Much valuable information is embedded in social media posts (microposts) which are contributed by a great variety of persons about subjects that of interest to others. The automated utilization of this information is challenging due to the overwhelming quantity of posts and the distributed nature of the information related to subjects across several posts. Numerous approaches have been proposed to detect topics from collections of microposts, where the topics are represented by lists of terms such as words, phrases, or word embeddings. Such topics are used in tasks like classification and recommendations. The interpretation of topics is considered a separate task in such methods, albeit they are becoming increasingly human-interpretable. This work proposes an approach for identifying machine-interpretable topics of collective interest. We define topics as a set of related elements that are associated by having posted in the same contexts. To represent topics, we introduce an ontology specified according to the W3C recommended standards. The elements of the topics are identified via linking entities to resources published on Linked Open Data (LOD). Such representation enables processing topics to provide insights that go beyond what is explicitly expressed in the microposts. The feasibility of the proposed approach is examined by generating topics from more than one million tweets collected from Twitter during various events. The utility of these topics is demonstrated with a variety of topic-related tasks along with a comparison of the effort required to perform the same tasks with words-list-based representations. Manual evaluation of randomly selected 36 sets of topics yielded 81.0% and 93.3% for the precision and F1 scores respectively. Microblogging systems are widely used for sharing short messages (microposts) with online audiences. They are designed to support the creation of posts with minimal effort, which has resulted in a vast stream of posts relating to issues of current relevance such as politics, product releases, entertainment, sports, conferences, and natural disasters. Twitter , the moslda)  ← entities(p) ∪ temporalExpressions(p)11:  unlinkedSpots[p] ← unlinkedSpots(p)12:  end for13: for eachpin P do14: le[p]← reLink15:  16:  # Add the newly linked entitiesle[p] ← le[p] ⋃ linkSpots17:  end for18: Phase 2—Identify topics  G ← relate(le)    ⊳ construct co-occurrence graph19: G′ ← prune    ⊳ prune the graph using τe20: gt ← subgraphsOfRelatedNodes    ⊳ sub-graphs of related nodes of G’21: observationInterval ← getObservationInterval(P)    ⊳ timestamps of the earliest and latest posts22: for eachvinG′ do23: types[v] ← getType    ⊳ determine the type of element (v)24:  end for25: Phase 3—Represent topics with Topico  for eachtopicingtdo26: sTopic ← sem-topic    ⊳ represent as s-boun-ti topic27:  T.add(sTopic)    ⊳ Add to topics for collection P28:  end for29: returnT30: P is a set of posts. The function entities(p) returns the entities within a post p. We denote a post and its corresponding entities as 〈p, l〉 where l are linked entities. Determining the candidate elements entails the use of an entity linker that links elements of microposts to external resources and a rule-based temporal term linker. We defined temporal term linking rules  ↣ [uri], where all the spots that are linked to an entity are shown as a list of lowercase terms and the entities are shown as uris. For example,  ↣ [dbr:North_Dakota] indicates the two spots north dakota and n. dakota that are linked to dbr:North_Dakota where some posts refer to the state North Dakota with its full name and others have abbreviated the word north as “n.”. The entity linking process may yield alternatively linked spots or unlinked spots. For example, the spot “Clinton” may be linked to any of dbr:Hillary_Clinton, dbr:Bill_Clinton, dbr:Clinton_Foundation or not at all (unlinked spot). Such candidate topic elements may be improved by examining the use of patterns within the collective information for agreement among various posters. Thus, the linked entities retrieved from all the posts are used to improve the candidate elements attempting to link previously unlinked spots or altering the linking of a previously linked spot (Lines 14-18). In our prototype, entities are retrieved using TagMe which links spots to Wikipedia pages. We map these entities to DBpedia resources which are suitable for the semantic utilization goals of our approach (see the The first phase determines the candidate topic elements that are extracted from each post (Lines 10-13). ng rules  to detec see the  section.The second phase decides which elements belong to which topics. We consider the limited size of microposts to be significant when relating elements since the user chose to refer to them in the same post. The more often a co-occurrence is encountered the more significant that relation is considered since the aim is to capture what is of collective interest. In this work, the term co-occurring elements/entities is defined to be the co-occurrence of the spots within a post to which these elements are linked.LEP = {〈p, l〉| p ∈ P∧ l = entities(p)}. Let the co-occurrence graph G =  where E = {|vi, vj ∈ V∧vi ≠ vj∧{vi, vj}⊆l∧〈p, l〉∈LEP}.To identify topics, we construct a co-occurrence graph of the candidate topic elements. Let Let dbr:Donald_Trump, dbr:Lester_Holt, dbr:Social_Profiling, dbr:Terry_Stop, dbr:Constitutionality. Within four posts, the co-occurrence between some of these entities ranged between 0.25 to 0.75. Thus, we have extracted a significantly rich set of information from the posts in terms of relating them to web resources which themselves are related to other resrouces via data and object properties.w(e)<τe) are removed. All vertices that become disconnected due to edge removal are also removed. The following equations describe how G =  is pruned to obtain the final co-occurrence graph G′ = :To represent collective topics (those of interest to many people) the weak elements are eliminated prior to identifying the topics (Line 20). The weak edges . The co-occurrence graph G′ is processed to yield sets of related topic elements, each of which will represent a topic (Line 21). The criteria for determining the topics (sub-graphs of G′) are (1) an element may belong to several topics since it may be related to many topics, (2) topics with more elements are preferable as they are likely to convey richer information, (3) topics with few elements are relevant if their relationships are strong .G = , τsc threshold: v ∈ V and freq(v) = |{〈p, l〉|v ∈ l∧〈p, l〉∈LEP}|. The maximal cliques algorithm  is used P (Line 22). Since our s-boun-ti topics represent persons, locations, and temporal expressions the entity types of elements are resolved (Lines 23-25). The type of temporal expressions is determined while they are being extracted. The types of other elements are identified with semantic queries. For example, if the value of the rdf:type property includes foaf:Person or dbo:Person its type is considered to be a person. To determine if an entity is a location, first, the value of rdf:type is checked for a location indicator . Locations are quite challenging as they may be ambiguous and be used in many different manners. Then, the contexts of the spots corresponding to entities are inspected for location indicators  within the post-collection. Again, we employ a threshold (τloc) to eliminate weak elements of location type. Finally, an entity v is considered a location if FBI in “FBI reports to both the Attorney General and the Director of National Intelligence.” is considered as an agent, whereas in “I’m at FBI” it is considered as a location.After the elements of the topics are determined, additional information necessary to represent the final topics is obtained. The observation interval is computed using the posts with the earliest and the latest timestamps in G′ (topics) along with the types of topic elements. The final phase is to represent these topics with the Topico ontology. Each t ∈ T is mapped to an instance of topico:Topic (Lines 26-29). The topics are related with their elements in accordance in accordance with their types. For example, elements of type person are associated with a topic with the topico:hasPerson property. The property topico:isAbout is used for all elements of type other than the types of person, location, and temporal expression. The observation intervals are associated with the topico:observationInterval property. The instantiated topics are referred to as s-boun-ti topics and are ready for semantic processing.At this stage, we have the sub-graphs of τc for topic similarity and T0 be the set of cliques (T0 ⊆ P(V′)). The set of merged cliques, T, is obtained by:tk = ti ∪ tj, ti, tj ∈ t0, vx ∈ ti, vy ∈ tj. Higher values for τc or Note that other graph algorithms could be used to obtain topics. Also, alternative pre- and post-processing steps could be utilized. For example, it may be desirable to eliminate or merge some topics to yield better results. For illustration purposes, let’s consider the consequence of using the maximal-cliques algorithm on pruned graphs. The maximal-cliques algorithm requires all of its elements to be related. The pruning of weak bonds introduces the potential of severing the relations necessary to be identified as a topic. In such cases, very similar topics may emerge, such as those that differ in only a single element. This conflicts with our desire to favor a variety of topics with higher numbers of elements. Not pruning the graph to prevent such cases would unreasonably increase the cost of computation since the original graphs are very large and consist of many weak relations. A post-processing step could be introduced to merge similar topics with the use of two thresholds: api for entity linking suggestions, the DBpedia and Wikidata for fetching semantic resources to be used as topic elements, and the Phirehose Library  dataset was collected to inspect the viability of topics emerging from tweets arriving at the same time but without any query (public stream). Note that the Twitter api imposes rate limits on the number of tweets it returns during heavy use. Although they do not disclose their selection criteria, the tweets are considered to be a representative set.For evaluation purposes t events . The Twipd1,pd2,pd3, and [vp]) are fairly similar. For all datasets, the percentage of unique contributors is generally greater than 70%, which is desirable since our approach aims to capture topics from a collective perspective.s-boun-ti topics are generated from collections of tweets. The debate datasets were segmented into sets of tweets posted within a time interval to capture the temporal nature of topics. Throughout the remainder of this paper, a collection of posts will be denoted as [dsid],  ↣ [dbr:Terry_stop], and  ↣ [dbr:Racial_profiling]. As seen, the topic captures the intended meaning regardless of how it was expressed by the contributors.The viability of our method requires the ability to link posts to linked data, thus the linked entities must be examined. From our datasets, some of the spots and its corresponding topic element that we observe are:  ↣   dataset, which were gathered with query terms the inauguration of Donald Trump as the U.S. President. It so happened that the Women’s March event that was to take place the subsequent day was trending and was related to the inauguration through those who posted tweets during this time. As a result, the topics included people such as Madonna and Michael Moore who were very active in the Women’s March. Besides, the locations London, France and Spain appeared in topics from tweets expressing support for the Women’s March. As a result, the topics that were captured rather accurately reflected what was on the mind of the public during the inauguration.Tweets can be very useful in tracking the impact of certain messaging since people tend to post what is on their mind very freely. Considering political campaigns, much effort is expended on deciding their talking points and how to deliver them. The ability to track the impact of such choices is important since it is not easily observable from the televised event or its transcripts. To give an example, during the 86pub]) would yield topics, which we expected they would not since there would not be sufficient alignment among posts. Indeed no topics were generated, however, some entities were identified. We speculate that in public datasets collected during major events, such as earthquakes and terrorist attacks, the strength of ties could be strong enough to yield topics, although this must be verified.Finally, we examined whether public streams  in terms of the effort required to perform various topic related tasks. The effort is described with the use of the helper functions shown in s-boun-ti topics and what would need to be done if wlb topics were used. For readability purposes, the queries are described in natural language. The sparql queries may be found in the supporting material.The main purpose of this work is to produce topics that lend themselves to semantic processing. To demonstrate the utility of Task 1: Who occurs how many times in the topics related to Hillary Clinton:This is a simple task that can be achieved by querying the topics for persons