patients were collected and 56 of these fulfilled the inclusion criteria (blood sampling and MRA). The included patients were more likely to have stroke as qualifying event , be on lipid lowering medication  and had a longer time between their clinical event and surgery . Characteristics of the included patients are presented in As presented in In the early stage plaque group, the median CQP value in the plaques was 30% (11–54). As reported earlier, plaques in the far wall and plaques from patients with previous ipsilateral stroke or TIA had higher CQP-values. In the 2, p = 0.001  vs. 0.8 (0.0–5.7), p = 0.036 compared with sections with no IPH. No associations were found between the number of vessels or vessel distribution between upstream and downstream regions and time since clinical event, lipid lowering medication, diabetes, age, sex or type of clinical event (data not shown).In the group with advanced plaque disease, the number of vessels in the endarterectomy specimens was increased in the upstream region compared with the downstream region, 4.6 (1.6–10.2) vs. 1.4 (0.3–5.7) vessels/mm = 0.001 . SectionAs presented in VEGF, VEGFR2, Notch1 and Dll4 were all significantly increased in the upstream region . VEGF, V2 (0.6–6.5) vs. 0.2 vessels/mm2 (0.0–1.3), p = 0.013  compared with preatheromas (AHA III), examined as median 2.4 vessels/mm = 0.013 . CD68 stin vivo in early atherosclerotic lesions as well as in ex vivo measurements from symptomatic patients with advanced atherosclerosis is increased in the upstream compared to the downstream region of the plaque. Furthermore, inflammatory markers were increased in the upstream region and we found an association between vessel density and inflammatory markers in both the upstream and downstream region. On the contrary, HIF1-α was increased in the downstream region and no association was seen with vessel density.Here, we show that vascularization measured Intra-plaque micro vessels might provide an entry point for inflammatory cells and also cause intra-plaque hemorrhage, which might aggravate inflammation, 9. FurtContrast agent uptake in carotid plaques measured with ultrasound correlates with histologically assessed plaque vascularization –23. We uVascularization was increased in the upstream regions in our study as well as in sections with intra-plaque hemorrhage. This finding are in line with previous reports of increased vascularization in the shoulder regions of both asymptomatic and symptomatic carotid plaques, 7 and fIn accordance with previous studies, 5, 6, wIn order to shed further light on the molecular mechanism that may be involved in plaque angiogenesis we estimated the expression of different key signaling pathways in upstream and downstream sections. VEGF and VEGFR2 are expressed in smooth muscle cells, macrophages and endothelial cells in human atherosclerosis. We saw We also measured Dll4 and NOTCH-1 expression since this system is a known crucial modulator of the angiogenic capacity of VEGF/VEGF-R2. Both DlThere are some limitations to this study. The cross-sectional design of the study prevents us from drawing conclusions on casual relationships. The presence of thrombosis in some plaques may have confounded the determination of the point of maximum stenosis by MRA. As we have reported previously, there wIn summary, we have shown that vascularization is increased in the more vulnerable upstream regions both in earlier phases of atherosclerosis as well as in advanced symptomatic plaques. In advanced plaques, vascularization correlates with markers of inflammation but not markers of hypoxia, which we interpret in favor of inflammation being the main driver of vascularization in these plaques while hypoxia might be more important in early lesions. The association with IPH may suggest a causative relation to plaque vulnerability.


