Hybrid approaches combine computational methods with experimental data. The information contained in the experimental data can be leveraged to probe the structure of proteins otherwise elusive to computational methods. Compared with computational methods, the structures produced by hybrid methods exhibit some degree of experimental validation. In spite of these advantages, most hybrid methods have not yet been validated in blind tests, hampering their development. Here, we describe the first blind test of a specific cross‐link based hybrid method in CASP. This blind test was coordinated by the CASP organizers and utilized a novel, high‐density cross‐linking/mass‐spectrometry (CLMS) approach that is able to collect high‐density CLMS data in a matter of days. This experimental protocol was developed in the Rappsilber laboratory. This approach exploits the chemistry of a highly reactive, photoactivatable cross‐linker to produce an order of magnitude more cross‐links than homobifunctional cross‐linkers. The Rappsilber laboratory generated experimental CLMS data based on this protocol, submitted the data to the CASP organizers which then released this data to the CASP11 prediction groups in a separate, CLMS assisted modeling experiment. We did not observe a clear improvement of assisted models, presumably because the properties of the CLMS data—uncertainty in cross‐link identification and residue‐residue assignment, and uneven distribution over the protein—were largely unknown to the prediction groups and their approaches were not yet tailored to this kind of data. We also suggest modifications to the CLMS‐CASP experiment and discuss the importance of rigorous blind testing in the development of hybrid methods. Proteins 2016; 84(Suppl 1):152–163. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. CASPCritical Assessment of Protein Structure Predictionsulfo‐SDAsulfosuccinimidyl 4,4′‐azipentanoateCLMScross‐linking/mass spectrometryFDRfalse discovery rateSAXSsmall angle x‐ray scatteringBS3Bis(sulfosuccinimidyl)suberate.Hybrid methods are emerging as new tools to model protein structure. These methods incorporate experimental data into computational protein structure approaches in an attempt to increase the accuracy of resulting models and the range of applicability. Hybrid methods can leverage experimental data that by itself would be insufficient to determine structures with satisfactory accuracy. However, when this data is complemented by computational approaches, it may suffice to aid conformational search to find good minima in the energy landscape, even in cases when purely computational methods would fail.et al.Experimental data sources for hybrid methods range from sparse NMR restraints,The importance of hybrid methods was acknowledged by the CASP committee in CASP10, when they introduced the “contact‐assisted” category.For CASP11, the Brock and Rappsilber laboratory proposed a new experiment to establish hybrid methods as a component of the CASP experiment. To address the time constraints of CASP, they proposed to use experimental data based on a novel protocol for photo‐cross‐linking and mass‐spectrometric analysis (CLMS).The employed photo cross‐linking/mass spectrometry approach in this experiment has a number of unique properties that makes it an excellent experimental data source for blind testing of hybrid methods. Cross‐linking and mass‐spectrometric analysis are relatively quick. The experiments reported in this article took approximately two weeks of experimental time and 4.2 days measurement time on average. This makes it possible to provide experimental data under the time constraints of the CASP experiment. The unique chemistry of photo‐cross‐linking reagents produce an order of magnitude more cross‐links than standard homobifunctional cross‐linking agents, such as BS3.important property is that the cross‐linking reaction can be performed prior to purification of the protein. Because cross‐links are already formed, the protein can be purified under non‐native conditions or the protein can be digested and the cross‐linked proteins can be enriched. Therefore, cross‐linking can be done in samples with low purity, in native environments,Another In this article, we describe the first blind test of a hybrid method in CASP with real, experimental data.We would like to point out that this was made possible by the efforts of the CASP organizing committee. The CASP committee identified and acquired suitable targets and published the resulting data on the CASP web page for the community of predictors. We would also like to thank the experimental groups that generously provided protein sample for this experiment. Please refer to the “Acknowledgements” section a full list of the involved researchers and affiliations.The goal of this article is to report on the experience and the logistics of the blind testing of hybrid methods. Furthermore, we report on the results of the experiment in CASP11 by presenting the cross‐linking results on four protein targets and briefly discuss the impact on modeling. The last goal of this article is to make recommendations to maximize impact of future instances of hybrid methods in CASP.Here, we only give a brief overview of the experimental cross‐linking/mass spectrometry method, describing those details required for understanding the remainder of this article. Full experimental details of the experimental protocol used in CASP 11, targeted to a mass spectrometry audience, can be found in a separate article (currently in preparation).Generally, protein residue pairs are covalently cross‐linked, effectively providing an upper bound of the linked residues that is partially determined by the length of the linker agent. The protein is then digested, which results into a peptide mix. Some of the peptides are cross‐linked if they have been in spatial proximity in the folded structure. The peptide mix is then subjected to mass spectrometric analysis. Peptide spectrum matching and database search reveals the cross‐linked residues. The output of this method is a list of cross‐linked residues which effectively provide distance restraints with an upper distance bound Fig. . The keyEach target was cross‐linked using the heterobifunctional, photoactivatable, chemical cross‐linker sulfosuccinimidyl 4,4′‐azipentanoate (sulfo‐SDA). The Rappsilber group first incubated sulfo‐SDA with the protein for 1 h and then photoactivated the sulfo‐SDA with UV light. The protein is then digested using different combinations of proteases.The digested peptides were loaded onto a liquid chromatography column to separate the peptides by hydrophobicity. The peptides were gradually eluted and sprayed into the mass spectrometer. This procedure reduces sample complexity during mass spectrometric analysis.For data analysis, the peak lists were searched against a database from the sequence of the CASP targets. The Rappsilber lab assumed the sulfo‐SDA linker reaction specificity to be lysine, serine, threonine, tyrosine, and protein N‐termini at one end and any amino acid residue at the other end. Lastly, the false discovery rate (FDR) was estimated using a modified target‐decoy search.The Rappsilber group compiled lists of residue‐residue cross‐links from FDR analysis and