who co-occur with Hillary Clinton (and counting them) as shown in Listing 1.Listing 1 Query: Who occurs how many times in the topics related to Hillary Clinton?SELECT ?person (COUNT(?topic) AS ?C)WHERE { ?topic topico:hasPerson dbr:Hillary_Clinton;  topico:hasPerson ?person. FILTER (?person NOT IN (dbr:Hillary_Clinton))}GROUP BY ?personORDER BY DESC(?C)The first three results (out of 41) are:Person        Count  dbr:Donald_Trump   "2205"^^xsd:integerdbr:Bill_Clinton    "338" ^^xsd:integerdbr:Tim_Kaine      "314"^^xsd:integers-boun-ti topics represent persons (with topico:hasPerson) retrieving those who co-occur with Hillary Clinton is trivial.Other persons include Barack Obama, Ruth Bader Ginsburg, Al Gore, Ronald Reagan, Anderson Cooper, Abraham Lincoln, and George Washington. Since wlb representations, it is necessary to determine the words that represent the person Hillary Clinton, other people, and count them. Thus, a type-resolution (tr) task to identify persons is needed which requires entity identification (ei) using an external resource (ex) with information about people.To achieve the same results with Task 2—When do the topics related to women’s issues occur?This type of query is useful to know when certain topics emerge and to track whether they trend, persist, or diminish within streaming content. The time of observation of subjects is significant since they tend to rapidly change on social media.abortion, rape, and women’s health. Retrieving the time intervals is straightforward, since the topico:observationInterval property captures this information. An inspection of the linked entities revealed that the posters used different terms related to these concepts, such as  ↣ [dbr:Rape].The query in Listing 2 identified 166 topics in 66 time intervals related to topics about Listing 2 Query: When do the topics related to women’s issues occur?SELECT DISTINCT ?startTime ?endTime WHERE { ?topic topico:observationInterval ?interval. ?interval time:hasBeginning ?begin. ?interval time:hasEnd ?end. ?begin time:inXSDDateTime ?startTime. ?end time:inXSDDateTime ?endTime. {?topic topico:isAbout dbr:Rape.} UNION {?topic topico:isAbout dbr:Abortion.} UNION {?topic topico:isAbout dbr:Women\’s_health.}}wlb representations, the terms related to the women’s issues and the time intervals (ti) corresponding to the time they appeared must be determined.To achieve the same results with Task 3—When do the top 50 issues related to topics including Hillary Clinton and/or Donald Trump appear?This type of query is relevant for tracking how a particular messaging resonates with the public, such as for political and marketing campaigns that involve immense preparation.topico:isAbout) in the topics related to dbr:Donald_Trump or dbr:Hillary_Clinton are retrieved. Then, when these issues occurred is determined. This query returned 3061 results, among which:The query in Listing 3 retrieves the top 50 issues (topic elements) associated with the topics including Donald Trump and/or Hillary Clinton and when they were observed. To do this, first, the top 50 issues . Hillary Clinton and/or Donald Trump for each two-minute interval during the 90-minute long debates .that indicates that the issue of Listing 3 Query: When do the top 50 issues related to topics including Hillary Clinton and/or Donald Trump appear?SELECT ?time ?issueOfInterest ?person {http://193.140.196.97:3030/topic/sparql>{SERVICE < SELECT ?issueOfInterest (COUNT(?topic) AS ?C) WHERE {  ?issueOfInterest topico:inTopic ?topic.  {dbr:Hillary_Clinton topico:isAPersonOf ?topic}  UNION  {dbr:Donald_Trump topico:isAPersonOf ?topic}}  GROUP BY ?issueOfInterest  ORDER BY DESC(?C) LIMIT 50}http://193.140.196.97:3030/topic/sparql>{SERVICE < SELECT ?time ?about ?person WHERE {  ?topic topico:hasPerson ?person.  ?topic topico:isAbout ?about.  ?topic topico:observationInterval ?interval.  ?interval time:hasBeginning ?intervalStart.  ?intervalStart time:inXSDDateTime ?time.  FILTER)} GROUP BY ?time ?about ?person}FILTER (?about=?issueOfInterest)}White_people and Black_people in Tax and Donald Trump only  and pd3 that are used for generating s-boun-ti topics . Topics were generated for alternative values of lda parameters for the expected number of topics: N = 2 − 10, 20, 30, 40, 50.Latent Dirichlet allocation (itterlda  with thes-boun-ti and lda are very different where lda topics capture terms expressed by contributors as words-list-based (wlb) topics, s-boun-ti topics map original content to instances in lod which are expressed with lod resources and the owl language [s-boun-ti a set of alternative words that are contributed may be mapped to the same semantic entity, capturing the intended meaning rather than how it was articulated.The topic representations of language . With s-rdfs:label) of the s-boun-ti topic elements. The union of the lowercase form of words in the labels of all elements is compared using Jaccard similarity with topmost 10 terms of lda topics (according to their distributions). We observed that there are cases that lda and s-boun-ti topic elements are the same but not matching due to some syntactic difference. For example, if an lda topic element is “emails”, and s-boun-ti topic element is dbr:Email, the strings “emails” and “email” do not match which results in lower similarity scores. To address similar issues, we assumed that the cases that one term is a substring of the other are matching. Each s-boun-ti topic is element-wise compared with lda topics that are generated for the same input set.To get a rough idea about the similarity of topics, we utilized the label , which could be very useful for those  who are following information from social media.To assess the proposed approach, lod, which have been improving in terms of quantity and quality during the span of this work, a most encouraging prospect. Potential improvements are elaborated in the following section.The proposed approach is a straightforward one aimed at gaining a basic understanding of the feasibility of mapping sets of tweets to semantically-related entities. If possible, this would facilitate a vast number of applications that harvest the richly connected web of data. Our observations lead us to believe that this is possible. Furthermore, this approach would improve by enhancing the techniques used to identify and relate topic elements, refining the topic representation, and with the increasing quality of data on lod to produce relevant machine-interpretable topics. We specifically focused on subjects of significant interest from a collective perspective. Topics of general interest lead to vast numbers of microposts. We generated semantic topics from a variety of tweets collections and represented them with an ontology that we developed for this purpose. The semantic topics were subjected to various tasks to examine their utility. The results show that relevant topics were identified for a diverse set of subjects. In the Experiments and results section, we presented the semantic topics generated from collections of tweets with emphasis on a complete set collected during the four major debates of U.S.elections . The utility of the resulting topics  was demonstrated through various tasks that facilitated the understanding of the issues relevant to the debate watchers, such as the persons, the locations, the temporal and other aspects of interest. Furthermore, issues at higher conceptual levels such as violence, ethnicities, and religions were revealed.In this section, we discuss some of our observations regarding the approach we proposed and present some future directions. The main objective of this work was to examine the feasibility of linking informal, noisy, and distributed micropost content to semantic resources in covid-19) that is widely reflected on social media. A preliminary exploration of topics generated from collections of tweets related to covid-19 also yielded relevant topics. In this case collections of tweets posted during the same time for 53 consecutive days were inspected to get a general sense of the issues of relevance. There were 140 people, 32 locations, 46 temporal expressions, 1097 issues that distinctly occurred in the topics. Among the occupations of people are politicians , journalists, singers, and athletes. The locations were dominated by China, Wuhan, Italy. While many locations were across the globe  others were regions within the U.S. . This is reasonable since the time intervals of the tweets correspond to midday in the United States and covid-19 cases were spiking in various parts of the country. There were also numerous temporal references, the most frequent ones being now, today, tonight, the months of January through May. The occurrence and the frequency of the specific temporal terms are significantly different from those encountered in the debate related sets, which did not have such a diverse set of temporal expressions . Such differences capture the nature of contributions where the temporal aspect of the pandemic is indeed of much more significance due to the interest in how fast the rate of cases change and speculation about when things would improve. The resulting topics were processed to see when various issues emerged. about topic elements (topico:isAbout) were observed daily. Upon observing the references to drugs, we checked if other drugs were also referenced simply by querying DBpedia if the element type is dbo:Drug. This identified the other drugs referenced in tweets as: BCG vaccine, Cocaine, Doxycycline, Favipiravir, Generic drug, Pharmaceutical drug, Polio vaccine, Ibuprofen, Paracetamol, Chloroquine, Azithromycin, Antiviral drug, Hydroxychloroquine. The most frequently referenced one was Hydroxychloroquine—a drug mentioned by the president of the United States several times. Obviously, tweets from such small intervals are insufficient to inspect such a vast issue. A more comprehensive examination with collections that covers all time zones would be required. Nevertheless, even with this small set, it is evident that this approach produces relevant topics.In our experiments, we observed that our approach produces relevant topics for diverse contexts. The topics of an entirely different context can be observed in a subject that is of great interest during the final preparations of this article, namely the coronavirus 2 (SARS-CoV-2) pandemic . In this work, we eliminate unlinked spots, which could be particularly problematic for spots with high frequencies since that indicates common interest. To alleviate this matter, such spots could be linked to an instance of owl:Thing indicating that there is some thing of significance whose type is unknown.Since semantic topics consist of topic elements, correctly identifying them is important. Here, the main challenges are the inability to link to anything at all and incorrect linking. Obviously, entity linking fails when suitable entities are not represented on d on lod  , cure (music band The Cure), and WHO (television character Dr. Who). We encountered such cases in our experiments, albeit not frequently, since the entity linker typically assigns low confidence rates for such links. Furthermore, our approach eliminates the links to entities that occur infrequently (see the Identifying topics section) in a collaborative filtering manner.A more troublesome issue stems from ambiguous terms, which is most prevalent in person names. For example, the spot Recently, word-embedding techniques that capture semantic similarity among terms are being applied to named entity recognition (NER) and disambiguation , 116, anIn our experiments, we focused on English tweets and named entity recognition so that we could interpret the results. Several tools work for other languages (including TagMe that we used in our prototype). Furthermore, the natural language processing community strongly emphasizes work on low resource languages, which is resulting in additional knowledge resources and tools. The goal is to work with multiple languages, which link to the same conceptual entity. Thereby, being able to glean information regarding content that is globally produced. This is important for many tasks of global interest, such as pandemic diseases, disasters, news, entertainment, and learning material.k-cliques  constrained by type rules could yield richer topics. However, caution must be exercised, since the volume of microposts and their limited context is likely to yield many potential yet unrelated candidates for k > 1, which is computationally challenging and costly. Note that the goal is not to simply increase the number of topics or their elements since we are aiming to reduce large sets of tweets to higher-level topics. Rather, the aim to increase the quality of topics by associating related elements.In this work, we chose maximal cliques to identify the topics so as to assure that all the elements are related by virtue of having been posted together. The co-occurrence graphs from which topics are extracted have relatively few nodes with high degree centrality  with the remaining node being relatively weak see . Thus, sapi and our computational resources. For the debates, this corresponded to the tweets posted withing 2-minute intervals. During heavy posting conditions the subjects change frequently and short windows are suitable since the topics change and