Baló’s concentric sclerosis (BCS) is a rare inflammatory demyelinating disorder of the central nervous system characterised by concentric layers of demyelination. It is unclear whether BCS is a variant of multiple sclerosis (MS) or a disease entity in its own right.To compare the cerebrospinal fluid (CSF) features of BCS to those of MS.Retrospective analysis of the CSF profile of all patients with BCS reported in the medical literature between 1980 and 2017.p < 0.000001). OCB frequency was low both in ‘historic’ patients  and in more recent patients . OCB-positive and OCB-negative patients did not differ significantly with regard to age, sex, disease duration, number of Baló-like lesions on MRI, number of relapses, treatment or final outcome. In accordance with the high rate of OCB negativity, Link’s IgG index was negative in 63% of all tested samples (p < 0.000001 vs. MS). CSF pleocytosis was present in 28%  and elevated CSF total protein levels in 41% (31/76) of samples.In total, the results of 146 lumbar punctures (LP) in 132 patients were analysed. The most striking finding was a lack of CSF-restricted oligoclonal bands (OCB) in 66% (56/85) of all LP in the total BCS group, in 74% (14/19) in the subgroup of patients with both MRI and histological evidence for BCS, and in 82% (18/22) in the subgroup of patients with highest radiological confidence . OCB disappeared in 1/2 initially OCB-positive patients. These findings are in stark contrast to MS, in which OCB are present in ≥ 95% of patients and are thought to remain stably detectable over the entire course of disease (OCB and IgG index frequencies in BCS are much more similar to those reported in neuromyelitis optica or myelin oligodendrocyte glycoprotein antibody-associated encephalomyelitis than to those in MS. Our findings suggest that in most cases, BCS-like lesions denote the presence of a disease entity immunologically distinct from MS. In addition, we provide data on the demographics, clinical course and radiological features of BCS based on the largest cohort analysed to date. Baló’s concentric sclerosis (BCS) is a rare demyelinating disorder of the central nervous system (CNS) characterised by concentric layers of demyelination as detected by MRI or histopathology, and named after the Hungarian pathologist József Baló 1895-1979) 895-1979 . BCS is In a number of studies, we have demonstrated highly significant differences in cerebrospinal fluid (CSF) features between patients with MS and patients with other autoimmune disorders of the CNS. In particular, we have found significantly lower frequencies of CSF-restricted oligoclonal bands (OCB) and other markers of intrathecal IgG synthesis in patients with AQP4-IgG-positive neuromyelitis optica (NMO) spectrum disorders (NMOSD), myelin oligodendrocyte glycoprotein antibody-associated encephalomyelitis (MOG-EM), acute demyelinating encephalomyelitis (ADEM) and paraneoplastic neurological disorders (PND) than in MS, as well as significant differences in intrathecal IgG composition and dynamics and in blood–CSF barrier function –14. ThisTo address the question of whether BCS is a variant of multiple sclerosis (MS) or an immunologically distinct disease entity in its own right, we therefore set out to systematically compare the CSF features of BCS with those in MS.We performed a systematic review of all BCS cases published in English, German, French, Spanish, Japanese, Chinese, Russian or Italian in journals indexed in the PubMed database of the US Library of Science at the US National Institutes of Health between 1980 and 2017. Relevant publications were identified using the following search expression: Balo[title/abstract] OR Balo’s[title/abstract] OR Balos[title/abstract] OR ‘concentric sclerosis’[title/abstract]. Further publications/cases were identified by screening the reference lists of all PubMed-retrieved publications and from the authors’ own files. Overall, 215 publications that made mention of Baló’s concentric sclerosis in the abstract and/or title were identified and information on the CSF findings of 132 patients with BCS was collected. Results from follow-up lumbar punctures (LP) were available from 12/132 (9%) patients . In total, results from 146 LP in 132 patients diagnosed with BCS were available for retrospective analysis.The following CSF parameters were assessed: CSF-restricted oligoclonal IgG bands (OCB); Link’s IgG index ; immunoglobulin G/A/M (IgG/A/M) CSF levels and IgG/A/M CSF/serum ratios (QIgG/A/M); measles/rubella/zoster reaction (MRZR) as defined by a positive reaction to at least two of the three viral agents; CSF albumin concentration and albumin CSF/serum ratio (QAlb); CSF white cell count (WCC); CSF cytology; CSF total protein concentration (TP); frequency of an albuminocytologic dissociation (ACD), i.e. CSF TP elevation in the absence of CSF pleocytosis; CSF myelin basic protein concentration (MBP); and CSF lactate concentration. A CSF