submitted them to the CASP organizing committee, which released them to the prediction groups.First, we report on the organization and execution of the experiment to provide the reader with the setup of the hybrid method/cross‐linking experiment in CASP11. We proposed the CLMS assisted structure prediction experiment mid‐March 2014.The CASP organizers identified and acquired suitable protein targets (no homologous structures could be identified by sequence similarity). The first positive response came from a PSI centre 12 days later on March 30th 2014. A total of nine proteins were sent to the Rappsilber laboratory between May 29th and June 9th 2014. From these nine proteins, the CASP organizing committee, in discussion with the Rappsilber group, selected four proteins that met the following criteria: 1) The protein is heavier than > 20 kDa, 2) it forms a monomer in solution and, 3) approximately 1 mg protein sample was available. Because of the relatively low spatial resolution (25 Å) of CLMS constraints, CLMS data is likely not informative for small proteins. Thus, the organizers excluded small proteins from consideration. Selected proteins needed to be monomers in solution to allow unambiguous assignment of cross‐linked peptides as intramolecular connections. At least 1 mg of protein sample should be available to have sufficient material for CLMS experiments. The final conclusion was made in a meeting between the CASP Organizing Committee and the Rappsilber lab Edinburgh on June 10th 2014. The selected targets for CLMS experiments were: Target 1, SP17834A‐RUMGNA_02398, Tx781; Target 2, SP16782A_BACCAC_02064, Tx808; Target 3, SP17984B‐SAV1486, Tx767 and Target 4, laminin, Tx812. All targets contain at least one hard template‐based modeling or free modeling domain.http://predictioncenter.org/).The Rappsilber group performed cross‐linking/mass‐spectrometry experiments for these targets in a timeframe of 48 days, starting on June 11 2014. Figure Cross‐linking data captures spatial proximity between residue pairs in the native structure. However, the cross‐linked atoms cannot be specified because the carbene species of activated diazirine group can react with any atom and current mass spectrometry technology does not routinely identify the linked atoms. Thus, it is not possible to specify a tight upper bound for the distance between two cross‐linked residues. The actual distance is affected by many factors, such as the side‐chain length, the cross‐linker length, and conformational flexibility of the protein. In CASP11, we used a conservative α‐carbon upper distance bound of 25 Å. Note that conformational flexibility of the protein in solution could result in correct cross‐link matches of residues that are further apart than 25 Å in the native structure.http://www.predictioncenter.org/casp11/domains_summary.cgi) with ‐D1 and ‐D2 for the first and second evaluation domain, respectively. Figure We first qualitatively analyze the CLMS data of the four CASP11 target proteins and their evaluation domains. Note that CASP predictions are usually analyzed the basis of evaluation domains that are identified by the assessors. We refer to the official CASP11 domain assignments . It is obvious that CLMS constraints between β‐strands are not informative, because adjacent β‐strands have a distance of ∼5 Å. However, it is quite surprising that there is an apparent bias to cross‐link coverage. Specifically, β‐strand cross‐links are often not observed at all. There is no entirely clear reason for this finding at this stage.Surface accessibility and environmental reactivity influences the formation of cross‐links and maybe a different cross‐linking chemistry needs to be developed to reduce this influence and to obtain a more even coverage.The distribution of digestion cleavage sites also contributes to uneven coverage of cross‐links. This is evident for Tx781, for which little cross‐link information is found up to residue 180 see Fig. . There aIn the following analysis, we quantify the distance information in the CLMS data. Figure Figure In summary, cross‐links from high‐resolution CLMS contain structural information that is obtainable in approximately 4.2 days of data acquisition for a single protein target. However, we hypothesize that the exploitation of CLMS data by modeling groups could be optimized along three dimensions: 1) Designing error‐tolerant modeling algorithms such that the negative impact of CLMS noise or ambiguous/false assignment is minimized; 2) better estimates or acceptance of higher error at low upper distance bounds of cross‐linked residues; 3) exploiting the geometry of protein models because cross‐links from soluble linking reagents are formed along the surface of the protein structure.http://predictioncenter.org/). We then compared the model quality of predictions submitted to the regular experiment  and to the CLMS assisted experiment . Since we would like to capture the impact of CLMS data, we only compared the best predictions from groups that submitted predictions to both experiments [19 groups, Fig. We analyzed the predictions submitted during the CASP11 experiment to measure the impact of CLMS data on model quality. We downloaded the predictions and summary tables from the CASP11 website . However, this is most likely due to the fact that many more groups submitted predictions to the regular experiment (143 groups) than to the CLMS assisted experiment (19 groups). As a result, the chances of finding a higher GDT_TS prediction in the regular experiment was much higher than in the CLMS experiment. In addition, we would like to point out that all measures of model quality lack precision in the analyzed model quality range and that the sample size in this experiment is too small to draw strong conclusions about the effect of CLMS data on prediction quality in CASP11. However, our earlier study showed that high‐density CLMS data enables the reconstruction of the domain structures of human serum albumin, albeit by using CLMS data from 2.1‐2.6 times more acquisitions.It should also be mentioned that CLMS data—at least in this very first inclusion in CASP—did not yet provide a significant advance for the field as a whole. The best models chosen from We believe that this initial experiment demonstrated that CLMS‐driven hybrid methods can be tested in the CASP context. As the experimental protocols are refined and predictors start developing tailored prediction approaches, hybrid methods may provide significant improvements over purely computational approaches in future rounds of CASP.The CASP11 CLMS experiment was a success in the sense that it performed a first, truly blind test of a hybrid method for protein structure prediction in a very short timeframe, demonstrating that experimental data acquired in a short time can be used in protein structure prediction, even if the predictions itself were not improved in CASP11.There were also some lessons learned during the experiment. They should be mentioned to make the reader aware of the challenges of blind testing of hybrid methods as well as to enable improvements for future iterations of CASP.The logistical challenges of an experimental‐computational experiment are much higher than for a pure computational, because it involves