the number of tweets to detect collective interest is sufficient. During slower posting conditions the subjects don’t change as fast, thus sets collected over longer durations are appropriate. Dynamically varying collection durations based on how frequently the subjects of topics change over time would be valuable. Topico is capable of representing such intervals, however, they must be determined through time series analysis.The size of the post collections we used was limited by the rate limits of the Twitter streaming Topico specifies an elementary set of topic element types, namely person, location, temporal expressions, and other entities  as well as refine existing types  [Experiments and results illustrate the kinds of topic-related tasks that could be of interest to campaign managers, journalists, and political enthusiasts. market) . While cExperiments and results section. The vision is to deliver this power to an end-user who is following the rapidly flowing distributed microposts. Towards this end, higher-level tasks should be defined such as similarity, sentiment analysis, and recommendations. Tracking topics, such as when they emerge, if they persist, if and when they spike, and if they exhibit some pattern is useful information. Reports that provide statistical information will enable those who are interested in the topics to take action. Other interesting tasks are tracking the evolution of and predicting topics. One of the future directions is an explorer for s-boun-ti topics which requires the generation of human-interpretable topics. Using such an explorer users could search and browse topics, view ranked topics, graphs, and charts that provide relational and temporal information (trends), view social network analysis. They must be able to view the results of the processing of semantic topics, which may be predefined domain-specific. Such a system should recommend topics and topic related observations such as trends, newly emerging issues. Furthermore, multimedia presentations that depict the lifecycles of topics persist over a given time can be generated with dynamic summarization techniques [The purpose of focusing on semantic topics is for their semantic processing potentials. We demonstrated how semantic topics can be utilized through various semantic tasks in the chniques , 121.Eventually, a tool that is customizable with domain-specific knowledge resources for detecting and processing topics. The specific nature of the subject and desired processing will vary depending on the context. A domain-specific topic detection system customized for diseases with knowledge bases like the International Classification of Diseases (ICD)  for disefederated queries that search across distributed resources. Unfortunately, the performance of federated queries can be quite inefficient. The order in which queries are executed must be carefully designed to achieve reasonable response times. Generally, electing to execute the more restrictive queries prior to others which restricts the search space is considered a good approach. Finally, generating streams of semantic topics could be facilitated with stream reasoning [c-sparql [c-sprite [One of the most interesting potentials of semantic resources is revealed via easoning  and querc-sparql  and c-spc-sprite .lod resources. To this end, an ontology (Topico) to represent topics is designed, an approach to extracting topics from sets of microposts is proposed, a prototype of this approach is implemented, and topics are generated from large sets of posts from Twitter.This work investigates the viability of extracting semantic topics from collections of microposts via processing their corresponding linked entities that are lod resources could be utilized in generating semantically represented and machine-interpretable topics. The proposed approach extracts a significantly rich set of information from the posts in terms of relating them to web-resources which themselves are related to other resources via data and object properties. We demonstrate the benefits of using lod and ontologies while identifying as well as utilizing the topics. During the identification phase, we were able to identify candidate elements and resolve their types. The ontologically represented topics consisting of entities enabled processing opportunities that revealed information about collections of microposts that are not readily observable even if each post were to be manually inspected. Also, we notice an increase in the quality of the generated topics over time thanks to the efforts related to the continued expansion and correction of lod resources.The main inquiry of this work was to examine whether an approach based on linking microposts to F1 of 93.3%) and regularly performed manual inspections show that the identified topics are relevant. In summary, we are encouraged by the results we obtained and list many research opportunities to improve the topic identification approach and process topics in general and in domain-specific manners.Our main goal of producing machine-interpretable topics was for their utilization in further processing. We demonstrated such utilization with several examples of various levels of complexity, where information that is not readily available in the original posts is revealed. A user evaluation (with 81.0% precision and S1 Appendix(PDF)Click here for additional data file.S1 FigSee tweets and See word cloud links show the related tweets and a word cloud generated from them. The Entity frequencies link shows the list of linked entities and their frequencies. All resources are reachable trough web-links for inspection.The (PNG)Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S1 Table(PNG)Click here for additional data file.S2 TableBefore and Pruned show the impact of pruning the graph. The columns labeled Topic show how many were retained in the topic.This table shows the percentage of tweets in the post sets that produce the vertices (topic elements), edges (co-occurring elements), and topics. The columns labeled (PNG)Click here for additional data file.S3 Table(PNG)Click here for additional data file.


Striatal tonic dopamine increases rapidly during global cerebral hypoxia. This phenomenon has previously been studied using microdialysis techniques which have relatively poor spatio-temporal resolution. In this study, we measured changes in tonic dopamine during hypoxia (death) in real time with high spatio-temporal resolution using novel multiple cyclic square wave voltammetry (MCSWV) and conventional fast scan cyclic voltammetry (FSCV) techniques.MCSWV and FSCV were used to measure dopamine release at baseline and during hypoxia induced by euthanasia, with and without prior alpha-methyl-p-tyrosine (AMPT) treatment, in urethane anesthetized male Sprague-Dawley rats.n = 5; mean ± SEM). Following intracardiac urethane injection, the tonic levels increased to a peak concentration of 1753.8 ± 95.7 nM within 3.6 ± 0.6 min (n = 5), followed by a decline to 50.7 ± 21.5 nM (n = 4) at 20 min. AMPT pre-treatment significantly reduced this dopamine peak to 677.9 ± 185.7 nM (n = 3). FSCV showed a significantly higher (p = 0.0079) peak dopamine release of 6430.4 ± 1805.7 nM (n = 5) during euthanasia-induced cerebral hypoxia.Baseline tonic dopamine levels were found to be 274.1 ± 49.4 nM (in vivo during hypoxia. We found a 6-fold increase in peak dopamine levels during hypoxia which was attenuated with AMPT pre-treatment. These changes are much lower compared to those found with microdialysis. This could be due to improved estimation of baseline tonic dopamine with MCSWV. Higher dopamine response measured with FSCV could be due to an increased oxidation current from electroactive interferents.MCSWV is a novel tool to study rapid changes in tonic dopamine release  Transient hypoxic ischemia or hypoxic ischemia following death results in massive striatal dopamine release within minutes . Hypoxicin vivo microdialysis and voltammetry  dopamine levels with a high spatio-temporal resolution and displays improved selectivity over conventional background subtraction voltammetry techniques, such as fast scan cyclic voltammetry (FSCV). In this study we used MCSWV to measure the rapid changes in tonic dopamine levels following cardio-pulmonary arrest with intracardiac urethane injection. We also present a case of dopaminergic changes during spontaneous intraoperative hypoxia and subsequent resuscitation in an anesthetized animal.Recently, we have described a novel voltammetry technique called multiple-cyclic square wave voltammetry (MCSWV)  to examiad libitum. Animals from two studies, primarily designated to investigate the mechanism of deep brain stimulation are included in this report. These studies were approved by the Institutional Animal Care and Use Committee (IACUC), Mayo Clinic, Rochester. The NIH Guide for the Care and Use of Laboratory Animals guidelines  were followed for all aspects of animal care. Here, we focus on the observations made upon euthanizing the rodents at the end of these studies.Male Sprague Dawley rats  weighing between 250 and 360 grams (age 8–13 weeks) were used for this study. They were housed in pairs, and maintained in standard size cages with a 12-h light-dark cycle (lights on at 0600 h) in an AAALAC accredited vivarium . Food and water were available in vivo biofouling (Carbon fiber microelectrodes (CFMs) were prepared using standard methods described previously . T300 caofouling .The animals were anesthetized with urethane  and administered buprenorphine  for analgesia. Following anesthesia, they were immobilized on a stereotactic frame  with a rat incisor bar set at −3.3 mm DV. The skin overlying the animal’s head was shaved and an incision was made to expose the skull. A trephine bit was used to drill holes in the skull for placement of the CFM and stimulating electrodes. The CFM was placed in the dorsal striatum  . ConcentThe animals were divided into two groups as described below. This report discusses the changes in dopamine levels observed in the immediate period after euthanizing the animals with intracardiac (IC) injection of 1 mL urethane (280 mg/mL). The depth of anesthesia was assessed using the toe pinch maneuver to elicit reflex withdrawal of the limb to pain before administering euthanasia. Absence of the withdrawal reflex indicated deep anesthesia. This helped minimize distress to the animal from IC injection.Group 1: CFMs were implanted into the dorsal striatum and FSCV was performed to measure phasic dopamine release in response to electrical stimulation. Dopamine release during global cerebral hypoxia was recorded for several minutes after euthanasia. A triangular waveform with a holding potential of -0.4 V, peak potential of 1.3 V, scan rate of 400 V/s and a scanning frequency of 10 Hz was used for FSCV. The recordings were performed with WINCS Harmoni device and controlled using WincsWare in Harmoni software  . After eStaircase, EInitial (= EEnd) and τ at 25 mV, −200 mV, and 1.0 ms, respectively. The technique was implemented using a commercial electronic interface  with a base-station PC and in-house software written in LabVIEW 2016 . Data was processed using MATLAB . Dopamine levels were determined by calibration of CFMs with 1 μM dopamine solution after the experiment. Quadratic regression described previously was used to determine the tonic dopamine concentration , a catecholamine synthesis inhibitor (which acts via competitive antagonism of the enzyme tyrosine hydroxylase), was administered at a dose of 250 mg/kg i.p. at 90–120 min before IC urethane injection. For clarity, group 3 will be referred to as AMPT group and group 2 as non-AMPT group further in the manuscript.Non-parametric Mann-Whitney test was used to compare the means between following data sets – (a) peak dopamine release following euthanasia-induced cerebral hypoxia measured with FSCV vs. MCSWV (group 1 vs. group 2), and (b) peak dopamine release between animals treated vs. those not treated with AMPT (group 2 vs. group 3).Immediately following IC urethane injection there was rapid cessation in respiration and cardiac activity. n = 5 rats) was observed in this series. Five animals were used to determine the peak dopamine response with death. However, the recording was discontinued shortly after the dopamine peak in one of the animals. Therefore, data from only four animals with at least 5 min of recording post-dopamine peak was used to construct the figure. A peak dopamine release of 6430.4 ± 1805.7 nM dopamine with a range of 2523.1–12942.5 nM (n = 5 rats). Initial) to + 900 mV (Epeak) and back to –200 mV (EEnd) during a cycle lasting 90 ms. Five such cycles occur consecutively and the final voltammogram is obtained by subtracting the results of the 2nd cycle from 5th cycle. The third dimension of the pseudo-color plot is the oxidation current, visualized with a color-scale gradient. The fourth dimension of the pseudo-color plot is time which is captured in a video film. The oxidation peaks are seen in red while the reduction peaks are visualized in blue. Two peaks each for oxidation and reduction are seen with MCSWV. The oxidation and reduction peak on the left occur during ramp up of the staircase potential. The peaks on the right occur during ramp down of staircase potential. The oxidation