white cell count ≥ 5/μl was classified as increased. An age-dependent upper reference range for CSF L-lactate was applied . The upper reference limit for total CSF protein was set at 50 mg/dl [In addition, the following demographic, clinical and paraclinical parameters were assessed: sex, ethnic origin, age at onset, age at LP, time between last attack and LP, attack and long-term outcome, treatment status at the time of LP, number and location of Baló-like lesions, frequency and number of coexisting non-Baló-like lesions, initially suspected diagnosis, level of diagnostic certainty , and outcome at last follow-up .U test and Fisher’s exact test (two-tailed) were used to compare groups. Spearman’s r was used to test for correlations between groups. Due to the exploratory nature of this study, no correction for multiple testing was performed. All analyses were performed retrospectively; accordingly, no CSF or serum samples were obtained for this study.The Mann–Whitney The sex ratio  was 1:1.54. The median age of disease onset was 32 years (range 4–57). In 68 patients, the diagnosis of BCS was based upon typical radiological features; in 35, on typical histopathological features; and in 25, on both typical radiological and typical histopathological features. Histopathological evidence was obtained from biopsy samples in 15, from autopsy samples in 42 and from both biopsy and autopsy samples in 3 patients. In 43 cases, a single Baló-like lesion was present at the time of LP as detected by MRI or, if MRI was not available, histology, and multiple  Baló-like lesions were noted in 53 cases. Baló-like lesions were located in the supratentorial brain at the time of LP in 93 cases, in the infratentorial brain in 3, in both the supra- and infratentorial brain in 5 and in the supra- and/or infratentorial brain and the spinal cord in 4. Additional non-Baló-like CNS lesions were present in 58/94 (61.7%) patients at the time of LP. Before the diagnosis of BCS was made, MS was initially suspected in at least 8 patients, stroke in 5 and other diagnoses such as ‘tumefactive demyelinating disease’, ‘brain tumour’ or ‘brain abscess’ in 8 (no data in the remainder). Antibodies to aquaporin-4 (AQP4-IgG) or myelin oligodendrocyte glycoprotein (MOG-IgG) had been tested in 20 and 3 patients, respectively, and were negative in all. The median age at first LP was 32 years (range 4–60). The exact time between onset of the most recent attack and LP was given for 54 LP and was ≤ 30 days in 42 cases; the median interval was 15 days (range 1–190 days). In 58 additional cases, the exact time interval was not specified but LP was performed during an acute attack and thus probably within 30 days of attack onset. At last follow-up , more than one attack had occurred in 27/102 (26.5%) patients. Fifty-eight LP were performed prior to treatment with steroids or immunosuppressants, 4 LP in patients who had been treated with steroids shortly beforehand, 1 LP in a patient treated with immunosuppressants and 1 LP in a patient who had been treated with PEX and steroids 6 months previously ; in the p < 0.000001 when compared to a reference work on OCB in MS [The most striking finding was a lack of CSF-restricted OCB in the majority of cases: 65% (52/80) of all patients were negative for OCB N=51) or only transiently positive (N=1) (Table  or only CB in MS ). Of notp < 0.00001). Moreover, so far, an effect of IS on OCB positivity in MS has been shown only for natalizumab [Immunosuppressive treatment may theoretically affect OCB frequency. However, the difference between BCS patients and MS in terms of OCB positivity was still highly significant after exclusion of all patients treated with immunosuppressants (IS) at the time of LP .N = 25); all of these lesions showed three or more layers of demyelination. In this subgroup of cases with particularly high radiological confidence, the lack of OCB was even more pronounced than in the total cohort .Due to the retrospective nature of the study, MRI quality varied considerably among cases. To exclude the possibility that the low rate of OCB was due to unintentional inclusion of patients with diagnoses other than BCS, we defined (in a fashion blinded to OCB results) a subgroup of cases with available high-quality MRI depicting textbook-like BCS lesions .N = 64), the differences did not reach statistical significance. Moreover, as a