the treatment of physical protein sample. First, the actual proposal for CLMS in CASP was made quite late, which meant that the organization of the experiment had to be made on the fly and whilst the prediction season was already underway. This reduced the time frame for data generation for the experimental group. Since the detection of cross‐link peptides is stochastic and can be improved with additional acquisitions,The Rappsilber group also faced the problem of protein sample deterioration. In the case of Tx781 it is likely that the protein sample deteriorated during transit whilst being held in UK customs due to a VAT exemption query.Approximately 4.2 days of data acquisition was performed for each CASP11 target. In addition, the prediction groups only had short time windows for prediction with CLMS data (12–15 days).The photo‐CLMS approach generates a novel type of experimental data with properties that are mostly unknown to CASP participants. The coverage, distribution, sparseness, and resolution of the CLMS data are important properties that can be used to develop effective algorithms for protein structure modeling with this type of data. Thus, we speculate that most prediction groups would have been more successful when they would have known the nature of the CLMS data beforehand.In this section, we would like to suggest some measures that would maximize the impact of the hybrid method blind testing in the CASP setting.The careful selection of protein targets will be an important factor in future experiments. We believe that targets should be selected with two goals in mind: 1) Testing the ability of computational methods to model structure with CLMS data for proteins that are well suited for CLMS experiments, and 2) testing a broad variety of different folds to explore biases and issues of CLMS data with certain fold types.From our experience, we think that α‐helical proteins with ∼200 residues or more seem to be the most suitable targets for current CLMS experiments. The α‐helical structure does not seem to bias cross‐link formation. In addition, α‐helical usually have a larger diameter than β‐sheet proteins, which makes CLMS constraints more informative.Proteins should have sufficient lysines (primary target of CLMS reagents).Proteins should have well distributed digestion sites to ensure uniform coverage with cross‐links, and that digested peptides can be detected by the mass spectrometer because they are not too big. Note that these are current technical limitations that are already actively worked on, and might be overcome eventually. However, for now it would be necessary to actively select targets that are amendable to current CLMS technology.et al.ab initio structure prediction.Targets should be from the free‐modeling or hard template based modeling category. This would test whether CLMS data is also useful to disambiguate templates  two ways: 1) Using CLMS data to extract better information from databases, and 2) Leveraging CLMS data in conformational sampling.Even if the spatial resolution of CLMS data continues to be too low to restrict the conformational space effectively, the data will contain at least some information about the topology of the protein, because adjacent secondary structures (at least α‐helices) should have cross‐links between them. This information could be helpful to select fragments with backbone conformations closer to native. Another possibility for CLMS data is to assist template selection and template alignment of hard template‐based targets.Conformational sampling with CLMS data is difficult because of the low spatial resolution of CLMS constraints, which might not sufficiently limit the conformational space. However, tailoring algorithms to the nature of cross‐linking data would compensate this issue to some degree. Cross‐links from soluble linkers are formed along the protein surface and therefore scoring functions should take the surface of the protein into account when modeling CLMS constraints. Xwalk uses breath first search on a surface grid to determine the shortest path between two surface points.A different route to increase information content would be to use a lower Euclidian distance bound in modeling. This would increase the number of long‐distance links, but the links that satisfy the distance bound will be more informative. Developing algorithms that are robust to noise will not only be helpful for CLMS data, but also for structure modeling with residue‐residue contacts.Furthermore, different length of involved side‐chains could be exploited to develop residue specific Euclidian upper distance bounds. However, photo‐cross‐link reagents are highly promiscuous and the fragmentation of peptides is sometimes incomplete. This introduces ambiguity into the site assignment of cross‐linked residues, which needs to be taken into account to develop such residue specific CLMS constraint functions.Lastly, protein structure prediction algorithms need to carefully weight CLMS constraints with other information from templates, fragments, the energy functions, contacts, and maybe other data from experimental methods.Additionally, we think that the CLMS‐CASP experiment would benefit from the participation of additional experimental groups. Further experimental groups could blind test their own methods, or rely on the sulfo‐SDA approach described in this article. The latter requires the dissemination of the experimental protocols and software for cross‐link data analysis. Further issues need to be tackled, such as the establishment of the CLMS protocol in a new laboratory that might require proper calibration of mass spectrometers and/or modifications to the cross‐link search software. Experimental groups need to invest a significant amount of staff time and consumables into the experiment, which leaves the open question of how such experiments will be funded in the future. We suggest that experimental groups should be offered authorship in the resulting CLMS‐CASP papers to compensate them for their investment and enable them to request funding for future rounds. Recruitment of more experimental groups is probably the most challenging task toward an improved CLMS‐CASP experiment. However, the experiment would benefit from more experimental groups by a higher number of targets that can be processed. Additionally, the CLMS community would have an opportunity to blind test their tools, which have a wide variety among different CLMS groups. Thus, the independent assessment of CLMS pipelines would have high value to advance this field of study. Finally, it would be highly beneficial to recruit groups that can deliver other types of experimental data for the CASP experiment.The CASP format introduced rigorous standards into the field of protein structure prediction and can possibly introduce such standards for hybrid methods. The blind testing of computational and experimental methods is important to assess the state of the art of hybrid methods.However, the most important feature of hybrid methods cannot be tested in CASP in the current setup: The determination of protein structures which cannot be crystallized and solved by NMR. Obviously, because these targets are elusive to traditional structure determination, there would be no