peak on the left was found to be most sensitive for detecting the dopamine signal as it had the highest current value. This peak was used for determining dopamine concentrations.n = 5 rats). The post-mortem dopamine release measured with MCSWV was significantly lower than that measured with FSCV (p = 0.0079). The pseudo-color plot in n = 5 rats). At 15 min post-injection, dopamine levels were found to be 204.2 ± 72.2 nM (n = 4 rats), dropping to 50.7 ± 21.5 nM (n = 4) at 20 min. n = 3), compared to animals who were not treated (p = 0.035) as seen in Following IC urethane injection, the tonic concentration of dopamine rapidly increased to a mean peak value of 1753.8 ± 95.7 nM . Following cardiac injection of urethane, tonic levels of dopamine peaked at 1753.8 ± 95.7 nM (n = 5 rats), showing an average 6-fold increase over baseline  and temporal resolution (≈10 s) using MCSWV. While this phenomenon has been extensively reported in the literature, to the best of our knowledge this is the first report describing real-time baseline . Previoubaseline . Microdibaseline . It has baseline . Upon deFollowing euthanasia, the rise in tonic dopamine levels is very rapid taking on average 3.6 min to reach a peak concentration from baseline. This is in line with two previous studies which have reported a 5–6 min period for dopamine levels to reach a peak following euthanasia . Ahn et Dopamine within dopaminergic neurons exists in readily releasable and reserve storage pools. These pools are differently sensitive to various pharmacological manipulations. We used AMPT to deplete dopamine in the newly synthesized, readily releasable pool . FollowiGiven the well-established evidence for the sensitivity of dopaminergic neurons to hypoxia and previous studies demonstrating this phenomenon in both a hypoxia model, as well as with death, strongly suggest hypoxia as the likely explanation for massive dopamine release observed in our study . Three pWe observed a sharp spontaneous rise in dopamine in one of our animals coinciding with an episode of a decrease in respiration rate and labored breathing . After tThe phenomenon of massive dopamine release in hypoxia followed by resumption of tonic levels drew our attention to a little discussed clinical and forensic problem, namely auto-erotic asphyxiation (AEA) behaviors . IncreasDopamine release in hypoxia is known to have neurotoxic effects on dopaminergic, as well as other striatal neurons . Animal In conclusion, we have demonstrated MCSWV as a novel tool to study real-time rapid changes in tonic dopamine dynamics during hypoxia. This conclusion is supported by the observations we have made during euthanasia-induced hypoxia, which are in line with previous studies utilizing microdialysis and FSCV. We found that following euthanasia with IC urethane injection, there is a 6-fold rise in tonic dopamine concentrations which declines to less than 20% of the baseline concentration at 20 min. This post-mortem dopamine release is reduced by depletion of newly synthesized, readily releasable dopamine pool using AMPT. The high spatial and temporal resolution of this technique makes it an ideal tool to investigate dopaminergic release dynamics involved in the mechanism of deep brain stimulation, drugs of abuse, motivated behaviors, and pharmacological agents targeting dopaminergic systems.All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Mayo Clinic, Rochester.KL, YO, CB, and DJ conceptualized the study. AB, AR, JR, and JP conducted the experiments and collected the data. AB, YO, DJ, and HS designed the analyses. AB conducted the analyses. AB, AR, KL, CB, YO, and HS wrote the manuscript. KL and YO supervised all aspects of this work. AB and RO drafted the figures. All authors commented on and accepted the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molecular diagnostic testing of the 11p15.5-associated imprinting disorders Silver-Russell and Beckwith-Wiedemann syndrome  is challenging due to the broad spectrum of molecular defects and their mosaic occurrence. Additionally, the decision on the molecular testing algorithm is hindered by their clinical heterogeneity. However, the precise identification of the type of defect is often a prerequisite for the clinical management and genetic counselling. Four major molecular alterations  have been identified, but their frequencies vary between SRS and BWS. Due to their molecular aetiology, epimutations in both disorders as well as upd(11)pat in BWS are particular prone to mosaicism which might additionally complicate the interpretation of testing results. We report on our experience of molecular analysis in a total cohort of 1448 patients referred for diagnostic testing of BWS and SRS, comprising a dataset from 737 new patients and from 711 cases from a recent study. Though the majority of positively tested patients showed the expected molecular results, we identified a considerable number of clinically unexpected molecular alterations as well as not yet reported changes and discrepant mosaic distributions. Additionally, the rate of multilocus imprinting disturbances among the patients with epimutations and uniparental diploidies could be further specified. Altogether, these cases show that comprehensive testing strategies have to be applied in diagnostic testing of SRS and BWS. The precise molecular diagnosis is required as the basis for a targeted management (e.g. ECG (electrocardiogram) and tumour surveillance in BWS, growth treatment in SRS). The molecular diagnosis furthermore provides the basis for genetic counselling. However, it has to be considered that recurrence risk calculation is determined by the phenotypic consequences of each molecular alteration and mechanism by which the alteration arose.The detection rates for the typical molecular defects of Beckwith-Wiedemann syndrome or Silver-Russell syndrome  are lower in routine cohorts than in clinically well-characterised ones.A broad spectrum of (unexpected) molecular alterations in both disorders can be identified.Multilocus imprinting disturbances (MLID) are less frequent in SRS than expected.The frequency of MLID and uniparental diploidy in BWS is confirmed.Mosaicism is a diagnostic challenge in BWS and SRS.The precise determination of the molecular defects affecting is the basis for a targeted clinical management and genetic counselling. H19/IGF2:TSS-DMR) is paternally methylated and regulates the expression of the maternally expressed H19 gene and the paternally expressed IGF2 gene. In contrast, the IC2 (KCNQ1OT1:TSS-DMR) is maternally methylated, resulting in the expression of the KCNQ1OT1 gene from the paternal and of CDKN1C from the maternal allele. Disturbances of both regions alter human growth and are associated with two imprinting disorders, Beckwith-Wiedemann and Silver-Russell syndromes . The characteristic feature of BWS is overgrowth; additionally, patients often exhibit body asymmetry, macroglossia, and umbilical wall defects  7q11.23q36.3(76550200-159119220) hmz). The 14q32 alteration subgroup (6.6%) comprised three carriers of a upd(14)mat, three patients with epimutations, and one patient in which the discrimination between upd(14)mat and epimutation was not possible.KCNQ1OT1 copy, and two gains of maternal 11p15.5 material affecting both IC1 and IC2. Two further rare findings (upd(6)mat) and a 7q32 deletion affecting the MEST gene were detected as well.An LOM of the IC2 which is typically associated with a BWS phenotype was observed in three cases referred for SRS testing (2.8%). CNVs of or within 11p15.5 was identified in another three cases (2.8%); they comprised a deletion of the paternal n = 103) exhibited a molecular alteration at least in 11p15.5. With 37.9%, LOM of the IC2 was the most frequent finding; additionally, 11.7% of patients with IC2 LOM were found to have MLID. upd(11)pat accounted for 28.2% of patients; in two further cases, a uniparental diploidy was present. IC1 GOM occurred in 12.6% of patients, but in three cases, IC1 LOM corresponding to a molecular diagnosis of SRS was identified. CNVs of or within 11p15.5 were present in 4.9%. The latter comprised a deletion in the IC1 on the maternal allele, and three duplications affecting the paternal IC1 (and IC2) copies.Out of the 235 newly ascertained patients for which BWS testing was requested, 43.8% of patients . He carPrenatal testing for SRS was requested only for 5 pregnancies due to intrauterine growth restriction, and all tests were negative. In the prenatal BWS cohort, two out of 39 samples were positively tested pat). The reason for prenatal testing in these two cases was abnormal ultrasound findings consistent with BWS; further details were not provided.CDKN1C testing was requested after exclusion of the major 11p15.5 alterations, among them were three prenatal cases. After a negative MS MLPA result, a pathogenic variant was identified in a male foetus with a large omphalocele who died during the delivery after premature rupture of the membranes in gestational week (gw) 23 . This frequency of 14q32 disturbances among patients referred for SRS testing could be expected as there is a growing number of reports on the clinical overlap of TS14 and SRS at least in the neonate and early childhood period mat as the major alterations is similar in both cohorts ; it becomes obvious that 14q32 alterations affecting the of cases %. This fof cases %. This fMEG3:TSS-DMR is easily possible as this DMR is already included in the commercially available ME032 assay which also addresses upd(7)mat. The advantage of the use of (multilocus) MS MLPA assays is also obvious from the additional findings we obtained in our cohort  show, tIn BWS, the frequencies of the molecular subgroups corresponded to that of previous studies e.g. , 21, 22), 2221, 2MLID is a shared subgroup among SRS and BWS patients, and it is characterised by an aberrant imprinting of loci in addition to the disease-specific epimutations, LOM of IC1 and IC2, respectively. Whereas the frequency of approximately 25% of MLID among the IC2 LOM patients with BWS phenotype confirms the data from other studies , 25; theAs recent data about the aetiology of MLID show, these patients should be identified as their mothers might carry the so-called maternal effect variants which make them prone for reproductive failures. These maternal effect variants account for up to 50% of MLID cases  and affeIn addition to the broad spectrum of 11p15.5 alterations, the detection of molecular findings which are not expected from the clinical reason for referral is another challenge in the diagnostics of SRS and BWS. As we could show with our new data, both IC2 LOM can be identified in patients referred for SRS testing, and vice versa IC1 LOM for BWS. In fact, different reasons for these at first glance, unexpected findings have been suggested ; among tIn addition to epimutations and uniparental disomy, CNVs also contribute to a significant number of cases (up to 5% in BWS) Table . In geneIn case of UPD detection, analysis referring to familial inheritance is not indicated in case of upd(11p)pat as this mutation nearly always occur de novo, and also for upd(7)mat, familial transmittance is extremely rare and should be discussed only in case of a family history indicating a chromosomal disturbance  .CDKN1C. Variants in this gene account for nearly 5% of patients with BWS, and it is even higher in familial cases, as confirmed in our family with a CDKN1C frameshift variant  and upd(11p15)pat. The mosaic distribution of these alterations can be very tissue-specific; therefore, an inconspicuous testing result in one tissue does not exclude epimutation or upd(11p15)pat in another cell system. To circumvent these false-negative results, the use of different methods and analyses of different tissues might be discussed , 8. HoweThe precise molecular diagnosis is required as the basis for a targeted management of SRS and BWS patients. For SRS, major questions in respect to therapy and prognosis refer to growth hormone treatment, pubertal development, feeding, and neurodevelopment , 35–37. Tumour monitoring is in the focus of clinical management BWS, and a correlation between molecular subgroups and tumour risks is obvious (for review: ). HoweveCDKN1C, and further differential diagnosis genes) helps to delineate the recurrence risk. Familial-inherited CNVs might predispose to different clinical pictures, depending on the nature of the CNV, its gene content, and the sex of the transmitting parent in case of imprinted regions. Finally, the history of the family of SRS and BWS patients should be carefully documented, as congenital dysmorphisms and malformations as well as reproductive failures might help to identify the molecular cause of the disease.In general, the precise definition of the molecular subtype is required for specific genetic counselling. The identification of a monogenetic cause  techniques (e.g. next-generation sequencing–based) will increase the specificity and sensitivity of diagnostic tests and thereby further improve the diagnostic yield and accuracy of the assays. However, the growing number of different molecular alterations resulting in similar phenotypes (e.g. SRS and TS14) puts the definition of the different disorders as clinical entities in questions and requires a discussion on their redefinition.