potentially important bias, most fatal cases were reported in Chinese, Philippine and Japanese patients in the 1980s and early 1990s, when OCB were not yet generally determined in those countries. However, if only cases published during the past 20 years (1998 to 2017) were considered, again, more OCB-negative than OCB-positive patients had an unfavourable outcome .We were then interested in whether OCB may define distinct subgroups of BCS patients. However, no significant differences were found between OCB-positive and OCB-negative patients with regard to sex , median age at onset (32 vs. 30 years), median age at LP (32 vs. 32 years), disease course at last follow-up (relapsing in 36 vs. 34%), number of Baló-like lesions (more than one in 48.1 vs. 55.6%), location of Baló-like lesions  and co-existing non-Baló-like lesion (present in 48.1 vs. 55.6%). While more patients in the OCB-negative group had an unfavourable outcome .Conversely, no statistically significant differences in OCB frequency were found between male and female patients (31.3 vs. 37.5%), between children/juveniles and adults (50 vs. 32.9%), between patients with a relapsing and patients with a non-relapsing disease course at last follow-up (36 vs 34%), between LP in patients with a single Baló-like lesion and in those with multiple Baló-like lesions (36.8 vs 32.4%) or between LP in patients with additional, non-Baló-like lesions and in those without such lesions (36.4 vs 27.6%). A non-significant trend towards a lower frequency of OCB in patients with no/almost no recovery or fatal outcome compared with patients who had partial to full recovery at last follow-up was noted  [In accordance with the widespread lack of IgG OCB, Link’s IgG index was positive only in 22.6% (7/31) of all samples tested for that parameter. This is in contrast to the reference study in MS, which reported an elevated IgG index in 86.4%  .N = 267; p < 0.000001) [p = 0.013).The CSF WCC was elevated in 27/96 (28.1%) samples. This compares to a CSF pleocytosis rate in MS of 58.4% reported in a reference work on CSF in MS (.000001) . Among pp < 0.0005 vs. BCS). The majority of patients with ACD and available data (90.9% or 10/11) were OCB negative. CSF MBP concentration was determined in seven samples and was elevated in three (42.9%).TP was elevated in 40.8%  of all CSF samples. Most patients with elevated TP and available data were OCB-negative (77.8% or 14/18). A so-called ACD, i.e. an increase in the CSF total protein concentration without an accompanying increase in the number of CSF white cells, was found in 17/72 (23.6%) samples. ACD is typically found in Guillain-Barre syndrome but not usually in MS  were reported only in single patients or not at all, preventing systematic analysis of these CSF parameters.43.2% (35/81) of all CSF examinations showed neither pleocytosis nor elevated TP, and 15.1% (11/73) showed neither CSF pleocytosis or TP elevation nor intrathecal IgG synthesis. Of note, 12 further LP were classified as ‘normal’ by the authors, but no more detailed information was provided. While these LP could not be included in the statistical analysis, they suggest that the low rate of WCC pleocytosis, OCB positivity and IgG index elevation observed in our study may even underestimate the real lack in CSF abnormalities in BCS and, in consequence, the real degree of difference between BCS and MS in terms of CSF pathology.p < 0.000001 compared with MS) and that Link’s IgG index, a quantitative marker of intrathecal IgG synthesis, is negative in more than 75% of cases (p < 0.000001 compared with MS). Given that intrathecal IgG synthesis is considered a diagnostic mainstay of MS [Traditionally, BCS is considered a variant of MS. Here, we demonstrate highly significant differences in CSF profiles between patients with BCS and patients with MS. In particular, we found that CSF-restricted OCB, a qualitative marker of intrathecal IgG synthesis, are missing in almost two-thirds of patients with BCS , further corroborating the notion that BCS and MS are, in most cases, distinct disorders.In addition, we found a significantly lower frequency of CSF pleocytosis in BCS than in MS reference cohorts  and CSF lactose levels, were reported in only a few patients, preventing statistical assessment.In the effort to better understand the significance of OCB in BCS, we looked into possible differences between