structure to evaluate the submitted models in CASP. Thus, using structural models to assess the function of a protein or answer scientific questions would increase the value of hybrid model testing in CASP.We envision a hybrid method format that does not aim to evaluate the models against experimental structures, which obviously have been amendable to traditional methods. Instead, the goal would be leverage the expertise of the CASP community to convert experimental data to the best structural models available, which are then made public for life‐scientists. Life scientist can verify these structural models by experiment, which would deepen our understanding of these protein systems. Note that our proposal is in line with the direction that CASP assessments are taking. For example, the CASP11 assessors included an assessment of function based on free‐modeling predictions into the experiment; this was successful in two cases.We now sketch the setup of this altered experiment. This experiment requires the identification of protein systems for which no determined structures exist and for which models would be most useful to the life science community. This could be accomplished by a specialized board of scientific advisors or an open call in which proposals are reviewed and evaluated. Many experimental groups should be able to provide protein samples, which can then be distributed to groups that are able to provide experimental data. Ideally, this experimental data would be diverse, such as EM, EPR, NMR, and CLMS. Then, the modeling experts of the CASP community would be able to submit structural models. Assessors could use their proven expertise to select the most promising models, which are then published on a web site for life scientists, together with statistics such as the local modeling error. This would generate truly new structural information, leveraging the expertise of experimental groups and the CASP community. Life scientists can these structures to plan mutagenesis experiments or speculate about the molecular mechanisms of this protein.The modeling community must address several challenges toward this kind of experiment. First, policies for structure prediction depositories must be developed and implemented. The inclusion of experimental data poses additional challenges, such as depositing policies for diverse experimental data. The wwPDB hybrid method task force recently worked out some recommendations for hybrid method repositories and it would be interesting to explore to what degree these recommendations are in line with the planned hybrid method efforts in CASP.We presented the results of the first cross‐link assisted structure prediction experiment in CASP11. This is the first time in the 22 years of CASP history that the CASP experiment is assisted with actual, experimental data. The experiment was blind to the experimental group and to the prediction groups. For three out of four targets, experimental CLMS data could be acquired that contained accurate structural information in the form of distance constraints between residue pairs with an upper bound of 25 Å. Overall, the CLMS data did not lead to a pronounced improvement in backbone quality of CLMS‐guided predictions.An experiment that involves the acquisition and release of experimental data faces new issues that need to be addressed by future CASP rounds. We made recommendations for improved CLMS‐CASP experiments that could lead to a larger impact of CLMS data in future CASPs. The rigorous execution and assessment of experimentally assisted predictions in CASP could be of high value to advance the field of hybrid methods.


Pest insects lead to excessive agricultural and therefore economical losses on crops worldwide. These insects have to withstand toxic molecules that are inherent to plant defences, as well as those that are produced and introduced by humans in the form of insecticides. In recent years, research on insect–microbe symbioses has recognized that microbial symbionts may play a role protecting against these toxins, leading to a form of defensive symbiosis between the pest insect and different types of microorganisms that we term detoxifying symbioses. In this minireview, we will highlight well‐characterized and emerging insect model systems of detoxifying symbioses and assess how the microorganisms influence the host's success. In agriculture, many insects are beneficial because they pollinate crops, disperse seeds or prey on herbivores , cocaine , crop rotation, chemical insecticides or biological pest control exploiting predators and parasitoids Douglas, . The forBuchnera aphidicola. Primary symbionts that are important for proper functioning of the host are generally maternally transmitted and have remained unculturable even with modern culturing techniques. This fastidious nature is caused by drastic reduction in genome size  are a notorious pest of peas, soya beans and other legumes, and received international notoriety after spreading from Asia to the United States in 2009  avoid calcium oxalate containing wild‐type Medicago truncatula leaves in the presence of mutants that lack calcium oxalate. Breakdown of oxalate was already reported in gut bacteria found in humans, pigs and sheep  that contains genes involved in amino acid metabolism, indicative of obligate symbionts of sap‐feeding insects. Just like aphids, the stinkbug M. punctatissima feeds on phloem sap that contains little amino acids and its diet is supplemented by microbes that provide essential amino acids  had their obligate symbionts experimentally exchanged, their performances on a crop legume were also reversed  and pine weevils (Hylobius abietis), two serious pests in the cultivation of pine seedlings and conifers causing losses of up to 80%  was closely related to the Rhodococcus erythropolis DCL14 that produces an enzyme responsible for monoterpene degradation  from the gut were able to degrade the ITC to less harmful compounds. Genome sequencing of these four isolates results in the assembly of several near‐identical plasmids recurring in the different genera, suggesting horizontal gene transfer. More specifically, the presence of tra genes indicates the occurrence of conjugation events. One plasmid, designated as Drgb3, contains the gene saxA  is the primary pest for coffee bean farmers across the world. Over 20 million coffee‐growing families are economically affected by this pest, and infestation levels are particularly high in plantations in Tanzania (90%), Malaysia (50–90%), Uganda (80%), Colombia (60 %), Mexico (60%) and Jamaica . These findings suggest that insecticide resistance might develop in a field even in the absence of pest insects, which could then quickly establish in a single insect generation  is a major global pest of cruciferous crops, estimated to cost approximately 4 billion euros per year in lost production and management (Zalucki et al., D. radicum, P. xyolostella produces an enzyme that prevents formation of dietary isothiocyanates (Ratzka et al., P. xylostella is not only able to overcome host defences, but it has even been shown to be highly resistant to a large variety of chemical insecticides and it is one of the only three insect species to have developed resistance to Bacillus