Jacob is a synapto-nuclear messenger protein that couples NMDAR activity to CREB-dependent gene expression. In this study, we investigated the nuclear distribution of Jacob and report a prominent targeting to the nuclear envelope that requires NMDAR activity and nuclear import. Immunogold electron microscopy and proximity ligation assay combined with STED imaging revealed preferential association of Jacob with the inner nuclear membrane where it directly binds to LaminB1, an intermediate filament and core component of the inner nuclear membrane (INM). The association with the INM is transient; it involves a functional nuclear export signal in Jacob and a canonical CRM1-RanGTP-dependent export mechanism that defines the residing time of the protein at the INM. Taken together, the data suggest a stepwise redistribution of Jacob within the nucleus following nuclear import and prior to nuclear export. Bothesponses –5. In adesponses –9 and scesponses , 11. Theesponses , 5 and oesponses , 13.N-methyl-d-aspartate glutamate receptors (NMDAR) and require an importin-mediated active retrograde transport along microtubules. One of these messengers is Jacob, a protein that encodes and transduces the synaptic and extrasynaptic origin of GluN2B NMDAR signals to the nucleus [Protein transport from synapse to nucleus is an established means for the regulation of gene expression , 15. Sev nucleus , however nucleus , 21, 22.Wistar rats were bred and housed in the animal facility of the Leibniz Institute for Neurobiology, Magdeburg. Experiments were conducted following ethical animal research standards defined by the German Law/European directive and approved by the Landesverwaltungsamt Saxony-Anhalt . The competent authority follows the advice of an official animal welfare committee of the Federal State of Saxony-Anhalt, Germany.For EM experiments, male adult Wistar rats were deeply anaesthetized using a mixture of Ketavet (Parke-Davis) and Domitor (Pfizer) and then transcardially perfused with a solution containing 0.9% NaCl for 1 min followed by 4% paraformaldehyde (PFA) and 0.05 glutaraldehyde in 0.1 M PBS for 20 min. Brains were removed and post-fixed in 4% PFA solution for 20 h. For pre-embedding immunogold labeling, brains were rinsed in 0.1 M PB and freeze-protected with 1 M sucrose in 0.1 M PB. Tissue was frozen at − 40 °C in iso-pentane and 25 µm frontal sections were prepared. For cryo-immunogold labeling, brains were rinsed in 0.1 M PB and sliced at 700 µm using a vibratome. Brain tissue was freeze-protected with 2.3 M sucrose. Samples of cerebral cortex (1 × 2 mm) were mounted on cryo holders and frozen by immersion in liquid nitrogen.2O; Sigma). Development was stopped by several washes in 150 mmol/L aqueous sodium nitrate. After three additional washes in 0.15 mol/L acetate buffer (pH 5.6), silver deposits were stabilized by a modified gold-toning procedure [For immunoelectron microscopy, freely floating sections were rinsed in 0.01 mol/L phosphate buffered saline (PBS) at pH 7.4, treated for 15 min with 1% sodium borohydride in PBS and thoroughly washed in PBS. Thereafter, sections were pre-treated for 30 min in a blocking and permeabilizing solution consisting of 10% normal goat serum , 0.1% saponine, in PBS at 22 °C. Rabbit anti-Jacob antibody (1:1000) was applied for 36 h in PBS containing 10% NGS, 0.1% saponine and 0.1% sodium azide at 4 °C. Sections were then thoroughly rinsed in PBS, pre-treated for 1 h with 0.1% acetylated BSA  and 0.1% Tween 20 in PBS and exposed for another 24 h to the secondary antibody, 1 nm colloidal gold-coupled goat anti-rabbit IgG  diluted 1:50 in the same solution. After several PBS washing steps for at least 2 h, sections were post-fixed for 15 min with 2% glutaraldehyde at 22 °C and then thoroughly rinsed in 150 mmol/L aqueous sodium nitrate. Gold particles with a diameter of 1 nm were silver enhanced for 1–1.5 h using a commercial developer  supplemented with one-third (v/v) gum arabic , hRanT2Rat hippocampal primary neuronal cultures were prepared from E18-E19 as described previously  and keptAnisomycin (7.5 μM), 4-AP (2.5 mM) and leptomycin-B  were purchased from Sigma, D-APV (20 μM) and ifenprodil (5 μM), bicuculline (50 μM) were purchased from Tocris, and NVP-AAM077 (50 nM) was a kind gift from Dr. Auberson (Novartis). Treatments of hippocampal neurons with D-APV, 4-AP/bicuculline were performed in the presence of anisomycin. Cultures were fixed 30 min after D-APV, 4-AP/bicuculline or leptomycin-B treatment. Ifenprodil and NVP-AAM077 were applied for 28 h and all neurons were immunostained against Jacob and co-stained with anti-MAP2 antibodies. Hoechst or DAPI were used as a nuclear counterstain. Jacob immunoreactivity was measured as mean grey value in arbitrary units of pixel intensity using the Image J software and normalized to untreated conditions.Custom-made, affinity-purified antibodies generated against two short amino acid stretches of rat Jacob  have been described previously , 22. The4Cl for 1 h at RT. Incubation with primary antibodies was done in a blocking buffer at 4 °C overnight. Secondary antibodies were diluted in a blocking buffer 1:500 and applied for 1 h at RT. After washing in PBS, either DAPI or Hoechst was added for 10 min and coverslips were then rinsed in water and mounted with Mowiol 4–88 .For immunocytochemical analysis, dissociated neuronal cultures were fixed with 4% PFA solution for 5 min at room temperature and after several washing steps with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Next, cells were incubated with blocking buffer containing 2% Glycine, 2% BSA, 0.2% gelatin and 50 mM NHFor proximity ligation assay (PLA), neuronal cultures were treated as described above and after incubation with primary antibodies overnight at 4 °C, coverslips were washed with Duolink wash buffer A (Sigma-Aldrich) and incubated with Duolink PLA probes PLUS and MINUS in a humidity chamber for 1 h at 37 °C. For ligation of the probes, coverslips were washed with buffer A and then incubated with ligase for 1 h at 37 °C. For amplification step, coverslips were washed with buffer A and then incubated with polymerase and Duolink FarRed Detection Reagent for 100 min at 37 °C. Coverslips were washed two times with Duolink wash buffer B for 5 min and one time with 0.01 × buffer B for 1 min. Prior to incubation with other antibodies for immunocytochemistry or DAPI counterstaining, coverslips were washed with PBS and afterwards, mounted with Mowiol 4-88.Images were acquired with a 63× (Leica) objective lens along the z-axis with 300 nm z-step in a 512 × 512 and 1024 × 1024-pixel formats at 8-bit and 16-bit image depth with at least two times frame average at 400 Hz laser frequency using either the SP8 -or SP5 CLSM system  equipped with white light laser (WLL). Nuclear Jacob immunoreactivity was measured as the mean grey value in arbitrary units of pixel intensity using Fiji software. For Jacob-LaminB1 co-expression analysis and PLA, raw confocal and STED images were deconvolved using Deconvolution wizard , where we first calculated the theoretical point spread function (PSF) based on optical microscopic parameters provided by original *.lif-files, and subsequently used optimized iteration mode.STED images were acquired on a SP8 CLSM setup (Leica) using a 100x/1.4 oil objective . The STED laser (775 nm) was set to maximum power and used to deplete the white light laser excitations at 561 nm and 633 nm with optimized STED laser delay time. Gating of hybrid detectors was set to 0.5–6 ns. The line profiles from STED images were generated from an average of two optical sections.Hippocampal primary neurons were transfected at DIV9 with the plasmid expressing WT-Jacob-GFP using lipofectamine 2000. Time-lapse imaging was performed as described previously , 30. Z-sHEK-293 T cells were co-transfected via the calcium phosphate precipitation method with the following plasmids: WT-Jacob-Myc and TagRFP-LaminB1 or tagRFP control. 