OCB-positive and OCB-negative patients with BCS with regard to demographics, disease course, MRI findings and disease outcome. However, no marked differences between the two groups were found in terms of age at onset, age at the time of LP, sex ratio, disease course, lesion location or the number of Baló-like lesions on MRI. Conversely, the rate of OCB-positive samples did not differ significantly between male and female patients, relapsing and non-relapsing patients, children and adults, or patients with a single Baló-like lesion and patients with multiple Baló-like lesions. However, a non-significant trend towards an association of OCB negativity with more severe disease outcome was found.We first hypothesised that the presence or absence of intrathecal IgG synthesis might depend on disease activity, as has been demonstrated in AQP4-IgG-positive NMOSD . InteresAlternatively, BCS lesions may not denote a single disease entity but may be a  final pathway of various demyelinating diseases, e.g. reflecting the presence of intralesional hypoxia as recently proposed. If so, BCS lesions could occur in the context of both conventional (OCB-positive) MS and other (mostly or exclusively OCB-negative) inflammatory demyelinating conditions such as MOG encephalomyelitis, ADEM, NMOSD or other as yet unidentified disorders. In potential keeping with that hypothesis, there was substantial heterogeneity among BCS cases in terms of disease course (relapsing vs. monophasic), MRI presentation  and outcome . Moreover, some patients with BCS had initially been diagnosed with MS, ADEM or NMOSD. Finally, at least some patients with BCS may suffer from what has been called ‘pattern III’ MS. ‘Pattern III’ MS is diagnosed on the basis of histopathological features, some of which suggest ischemia-mediated tissue damage, and has only recently been proposed to denote a disease entity distinct from classical MS based on our observation that patients with ‘pattern III’ lesions are typically negative for OCB . While pInterestingly, a recent study reported a loss of aquaporin-4 (AQP4) and other astrocytic proteins in BCS, which in some lesions preceded the loss of myelin, suggesting that BCS might, at least in a subset of cases, be primarily an astrocytopathy, just as AQP4-IgG-positive NMO . HoweverOur study has both strengths and limitations. Among its strengths, we count the very high number of LP examined  and the fact that our results were both highly significant and robust when retested in subgroups with particularly high diagnostic certainty (see the ‘On the other hand, one may count the retrospective nature of our study and the fact that the patients included in the analysis were seen at many different centres among the potential limitations. However, it is virtually impossible to perform a large prospective single-centre study on BCS given the condition’s very low prevalence. Moreover, the study’s retrospective ‘multicentre’ setting reduced potential risks resulting from centre-specific selection bias, a problem inherent to single-centre studies. Second, no CSF results were given in numerous reports on BCS in the previous literature. This might have introduced a bias towards cases with pathological CSF findings. However, this would imply that our results actually underestimate the observed lack of OCB, IgG index elevation and WCC pleocytosis in BCS and would thus make our findings even more relevant.The present study focused on immunological parameters routinely tested in patients with MS and BCS. Further immunological studies aiming at investigating the differences between BCS and MS in terms of immunopathogenesis in greater detail are now required.It is reassuring that our results are in agreement with a previous much smaller study performed on behalf of the MAGNIMS network . FollowiIn summary, our study demonstrates significant differences in CSF findings between patients with BCS and patients with MS, especially in terms of intrathecal IgG synthesis. This suggests differences in immunopathogenesis between BCS and MS and supports the notion of Baló-like lesions denoting the presence of a disorder immunologically distinct from MS in most cases. This could have clinically important diagnostic, nosological, prognostic and therapeutic implications. Of note, the OCB frequency in BCS is more similar to that reported in NMOSD and MOG-EM than in classic MS Fig. . Further