thuringiensis‐based insect control methods (Furlong et al., P. xylostella is at least in part attributed to the insect's own physiology, and includes altered target sites for carbamates and organophosphates, metabolism of parathion via glutathione S‐transferases and detoxification of pyrethroids via microsomal P‐450 monooxygenases (Ramya et al., Bacillus cereus colonizing the moth's gut were able to break down the insecticide indoxacarb for use in metabolism and growth (Janmaat and Myers, et al., et al., in vivo breakdown of either insecticide by P. xylostella gut bacteria has not yet been demonstrated, so cause and effect remain unclear. Even if detoxifying symbiosis does not play a role in this association, further study can still be fruitful because organophosphorus‐degrading bacteria by themselves may prove of value in ecological and industrial applications (for an overview we refer to the review of Singh, The diamondback moth et al., et al., In agriculturally important pest insects, the microbiome offers potential for improvement of current methods in pest management. Primary opportunities include the prediction of host traits and thereby the efficacy of control strategies, targeting of the microbiome for direct pest control or targeting the microbiome to reduce the vector competence of the pest Douglas, . Even thAn increased demand for novel insect pest management created by growing human populations and global climate change is anticipated, and symbiotic microorganisms offer one potential route to meet this demand Douglas, . Of the The authors have no conflict of interest to declare.


Recently, we found abundant innervation of antigen presenting cells that were reached and enclosed by single neurites. These neurally hard‐wired antigen presenting cells (wAPC) could be observed in the T‐cell zone of superficial cervical lymph nodes of rats and other mammalians, including humans.As a consequence, we investigated lymph nodes at many different anatomical positions as well as all primary and secondary lymphoid organs (SLO) in rodents for a similar morphology of innervation regarding antigen presenting cells known in those tissues.As a result, we confirmed wAPC in lymph nodes independent from their draining areas and anatomical positions but also in all other T‐cell zones of lymphoid organs, like Peyer's patches, NALT and BALT, as well as in the thymic medulla. Other cells were innervated in a similar fashion but with seemingly missing antigen presenting capacity. Both types of innervated immune cells were observed as being also present in the dermis of the skin. Only in the spleen wAPC could not be detected. Beyond this systematic finding, we also found another regular phenomenon: a dense network of neurites that stained for neurofilament always in antigen entrance areas of lymphoid organs . Lastly, also thymic epithelial cells (TEC) restricted to the corticomedullary junction of the thymus showed similar neurofilament staining.Therefore, we propose much more hard‐wired and probably afferent connections between lymphoid organs and the central nervous system than is hitherto known. The immune and the nervous system are present in nearly all body tissues and organs. And as one main function of both systems seems to be controlling homoeostasis in many directions, one could ask how they control each other. This question and the necessary bi‐directional communication pathways between the immune and the nervous system now engaged researcher interest in many different fields. Besides the long known and well established pathways like the endocrine communication via the neuroendocrine system and the Hypothalamic‐Pituary‐Adrenal‐Axis, much less has been found concerning hard‐wired pathways where neurites innervate lymphoid tissues directly. Today, sympathetic innervation has been shown for most lymphoid organs, whereas the parasympathetic efferents could only be detected in some of these immunologically relevant organs. And looking at the possible afferent pathways, only rudimentary knowledge could be stated with respect to possible autonomic or somatic afferents To get an overview of possible APC innervation in most lymphoid organs, we included inductive SLO that show division in B‐ and T‐cell zones like lymph nodes of different draining areas , spleen, Peyer's patches, NALT and BALT, because we found the wAPC exclusively located in the T‐cell zones of lymph nodes. T‐cell zones in the different lymphoid tissues were always visualized by CD3 staining. And although there is no clear definable T‐cell zone in the dermis below skin and mucosa we included that tissue, too, as it is located at a similar first place of antigen encounter. Additionally we tested thymus and bone marrow as PLO. As a first step, the tissues were stained for neural structures with neurofilament antibody SMI 312). In the case that we found innervated cells, we further tested MAP2 for indicating some dynamic properties as well as MHC II for antigen presenting capacities. Due to the fact described above, that APC in part show uniformity and in other parts heterogeneity of marker expression in different lymphoid tissues, we decided to only look for the most common APC markers: CD11b/c and CD103 as dendritic cell integrins, CD68 for macrophages and the signalling molecule CD172a. In the results section, for the sake of clarity, we will concentrate on systematics and outstanding findings; we will not describe every single marker result in detail for every lymphoid tissue (for details see Table 2. In theNasopharynx associated lymphatic tissue (NALT) and brain from 8 weeks old female C57BL/6J mice were purchased from Envigo RMS (The Netherlands).Bronchus associated lymphatic tissue (BALT), thymus, spleen, brain, lymph nodes , skin, bone marrow and Peyer's patches from the intestine from 12 weeks old female Sprague–Dawley rats were also purchased from Envigo RMS (The Netherlands).All organs and tissue specimens were removed post mortem and directly snap frozen in liquid Nitrogen.All experimental procedures were approved by the DEC‐Consult Animal Ethics Committee, Utrecht, The Netherlands, number HAR‐0005 according to all applicable rules, laws and regulations.For details of species, number of organs, slices and type of sections see supplementary Table S1.Twenty micrometer frozen sections  of all organ and tissue specimens were dried for 20 min. at room temperature and fixed in cold (−20°C) Aceton Methanol (1:1). Blocking of unspecific binding sites was performed by a 5 min incubation (room temperature) with Super‐Block  and a subsequent washing step for 5 min in PBS .Primary antibodies against rat tissue were rabbit anti‐MAP2  , mouse anti‐CD11b/c (OX42) , mouse anti‐CD103 (OX62) , mouse anti‐CD172a  or (OX41) , mouse anti‐MHC II (OX6) , mouse anti‐CD68 (ED1) , rabbit anti‐CD3 (SP7), , mouse anti‐CD169 (ED3) , mouse anti‐Keratin (80) , rabbit anti‐LYVE1  , anti‐ mouse‐Peripherin  and mouse anti‐NF (SMI312) .As primary antibodies against mouse tissue we used the same antibodies as listed above for the rat with the exception of anti‐CD11b/c, anti‐CD103 and anti‐CD172a because of species specifities. Instead, we used the following antibodies: mouse anti‐CD11b/c  , rabbit anti‐CD103   and mouse anti‐CD172a   .Primary antibodies