24 h post-transfection cells were harvested and lysed in 1 ml of RIPA buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl and 1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS), PIC and PhosStop for 1 h at 4 °C. The cleared by centrifugation cell lysate was incubated with anti-Myc antibody-coated magnetic beads  and immunoprecipitation was performed following the manufacturer’s protocol  with subsequent WB analysis.E. coli SHuffle (C3026) cells were transformed with either pMAL or LaminB1(PMXB10) vectors and E. coli ExpressLysY/Iq cells were transformed with CRM1 (PMXB10) vector. All bacterial cultures were grown at 30 °C until OD600 reaches 0.4–0.8 and subsequently 0.4 mM of IPTG was added for additional 16 °C. For purification of LaminB1 and CRM1, bacterial cell lysates were prepared in 20 mM Tris pH 8.5 500 mM NaCl, sonicated, centrifuged at 20.000g and then the supernatant was loaded onto chitin resin and bound to columns. After several washing steps, columns were incubated with the wash buffer containing 50 mM dithiothreitol (DTT) for 24 h at 10 °C. Proteins were eluted and run on the SDS-PAGE gel for the purity checking. For interaction assays between Jacob and LaminB1, and also between Jacob and CRM1, the matrix (amylose-MBP) alone or coupled with Jacob proteins (amylose-MBP-45-532-Jacob and amylose-MBP-45-532-Jacob-L476A) was washed with the buffer containing 20 mM Tris pH 7.5, 1 mM DTT, 3 mM EDTA, 100 mM NaCl, 0.3% TritonX-100, protease inhibitors . Subsequently, amylose resin was incubated with 20 μg of an untagged-LaminB1, untagged-CRM1 or with HEK293T cell extract expressing CRM1 overnight at 4 °C and all proteins were eluted and analyzed by WB.Proteins were bacterially produced and purified as previously described . In brieData in the manuscript is shown as mean ± S.E.M. Graphs and statistical analysis were made with GraphPad Prism (GraphPad Software). Statistical tests used are indicated in the figure legends. The number of subjects considered for statistical comparison is included in the graphs.Protein transport from synapse to nucleus and subsequent nuclear import of Jacob requires NMDAR activity. We therefore first asked whether the association of the protein with the nuclear envelope is regulated by neuronal activity. Immunofluorescence staining of hippocampal primary neurons with a previously characterized antibody , 21, 22 We have previously shown that Jacob harbours a bipartite nuclear localization sequence  located within exons 6 and 7 , diminishing the number of hydrophobic consensus residues within the specific motif, and found that mutant protein (Jacob-NES-L476A-GFP) is preferentially retained in the nucleoplasm following expression in hippocampal neurons  mutant of Jacob  is an inhibitor of nuclear export that can be used to study nucleo-cytoplasmic translocation. The cellular target of LMB is CRM1 . TreatmeIn previous work, we have found that GluN2B-containing NMDAR are instrumental for long-distance transport and nuclear import of Jacob , 16, 45.The nuclear lamina is a filamentous protein meshwork at the interface of chromatin and the nuclear membrane , 2. AparAlternatively, it is conceivable that the nuclear lamina merely provides a docking site for an intermediate step relevant for nuclear export of Jacob. Our data support two non-mutually exclusive scenarios. Since the interaction of Jacob with the nuclear lamina is direct, the association of CRM1 with the NES should be dispensable for binding of Jacob to LaminB1. It is, however, puzzling that in a cellular context the presence of the NES is required for the localization at the INM and that LMB results in dissociation of the overexpressed protein from the nuclear lamina. Thus, CRM1-binding to Jacob appears to promote the localization at the INM as an intermediate step before nuclear export. In this scenario, another factor would be required to actively release the complex from the nuclear lamina for nuclear export. On the other hand, it is possible that re-modeling of the Jacob complex is a prerequisite for nuclear export. Activity-dependent nucleocytoplasmic shuttling has been shown for many regulators of transcription and, in this context, nuclear residing time is a crucial factor, as is protein degradation. Processes that eventually contribute to the destiny of Jacob following nuclear export are likely regulated and will occur at the nuclear lamina. Finally, we show here for the first time that synapto-nuclear messenger proteins display transient localization and association with the nuclear periphery. The question arises whether this is a feature specific to Jacob. The current study paves the way to address these questions and further research is needed to explore the underlying molecular mechanisms.We propose that the nuclear function of the synapto-nuclear messenger protein Jacob relies on redistribution processes that will likely occur in a stepwise manner. A transient association with the inner nuclear membrane is regulated by multiple factors: NMDAR activity and nuclear import, direct interaction with LaminB1, and NES-CRM1-dependent mechanisms.Additional file 1: Movie S1.Additional file 2: Figure S1. The RanGTP-CRM1-dependent mechanism is critical for Jacob`s subcellular distribution and association with the nuclear rim. A. Association of WT-Jacob-GFP with the nuclear rim was quantified based on LaminB1 stainings. The threshold was applied to LaminB1 image, the ring was interrupted (*) and ROIs were outlined using the wand tool . Laser settings and signal amplification parameters throughout the image acquisition of overexpressed Jacob were kept constant. Only those cells that fitted into the dynamic range were selected for quantification. Scale bar indicates 10 μm. B. Graph represents relative amounts of WT-Jacob/mNES-Jacob at the nuclear rim when co-expressed either with RanQ96L or RanT24N normalized to control (mCherry).Additional file 3: Movie S2.


In recent years, substantial advances have been made in noninvasive cardiac imaging, including cardiac computed tomography (CT) and cardiovascular magnetic resonance (CMR). The purpose of this study was to prospectively compare the diagnostic performance of contrast-enhanced whole heart coronary CMR angiography (CCMRA) to dual-source coronary CT angiography (CCTA) for the diagnosis of significant coronary stenoses (≥50%) in patients with known or suspected coronary artery disease (CAD) referred for conventional x-ray coronary angiography.Our objective was to directly compare the diagnostic accuracy of contrast-enhanced whole-heart CCMRA (CE-CCMRA) to dual-source CCTA (DS-CCTA) for the detection of CAD. We prospectively studied 57 symptomatic patients with suspected or known CAD who were scheduled for conventional x-ray coronary angiography. Significant CAD was defined as an x-ray defined diameter reduction of ≥50% in a coronary artery with a reference diameter of ≥1.5 mm.P > 0.05), 85% versus 90%(P > 0.05), 90.6% versus 93.5%(P > 0.05), and 89.4% versus 90%(P > 0.05), respectively. The area under the curve (AUC) was 0.89 (95% CI: 0.79 to 0.99) for CE-CCMRA and 0.92 (95% CI: 0.83 to 1.00) for DS-CCTA.CE-CCMRA and DS-CCTA were completed in 51 (89%) of 57 patients without complications. The acquisition times of CE-CCMRA and DS-CCTA, respectively, were 9.5 ± 3.1 min and 8.3 ± 1.4 s. On patient-based analysis, the sensitivity, specificity, positive and negative predictive value of CE-CCMRA and DS-CCTA were 93.5% versus 93.5%(DS-CCTA was found to be superior to CE-CCMRA in the diagnosis of significant coronary stenoses (≥50%) in patients with suspected or known CAD scheduled for conventional x-ray coronary angiography, owing to shorter scanning times and higher spatial resolution. However, CE-CCMRA and DS-CCTA have similar diagnostic accuracies. Along with economic changes and modernization in developing countries, including China and India, the prevalence of atherosclerotic cardiovascular diseases is also increasing rapidly. Cardiovascular disease mortality in such countries is much higher compared to that of most developed Western countries . ConventOver the past decade, substantial advances have been made in noninvasive cardiac imaging, including the introductions of coronary computed tomography angiography (CCTA) and cardiovascular magnetic resonance imaging (CMR), which have challenged the role of the invasive standard , 3. CurrThe purpose of this current study was to prospectively compare the diagnostic performance of DS-CCTA and CE-CCMRA for the diagnosis of significant coronary stenoses (≥50%) in patients with known or suspected CAD referred for conventional coronary angiography.This prospective study was approved by the local ethics committee; all patients gave written informed consent. From June 2014 until January 2015, 74 patients with known or suspected CAD were enrolled. The patients were scheduled for conventional x-ray coronary angiography. The exclusion criteria were: absence of a sinus cardiac rhythm, orthopnea, history of previous heart surgery, presence of coronary stent, and contraindications to CMR imaging  or CT with known allergy to iodinated contrast material, impaired renal function (creatinine > 1.4 mg/dl) and thyroid disorders.Patients with heart rates> 75 bpm received an oral β-blocker  before CE-CCMRA. In addition, 0.5 mg nitroglycerin was administered sublingually to all subjects before CE-CCMRA. A medical abdominal belt was wrapped tightly along the side of the ribs to reduce abdominal motion during deep inspiration. No β-blocker or nitroglycerin was administered to patients before DS-CCTA scans.2, acquired voxel size = 1.3 × 1.3 × 1.8 mm3 and interpolated to 0.65 × 0.65× 0.9 mm3) was employed. The three-dimensional k-space data were collected with a centric ordering scheme in the phase-encoding direction and a linear order scheme in the partition-encoding direction. In addition, a nonselective inversion pulse with an inversion time (TI) of 200 ms was applied before the navigator echo pulses to suppress the background tissues. In order to reduce the acquisition time, parallel imaging technique  was used in the phase-encoding direction with an acceleration factor of 2. A 0.2 mmol/kg intravenous injection of contrast agent  was injected into an antecubital vein at a rate of 0.3 ml/s, immediately followed by 20 ml saline given at the same rate. Data acquisition began 60s after the initialization of contrast agent administration.CE-CCMRA was performed using a 3 T whole-body scanner  with a 12-channel matrix coil . The R-wave acquired from a three-lead wireless vectorcardiogram was used to trigger the data acquisition. The CE-CCMRA scanning procedure was as follows: after initial localization of 2-chamber and 4-chamber views and left ventricular short-axis views, a retrospective electrocardiography (ECG)-triggered cine (a fast low-angle shot (FLASH) sequence with 80 cardiac phases) was performed in the 4-chamber plane to determine the quiescent period for coronary artery imaging [All patients underwent DS-CCTA . The DS-CCTA protocol included the following parameters: retrospective ECG gating; collimation, 32 × 0.6 mm; slice acquisition, 64 × 0.6 mm using the z-flying focal spot technique; gantry rotation time, 330 ms; pitch, 0.20–0.43 adapted to the heart rate; tube voltage, 100 or 120 kVp (depending on age and body mass index); and maximum tube current, 320 mAs per rotation. Spatial resolution was 0.6 × 0.6 × 0.6 mm. Temporal resolution was variable according to the heart rate of the patient.All DS-CCTA data were acquired in deep inspiration. The volume of iodinated contrast material  was adapted to the scan time. A contrast bolus  was injected in an antecubital vein with a flow rate of 4 to 6 ml/s followed by a saline chaser of 40 ml at the same rate. Contrast agent application was controlled by a bolus tracking technique. The region of interest was placed into the aortic root, and image acquisition was started 7 s after the signal density level reached the predefined threshold of 100 HU.All DS-CCTA coronary angiography datasets were reconstructed with a slice thickness of 0.75 mm and increment of 0.4 mm using medium-to-smooth convolution kernel (B26f), resulting in an inplane resolution of 0.6 to 0.7 mm and a through-plane resolution of 0.5 mm .Conventional x-ray coronary angiography served as the reference standard in this study. The diameter stenosis, as a percentage of the reference diameter, was determined in two orthogonal directions and the measurement was performed in the projection that showed the highest degree of stenosis, and was evaluated by two experienced invasive cardiologists without knowledge of the results of CE-CCMRA and DS-CCTA. All segments of the coronary artery tree with a reference diameter of 1.5 mm or greater were included in the study. A clinically significant stenosis was defined as stenosis of at least 50% of the vessel diameter.Transverse images were transferred to an off-line workstation . Images for each patient were analyzed in consensus by two cardiovascular radiologists with 16 and 20 years of experience. Two observers blind to the results of conventional coronary angiography evaluated the DS-CCTA and CE-CCMRA results using axial slices images, as well as multiplanar or curved reformatted reconstructions and maximum intensity projections. A 16-coronary-arterysegment model was used, based on recommendations from the American Heart Association . Image quality was assessed on a 4-point scale: 1 = poor (not assessable); 2 = fair (mild to moderate artifacts); 3 = good (minimum to mild artifacts); and 4 = excellent (minimum or no artifacts). Contrast-to-noise ratio (CNR) was evaluated in nondiseased proximal coronary segments , mid-left anterior descending coronary artery (LAD), left circumflex coronary artery (LCX), and right coronary artery (RCA)) and computed as the difference in mean signal intensity between the vessel lumen and the adjacent tissue, divided by the standard deviation of the background noise in the proximal aorta . The sevP value of 0.05 or less was considered a significant difference.Quantitative variables were expressed as the mean value± SD, and categorical variables as percentages. CNR were compared using paired t-tests. Diagnostic performances of DS-CCTA and CE-CCMRA used x-ray coronary angiography as the standard of reference, and were presented as sensitivity, specificity, positive predictive value and negative predictive value. They were presented with the corresponding 95% confidence intervals (CIs). McNemar’s chi-square test was used to compare the diagnostic accuracy between CE-CCMRA and DS-CCTA. Inter- and intraobserver variability for image quality grading and the detection of significant coronary artery stenosis was calculated using κ statistics, and Wilcoxon test was used to compare the image quality of CE-CCMRA and DS-CCTA. Differences in accuracy between CE-CCMRA and DS-CCTA on both a patient and vessel basis were compared using McNemar’s chi-square test. Receiver operating characteristic (ROC) curves were used to assess the diagnostic efficacy and the area under the ROC curves (AUC) were compared between the two imaging modalities..A 2-tailed n = 4), unstable angina(n = 5), atrial fibrillation(n = 3), allergy to iodinated contrast material(n = 3), impaired renal function(n = 2), and seven were unsuccessfully scanned.During the study period, 74 patients were eligible for inclusion Fig. . Seventen = 1) at the time of DS-CCTA. The success rate of DS-CCTA (98.2%) was higher than that of CE-CCMRA (91.2%). Thus, the final study group consisted of 51 patients  . Studies of six patients were discontinued because of irregular breathing patterns (n = 3) and suddenly discomfort (n = 2) during the CE-CCMRA scan, or insufficient intravenous contrast n =  at the tp < 0.001). The kappa values for interobserver agreement for image-quality grading were 0.86 and 0.83, respectively. Representative examples of normal coronary angiograms and coronary stenoses detected by CE-CCMRA and DS-CCTA are shown in Figs. The evaluation of segments by conventional x-ray coronary angiography, 3 T CE-CCMRA and DS-CCTA for 51 patients are shown in Table The diagnostic abilities of CE-CCMRA and DS-CCTA to detect significant stenoses in a patient-, and vessel-based analysis are detailed in Table In the patient-based analysis, DS-CCTA correctly recognized 29 of the 31 significant stenoses detected on x-ray angiography. CE-CCMRA also correctly identified significant CAD in 29 out of 31 patients. The sensitivity (93.5%) of both tests was equally high. In contrast, CE-CCMRA had a slightly lower specificity (85%) but no significant difference in the ability to detect coronary stenosis than DS-CCTA (90%). CE-CCMRA had a AUC of 0.89 (95% CI: 0.79 to 0.99). DS-CCTA had a AUC of 0.92 (95% CI: 0.83 to 1.00). In the vessel -based analysis, the sensitivity, specificity, PPV, and NPV for detecting patients with significant CAD by DS-CCTA scan were 94.7%(54/57), 90.7%(87/96), 85.7%(54/63), and 96.7%(87/90), respectively. In addition, the diagnostic performance of CE-CCMRA on a per-vessel basis was similar, with no significant differences. The AUC was 0.91 (95% CI: 0.86 to 0.97) for CE-CCMRAand 0.93 (95% CI: 0.88 to 0.98) for DS-CCTA.Differences were observed for the RCA, LM-LAD, and LCX arteries Table , Fig. 5.This present study describes on the first single-center direct comparison of CE-CCMRA and DS-CCTA to detect coronary artery stenosis in patients scheduled for conventional x-ray coronary angiography. Our study demonstrates that both DS-CCTA and CE-CCMRA can provide diagnostic accuracy in detecting significant CAD and are reliable for excluding significant CAD, without significant differences on a vessel- or a patient-based level. However, DS-CCTA provides better image quality than CE-CCMRA. We evaluated all segments being > 1.5 mm in diameter. Only 5% of the coronary segments were significantly lower than CE-CCMRA (12%). This reflected the currently better image quality of DS-CCTA. Better image quality is attributable to higher spatial resolution and CNR.In recent years, CCTA has become widely used, and studies have demonstrated its diagnostic accuracy –15. DS-CStudies during the past 20 years have shown that CMR technology allows noninvasive, radiation-free, comprehensive evaluation of CAD , 17. PreSeveral studies using the direct comparison of CMR and CCTA have been carried out with different sequence types employed at different field strength, and with different generations of multislice CT , 22–25. In this current study, the diagnostic ability to detect significant coronary stenosis (≥50%) in CAD by visual analysis was slightly, although not significantly, higher for DS-CCTA than for CE-CCMRA. Such a difference may be caused by the higher spatial resolution and CNR of DS-CCTA, which led to a visualization of more coronary segments. Therefore, from a clinical standpoint, both DS-CCTAand CE-CCMRA provide specific advantages, considering the benefits and drawbacks of these imaging modalities, and widely increase the clinical utility of these methods.This study also has some limitations. First, the study population from the single center was relatively small. We will continue our studies with a large patient population for further subgroup analysis. Another drawback is that, after contrast administration, DS-CCTAand CE-CCMRA are not suitable for repeat examinations when the scans fail. Moreover, the time for completing CE-CCMRA procedures still remains long, and CE-CCMRA has relatively low spatial resolution. Third, the evaluation of the diagnostic performance of 3 T CE-CCMRA in this study was performed using a 12-channel cardiac coil. Recently, a 32-channel cardiac coil has become commercially available, which would reduce the imaging time of cardiac imaging , and couIn conclusion, DS-CCTA is superior to 3 T CE-CCMRA in the diagnosis of significant CAD in patients with suspected or known CAD scheduled for conventional x-ray coronary angiography. DS-CCTA has shorter scanning times and higher spatial resolution. However, 3 T CE-CCMRA and DS-CCTA have similar diagnostic accuracies.