were diluted in PBS as described above and incubated for 60 min at room temperature or overnight at 4°C, followed by 15 min incubation of secondary antibodies.Secondary antibodies used were goat anti‐rabbit‐DyLight 549 , goat anti‐rabbit‐Alexa 488 , goat anti‐mouse‐DyLight 549  and goat anti‐mouse‐Alexa 488 .For multicolor staining, primary antibodies in certain cases were conjugated with: Cy3 , FITC , HiLyteFluor 647 , and HiLyteFluor 488 .Only in the case of mouse anti‐NF (SMI312) , we always used the primary antibody conjugated with HiLyteFluor 488 .Slides were mounted using Vectashield Hard Set mounting medium with DAPI .A Zeiss Axio Imager.M2m microscope  with Zen software was used for image collection.To be sure, that neurofilament antibody SMI312 stains neural structures in the PNS , staining controls were performed with tissue specimens containing distinguishable peripheral nerves  and brain .We performed isotype controls of every used primary immunglobuline (Mouse IgG MG1‐45) and Mouse IgM (MM‐30); BioLegend, Rabbit IgG (poly); Abcam plc, Cambridge, UK), with every used concentration (specific primary antibody) on every type of tissue with the standard staining protocol. Representative results are shown in Figures ‐45 and MTo exclude unspecific staining of secondary antibodies, staining procedures were performed without primary antibodies (replaced by PBS) on every single tissue type (Data not shown).For frozen bone marrow sections, the femur (rat) was fixed in 4% PFA for 24 h and then placed in 15 ml of 14% EDTA for 2 weeks (solution was refreshed daily). To complete the procedure, tissue was rinsed four times in ddH2O tissue and frozen in Tissue‐Tek® O.C.T.™ Compound  for further processing.For histological overviews we stained all tissue specimens with the standard procedure of Hematoxylin and Eosin. 20 μm cryo slides were dried at room temperature, followed by an incubation with Hämalaun  for 3 min at room temperature. Rinsing in aqua dest. for 1 min and for 10 min in tab water. Eosin  counterstaining was performed for additional 3 min at room temperature followed by dehydration ascending ethanol series  and finished with 2 × 2 min incubation in Histol .We recently found hitherto unknown neural structures stained by anti‐neurofilament in lymph nodes of rats like “neurally hard‐wired” APC in the T‐cell zone (wAPC) and dense innervation of the subsinoidal layer. Consequently, we now continued our search for similar neural structures in all other primary and secondary lymphoid organs (SLO). As a result above all, the density of neural structures not associated with blood vessels that could be stained by neurofilament in all lymphoid organs tested was much higher than in any other work we sifted in literature. The complexity of neural architecture in lymphoid organs therefore seems to be much higher than hitherto estimated.In our last work, we showed and characterized wAPC in superficial cervical lymph nodes of rats, humans and T‐cell deficient mice. Now we checked most other lymph nodes of rats with different draining areas, following a clear anatomical definition As expected, the T‐cell zone of this mucosal SLO also contained APC innervated like the wAPC in the lymph node whereas B‐cell follicles were omitted. Showing sometimes deviating innervation morphology, many wAPC seemed to be not fully enclosed by neural structures, appearing as if the projecting neurite was only densely associated with the APC, going over the cell body margins. Moreover, in this lymphoid tissue we also observed cells being innervated, but seemingly without antigen presenting capacity as they missed MHC II expression. Hence, in the following we will use a different term for all other immune cells in lymphoid organs that are reached by neurites and are innervated with a similar morphology like the wAPC, but without showing MHC II expression: “wired immune cell” (wIC). MAP2 expression, which in the lymph nodes is co‐localized with neurofilament expression in the majority of detected wAPC, could only be seen in rare cases of wAPC in the Peyer's patches. On the other hand, macrophage and dendritic cell markers showed a similar heterogeneous distribution along the detected wAPC like in the lymph node. As mentioned before, the top of the subepithelial dome right below the mucosa epithel also showed a neural nexus, the high density of neurites described above for the subsinoidal layer of the lymph node. The normal mucosa epithel without underlying Peyer‘s patches turned out to be negative for this phenomenon . As this was a form of redundant organization, it led us to the suggestion that the same organization could be present in the skin‐draining lymphatic pathway. As we already had positive results regarding the presence of wAPC and a neural nexus for skin draining lymph nodes in a second position to antigen contact, we decided to also check the first position in this pathway—the tissue layers of the skin. We concentrated on the dermis as the corresponding subepithelial connective tissue layer to the lamina propia containing the Peyer‘s patches below the intestinal mucosa epithel. Consistent with all the previous results, we found wAPC in the dermis, showing a similar morphology detected in Peyer‘s patches and lymph nodes as well as also many wIC without any detectable MHC II staining. Most of the wAPC and wIC were also positive for MAP2, indicating some dynamics, whereas dendritic cell markers again were expressed very heterogeneously as always in all examined lymphoid organs. Interestingly, we were also able to stain many single neurites—probably free nerve endings—in the dermis, whereas the previously described sensory nerve fibres in close association to Langerhans cells in the epidermal tissue layer Following looming systematics, in both SLO of the respiratory tract (NALT and BALT), wAPC were irregularly present in T‐cell zones, omitting B‐cell areas. Similar to the findings in the Peyer's patches, the innervation morphology of wAPC appeared heterogeneous as some cells were totally enclosed by the neural meshwork, while others seemed to only be partly covered or having just a close association to a surrounding neurite. Also supplementing the results in Peyer‘s patches, BALT and NALT tissues showed many innervated immune cells reached by neural structures similar to the wAPC, but without any demonstrable antigen presenting capacity by MHC II expression—cells we defined above as wIC. At this point it should be emphasized that here like in all other lymphoid organs investigated, of course there were always some APC showing no neural contact at all. Besides this results for innervated single cells, a neural nexus at the margin of BALT and NALT could be confirmed, therefore again appearing at antigen and APC entrance areas. Interestingly, we also observed a zone of lower cell density at the border of BALT, looking like a lymphatic sinus, comparable to the subcapsular sinus of the lymph node or to the marginal sinus in the spleen. And, although we detected this sinus in only a single case, the neural nexus was located exactly inside and below that layer Fig. A–D. LYVEThe spleen is responsible for processing blood‐borne antigens, while all other tested SLO so far are involved in lymphatic drainage. Interestingly, just in the spleen no wAPC could be detected in any part of this lymphoid organ. Very different were the observations regarding a neural nexus. Here, the neural nexus presented itself as a hitherto unknown density of single neurites exactly at the border between the red and the white pulp, also irregularly present at different white pulp areas in the tested spleen sections. Extrapolating that network of neurites around a whole white pulp to three dimensions, the neural nexus would build up a structure that forms some kind of hull around the lymphoid organ. With CD169 staining, we proved the location of the neural nexus in more detail, referring to other works and the special rat spleen anatomy After gathering results for all the different SLO, we searched for similar neural structures in association with immune cells in PLO. We found wAPC and other wIC in the thymic medulla, consistently in that area, where much antigen presenting capacity is necessary for negative selection. However, beside the visible neural structures that seem to innervate immune cells with or without antigen presenting capacity, neurofilament staining in the thymus depicted a network of another cell type. Additional keratin staining proved those cells to be thymic epithelial cells (TEC). Interestingly, while keratin marked all types of cortical or medullary TECs throughout the thymus, neurofilament positive TECs are restricted to the corticomedullary junction, the main entrance area for thymocytes. This neurofilament signal—different to the morphology of wAPC or wIC—not only exhibited all the cell bodies, but also followed most of the fine membranous extensions the TEC built up to connect to each other. It was not possible to figure out whether the neurofilament signal was located inside the TEC or outside and only in close association with their cell membrane, as otherwise could be clearly outlined for the wAPC or wIC. Respecting the different cell type as also the different innervation morphology, we will refer to that TEC showing neurofilament signals in the corticomedullary junction as “neural thymic epithelial cell” (nTEC) Fig. A–D. OtheTo complete the set of all lymphoid organs, we also tested the other PLO, bone marrow, for similar neural structures. Unfortunately, we were not able to identify any neural structure comparable to our results in all other lymphoid organs thus far. We summarized the findings according to the neural architecture in the different lymphoid organs in Table The aim of this work was to get an overview of primary and secondary lymphoid organs of Mammalia with respect to the presence of similar innervation morphology of single cells with APC character, recently found in the lymph nodes of rats—wAPC The results that wAPC are present in all lymphoid organs of lymphatic drainage pathways have interesting implications: Migratory APC normally have their first antigen contact just below or inside epithelial tissues like skin or mucosa. Such tissues at “first places” of antigen contact and processing (often named inductive tissues) included in our work have been Peyer‘s patches, BALT and NALT for mucosa and dermis for skin tissue. The corresponding “secondary place,” where migratory APC normally fulfil their antigen presenting tasks are the lymph nodes. Therefore we included superior mesenteric lymph nodes draining mucosa tissues and many other different lymph nodes draining the skin . Of course, not all lymphatic drainage pathways are already described in a consistent way, as the connection of BALT and NALT to draining lymph nodes is still debated as well as the definition of skin and its underlying dermis as a SLO The phenomenon of a high density of neurites we first observed in the subsinoidal layer directly below the subcapsular sinus in lymph nodes of rats nTEC appeared as neurofilament positiveTEC in the corticomedullary junction of the thymus. And as we have not been able to assign the neurofilament signal exactly to be outside or inside the TEC, two possibilities remain to be most likely: Either the nTEC express neurofilaments as their own intermediate filaments, or the nTEC are accompanied by neural structures, which express those neurofilaments in their neurites, as is the case with the wAPC. Anyway, in both cases the nTEC phenomenon probably will not imply a new type of TEC in general, it rather will outline an additional feature of known cortical TEC or medullary TEC. Looking at the two possibilities in more detail highlights different cellular capabilities that may be linked with them. If nTEC express neurofilaments as intrinsic intermediate filaments, despite the fact that all TEC have epithelial character, these—then more appropriately called “neuroepithelial”—cells may also have neuronal functions. On the other hand, if these nTEC are enclosed with a dense neural meshwork of neuronal origin with the neurofilaments expressed by neurites, we should likely talk about innervated TEC with their known functions, as we have already done with APC and other immune cells in different SLO. In the latter case the difference between the innervation morphology has to be considered: With the nTEC connected to each other, the neurofilament signal follows all those fine membranous extensions, whereas with the wAPC those extensions are always excluded. However, with both possible cases the nTEC phenomenon can be integrated in the looming systematic innervation patterns observed in SLO so far. It is precisely the corticomedullary junction that is described as the main gate for incoming or exiting immune cells like thymocytes and other myeloid cells Unfortunately, little is known about TNC aggregates beside the fact that they also contain TEC and seem to be involved in thymocyte differentiation Taking together the results for the neural structures identified in all lymphoid organs and also in different lymph nodes, one main difference can be observed: Compared to the regular appearance of the neural nexus and nTEC phenomenon, wAPC and wIC appearance, amount and densities were very heterogenous. For this variability, many different reasons are thinkable and may be true side by side. First, there is no exact knowledge about the relation and total amount of APC populations in different lymphoid organs like in different lymph nodes, so the result could simply portray different amounts of APC in total. Second, regarding different lymph nodes, most APC that present antigens there may do this successfully in the first draining nodes. Consequently, if wAPC innervation is a neuro‐immune connection involved in that task, less innervation because of surveillance is necessary in lymph nodes that are only the third or fourth station in lymphatic circulation. The same aspect could explain the probable absence of wAPC in the spleen handling blood‐borne antigens. As blood has a privileged role in being thoroughly separated from pathogens, less wAPC innervation may be necessary. Third, the negative result for wAPC innervation only in the spleen also follows our previously suggested function Obviously, looking at our results, the many different places of the whole antigen recognizing system seem not to be blind spots of the nervous system. All places show neural structures in a