The introduction of the 2016 WHO Classification of Tumors of the Central Nervous System has resulted in tumor groupings with improved prognostic value for diffuse glioma patients. Molecular subtype, primarily based on IDH-mutational status and 1p/19q-status, is a strong predictor of survival. It is unclear to what extent this finding may be mediated by differences in anatomical location and surgical resectability among molecular subgroups. Our aim was to elucidate possible correlations between (1) molecular subtype and anatomical location and (2) molecular subtype and extent of resection.We performed a systematic review of literature searching for studies on molecular subtype in relation to anatomical location and extent of resection. Only original data concerning adult participants suffering from cerebral diffuse glioma were included. Studies adopting similar outcomes measures were included in our meta-analysis.In the systematic analysis for research questions 1 and 2, totals of 20 and 9 studies were included, respectively. Study findings demonstrated that IDH-mutant tumors were significantly more frequently located in the frontal lobe and less often in the temporal lobe compared with IDH-wildtype gliomas. Within the IDH-mutant group, 1p/19q-codeleted tumors were associated with more frequent frontal and less frequent temporal localization compared with 1p/19q-intact tumors. In IDH-mutant gliomas, greater extent of resection was achieved than in IDH-wildtype tumors.Genetic profile of diffuse cerebral glioma influences their anatomical location and seems to affect tumor resectability. Extent of resection and molecular markers are prognostic factors in diffuse glioma.Molecular subtype of glioma affects tumor location and extent of resection.Extent of resection is a well-established prognostic factor in diffuse gliomas of all grades. With the introduction of the WHO 2016 classification of central nervous system tumors, the “layered” histological and molecular diagnosis of gliomas has become the new standard. In this systematic review, we demonstrate that a glioma’s molecular subtype affects tumor location and—to a lesser extent—extent of resection. Our findings underscore that the prognostic value of extent of resection cannot be studied fully without incorporating molecular subtype and location. Clinically, our analysis suggests that further research is needed to develop refined neurosurgical guidelines for diffuse glioma, which are stratified by molecular subgroup. Such research and guidelines would further launch oncological neurosurgery into the era of precision medicine.1 Based on their genetic profile, astrocytomas  and oligodendrogliomas  are now more homogeneously defined.2 IDH-wildtype tumors are either classified as diffuse astrocytoma or glioblastoma, depending on their histological grade.In 2016, the World Health Organization (WHO) Classification of Tumors of the Central Nervous System underwent major revision. For the first time, molecular characteristics are incorporated in the classification of brain tumors. This allows for more accurate, “layered” diagnosis, improved patient management, and more accurate estimation of prognosis and likelihood of treatment response.3–8 For this reason, studies based on the WHO 2007 criteria should be reevaluated in light of the new classification.Since the introduction of the revised classification, multiple studies have investigated outcomes of patients with diffuse gliomas and consistent conclusions are drawn: genetic subtype as outlined in the 2016 WHO classification is a stronger prognostic marker for survival than the earlier histopathological categorization.9–13 However, this may not necessarily apply to each molecular subtype of diffuse glioma. Theoretically, molecular characteristics can influence survival in two ways: either directly through intracellular pathways inducing relatively indolent or aggressive tumor behavior, or indirectly via EoR  has been established as a prognostic factor for survival in both low- and high-grade diffuse gliomas. via EoR . It is uResearch question 1: Is anatomical location of diffuse glioma (according to the WHO 2016 classification) correlated with molecular subtype?Research question 2: Is EoR of diffuse glioma (according to the WHO 2016 classification) correlated with molecular subtype?15 A comprehensive electronic search was conducted through PubMed in November 2017. The search comprised terms indicating glioma or a subtype hereof, terms regarding histological or molecular classification and terms either related to anatomical localization or EoR .n or EoR .Studies eligible for inclusion in this review were observational or interventional and prospective as well as retrospective. Only manuscripts written in English, German, French, or Dutch were considered for inclusion.1 or derivable into WHO 2016 classification) as well as anatomical localization and/or EoR were reported. Only data of adult patients (aged ≥ 18 years) were included in the review. Reviews and case studies were excluded. References of included studies were screened for additional studies eligible for inclusion. All steps of study selection and data extraction were performed by a junior researcher (B.d.L.) and reviewed by K.v.B. Discussion regarding inclusion or data extraction was solved in consensus meetings.Titles and abstracts were screened to identify potentially eligible studies. Full-text articles were selected based on the following inclusion criteria: (a) the patients involved were diagnosed with a grade two, three, or four diffuse glioma based on histopathological examination, and (b) molecular glioma subtype  were too heterogeneous in presentation to allow for formal meta-analysis. Study findings that could not be included in the systematic study were synthesized narratively. For both research questions, included studies were categorized by the molecular markers investigated and findings were described correspondingly.Literature searches for research question 1 and 2 yielded 2248 and 701 studies, respectively. After article screening and selection, 89 and 39 studies were selected to undergo further assessment. Hereof, 34 and 18 studies were considered eligible for inclusion based on quality criteria . AlthougA total of 20 studies were included in the systematic analysis of mutation status in relation to anatomical location. All were cohort studies. Data were collected retrospectively in eighteen studies and prospectively in two studies. Eleven studies focused on imaging, five on clinical or demographic characteristics and four on survival. The median number of participants in these studies was 147 (range: 25–406) and the median quality score was 10 out of 16 (range: 9–14).29 Regarding eloquent localization, one of three studies reports that IDH-mutant gliomas were significantly more often located in noneloquent regions compared with IDH-wildtype tumors.25 The other two studies did not reach statistical significance . Within the IDH-mutant group, 1p/19 co-deletion was associated with more frequent frontal and less frequent temporal locations compared with 19/1q-intact tumors. IDH-wildtype status was further associated with parietal tumor location (P = .007). A correlation between 1p/19q co-deletion status or combined mutational status and parietal localization was not found . IDH mutation, 1p/19q co-deletion, and combined mutational status did not affect eloquent localization of gliomas   .In the systematic analysis of mutation status in relation to EoR, nine studies were included. All were cohort studies. Data were collected retrospectively in eight studies and prospectively in one study. Four studies primarily focused on clinical or demographic characteristics, three on survival and two were imaging studies. The median number of participants per study was 141 (range: 22–709) and the median quality score was 9 out of 16 (range: 8–13). No formal meta-analysis was possible.37 In four studies, IDH-mutant tumors showed better resectability and the fifth study indicated greater EoR in IDH-wildtype tumors  gross total resections are more frequently achieved and these patients are less likely to undergo a biopsy compared with patients with IDH-wildtype, 1p/19q-intact gliomas. Data on other mutations, such as TERT and TP53, were insufficient to describe a correlation between anatomical location or EoR.Our findings of correlations between molecular subtype and EoR as well as between molecular subtype and anatomical location suggest that EoR may be influenced by molecular subtype, possibly through preferential anatomical locations of molecularly defined diffuse glioma . These a30 insula,39 brainstem, and other midline localizations.41 IDH wildtype status is also correlated with the presence of multifocal tumors.40 Furthermore, an association between presence of IDH mutation and surface localization was found.42 In accordance with our results, EoR was reported to be greater in IDH-mutant gliomas.43 A single study involving 200 patients describes not finding a correlation between IDH mutation status and EoR.44Regarding IDH mutation, these studies too report wildtype gliomas to be more frequently located in the temporal lobe,45 and in frontal location,46 as we found. A positive association between presence of a co-deletion and surface localization42 is reported, as well as absence of such a correlation.30 Regarding the relation between 1p/19q co-deletion and EoR, results are ambivalent as one study reports lower EoR in codeleted gliomas44 and another describes not finding an association.47Concerning 1p/19q co-deletion, studies report high incidences of codeleted gliomas in the right frontal lobe and anterior insula6 and less often in temporal areas. Moreover, they show that these tumors are more frequently completely resected and less often biopsied. Only three studies have focused on the relation between location and EoR. Analysis performed by Beiko et al.14 demonstrates that frontal location and IDH mutation are independent prognostic factors for complete resection of high-grade astrocytoma . Wijnenga et al.25 report that insular and eloquent localization are associated with greater postoperative tumor volume in diffuse low-grade glioma (P < .0001 for both factors), as well as increasing age and preoperative tumor volume . Moreover, they state that corrected for these factors, molecular subtype did not correlate with postoperative tumor volume. This supports the idea that the relation between molecular subtype and EoR is mediated by anatomical location. More research in adequate-sized (prospective) cohorts is needed to elucidate whether location—and possibly other known prognostic factors such as age—fully explains the association between molecular subtype and EoR, or whether other mechanisms and factors mediate this relationship.Of course, there may be factors other than localization and molecular subtype that also have an influence on EoR. The hypothesis of the relation between molecular subtype and EoR being confounded by anatomical location is endorsed by our own results as well as other studies’ findings. Our results demonstrate that IDH-mutant gliomas, especially those which are also 1p/19q-codeleted, are more frequently located in the frontal lobe, where higher resection percentages are achieved,39 found that in low-grade gliomas tumor location, tumor diameter on MRI, EoR, and IDH mutation were prognostic factors in univariable analysis , yet in multivariable analysis only IDH mutation held up as an independent prognostic factor (P = .001). Likewise, Wijnenga et al.25 found that in low-grade gliomas eloquent tumor localization, resection percentage, and molecular diagnosis are prognostic factors univariably  and in multivariable analysis only molecular diagnosis was an independent prognostic factor (P = .0001). Their results also do not demonstrate categorized EoR or postoperative tumor volume to be statistically significant prognostic factors for overall survival in IDH-mutant low-grade gliomas, regardless of their 1p/19q-status. This analysis was not feasible in the IDH-wildtype group due to small sample size. Findings of above-mentioned studies suggest that the prognostic value of EoR is better explained by molecular diagnosis whenever correction for this factor takes place. Beiko et al.14 studied high-grade astrocytomas and their univariable analysis demonstrates that in IDH-wildtype tumors, EoR was not a prognostic factor, though postoperative tumor volume was (P = .021). However, this did not hold up after correction for preoperative enhancement and age in the multivariable analysis. In the IDH-mutant group preoperative volume, postoperative volume and EoR were indicated as prognostic markers univariably , but only preoperative and postoperative volume remained significant in the multivariable analysis . By contrast, analysis performed by Patel et al.24 in low-grade gliomas demonstrates that EoR is a prognostic factor in IDH-wildtype tumors, but not in the IDH-mutant group . Current data are too heterogeneous and insufficient to draw any conclusions concerning relative prognostic value of EoR and molecular subtype.Prognostic value of molecular subtype and EoR has been well established. However, as we have demonstrated that a correlation between the two exists, a confounding effect may be in play. The key question is whether EoR holds up as a prognostic factor for survival after correction for contemporary molecular subtyping and subsequent glioma classification. Several studies have performed multivariable analysis including both EoR and molecular markers. Metellus et al.Studies on EoR of diffuse glioma should be stratified by molecular subgroups as outlined by the WHO 2016 classification. Until more of these studies become available, no clear recommendations can be given regarding surgical management for different molecular subgroups; until then, current guidelines favoring maximum safe resection should be considered as standard of care.The main conceptual strength of this review/meta-analysis is the systematic study of the interrelationships between tumor type, location, and EoR in the context of current-era, molecular marker-based classification of gliomas (WHO 2016). Methodological strengths of this review include its systematic set-up and search, and the standardized assessment of methodological quality of included studies. The review’s limitations are secondary to the limitations of the underlying studies, including potential selection bias due to mere inclusion of resected and biopsied gliomas—without regard for patients who are on a watchful waiting-strategy. Also, we did not analyze the degree of potential reporting and publication bias. Furthermore, the majority of included studies classified anatomical location by brain lobes. This is probably not sufficient when it comes to correlating anatomical location of brain tumors to their EoR. Location in or adjacent to eloquent regions is more likely to influence EoR. Only a limited number of studies looked into eloquence of tumor location, with heterogeneous and sparse definitions of eloquent location.Genetic profile of diffuse cerebral glioma influences their anatomical location. Available evidence strongly suggests that it also affects tumor resectability.Further research should focus on influence of molecular markers on occurrence of diffuse glioma in eloquent brain regions and analyze this in relation to extent of resection. Furthermore, multivariable regression analyses taking into account molecular subtype, extent of resection, and anatomical as well as eloquent localization should be performed in a diffuse glioma sample, as this would enable elucidation of the relative prognostic value of these factors. Future studies concerning extent of resection should stratify their results by molecular subgroups as outlined by the WHO 2016 classification. These lines of research should finally answer the question whether the surgical strategy for a diffuse glioma is dependent on the molecular subtype. If this is the case, then preoperative prediction of molecular subtype with advanced imaging becomes increasingly important.Tumor localization should be taken into account as a possible confounder in comparative prognostic research regarding diffuse cerebral glioma. Lastly, there is need for research into the relations between anatomical location, MRI characteristics, and extent of resection with the aim of finding out whether MRI characteristics are predictive of EoR and to what degree relative to anatomical location.Knowledge of prognostic value of molecular markers, extent of resection, and anatomical as well as eloquent localization will enable more specific formulation of recommendations for surgical management of diffuse glioma. These should ultimately be stratified by molecular subgroups as outlined by the WHO 2016 classification. This would further launch oncological neurosurgery into the era of precision medicine.vdz032_suppl_Supplementary_materialClick here for additional data file.