form of systematic and redundant organization in all lymphoid organs. Hereby, two phenomena can be differentiated, first the similar but irregular appearing innervation patterns of wAPC and wIC in T‐cell zones, and second a neural nexus in “gate” areas of lymphoid organs (including nTEC as the thymic counterpart). Summarizing this together with unpublished results regarding the afferent nature of this connection, an intriguing view of a sensory network arises:The whole immune response chain, from the early T‐cell selection processes in the thymus throughout the places of innate and adaptive responses up to effector regions, may be under dense surveillance of the central nervous system. This surveillance, then, seems to be concentrated on the one hand at the place of antigen processing or antigen presenting , with all that tissues systematically showing the innervation morphology of wAPC. On the other hand, neural surveillance seems to be concentrated at the “gates” for antigen or APC entrance , with each systematically showing the phenomenon of a neural nexus most likely containing many free neurites. Moreover, even exit places seem to be included (medullary sinuses to cord border). In this scheme, at least some of the many free nerve fibres in the dermis may likewise function for sensory purposes in the surveillance of the immune system, resembling the neural nexus of “true” lymphoid organs. Anyway, even if we strongly suggest those neural structures to be of an afferent nature, the other efferent possibility has to be considered. But however, afferent or efferent, the fact that all those neural structures can be stained with the same neurofilament antibody points to a common function accompanied by similar cytoskeletal structures.Preparation of bone marrow as whole slices is always a challenging task because of being able to cut the tissue after treating osseous structures without destroying the sensitive marrow tissue Finally, many questions remain:What type of immune cells are innervated in the different lymphoid organs?To get first evidence, all those innervated cells in the different lymphoid organs have to be checked for their expression profiles. Indeed this will be a mammoth task to accomplish. Not only do all lymphoid organs, by issues of their tissue architecture, differ in preparation methods, but also the correct markers for antigen presenting cells will differ from species to species, from organ to organ and even from parts of the parenchymal tissue.What is the origin of the innervating neurites and how do they “hard‐wire”?Only knowing the origin of the innervating neurites together with functional experiments will give a reliable answer to the afferent or efferent nature of the neural structures. It is also important to state that in a strictly neurobiological interpretation, innervation as an expression can only be used if synaptic vesicular transmission has been shown. And as we already searched for that transmission possibility but were not able to find any hint in recent work With more answers to these questions it may be that not only an “immune sense” will be confirmed, but also an “immune consciousness” will be unravelled.We recently identified innervated APC in the T‐cell zone of lymph nodes of rats. These wAPC thousandfold showed an innervation morphology as single cells, separately reached by single neurites and hard‐wired with a dense neural meshwork covering the cell body but not membranous extensions. Here, we continued our recent work in the search for those neurally hard‐wired connections of the nervous system to APC in lymphoid organs.First, we confirmed the presence of wAPC in all other lymph nodes at different anatomical locations, like also in all other secondary lymphoid organs except the spleen, but also in the dermis of skin and mucosa and in a primary lymphoid organ (PLO), the thymus.Second, in lymphoid organs and other tissues with first antigen contact, like Peyer's patches, NALT and BALT but also thymic medulla and dermis, we found likewise innervated cells but without clear MHC II expression—wIC. This wIC, together with the wAPC in these tissues, often showed deviating innervation patterns compared to the neural meshwork in the lymph nodes, but all have in common a very close association of the innervating neural structure to only the cell membrane of the cell body, not following membranous extensions.Third, we showed that most known “gates” of the immune system, no matter if for antigens or APC, for entrance or exit—like the subsinoidal layer of the lymph nodes and spleen, the subepithelial dome of Peyer's patches, the margins of NALT and BALT, and probably also the dermis—stained for a high density of single neurites, a phenomenon we called neural nexus and which we suggest contains many free nerve endings.Fourth, in the thymus there seem to be an also hitherto unknown staining of neurofilaments closely associated with thymic epithelial cells and restricted to the corticomedullary junction, which leads us to name these cells nTEC. Whether nTEC express neurofilaments in an intrinsic fashion, or if their membranes are closely covered by neurites could not be resolved.wAPC and wIC, therefore, should define an abundant innervation morphology of probably many different antigen presenting cells and other cells of the immune system, possibly including stromal cells with all their differing expression profiles, whereas neural nexus should characterize a dense network of neurites thought to contain also free nerve endings at most, if not all, “gates” of the lymphoid organs.For the aforementioned reasons, and actual research with unpublished results, we strongly suggest wAPC and wIC, neural nexus and nTEC to be part of an afferent and sensory system for the surveillance of the immune system.The functional aspect of this fascinating system distributed through the whole immune system of mammalia awaits further discoveries and is central to our lab's research.This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.Additional supporting information may be found in the online version of this article at the publisher's web‐site.Figure Sa. A: Location of BALT tissue (arrows) in the bronchial airway walls, associated with some alveolar tissue (Av) and preferentially located at bronchial (Br) bifurcations.Figure Sb. A: Position of two NALT aggregates marked by arrows in the floor of the dorsal nasal cavity below the stratified squamous epithel (Ep) and the dermis (Dm).Figure Sc. A to D: Counterstaining with anti‐peripherin, a marker for peripheral nerves and anti‐neurofilament clearly shows a partial overlap but also a partial co‐staining of both markers in a peripheral nerve in the hilus region of a superficial cervical lymph node.Figure Sd. The typically beaded appearance of a huge peripheral nerve passing nearby an axillary lymph node can be seen. Lymph nodes of Sprague–Dawley rats stained with monoclonal anti‐neurofilament (green) and DAPI (blue).Figure Se. The typically beaded appearance of peripheral nerves drawing through the palatal area and surrounding the NALT can be observed. NALT (white arrow) surrounded by the palatal area of C57/BL/6 mice stained with monoclonal anti‐neurofilament (green) and DAPI (blue).Figure Sf. A to D: Counterstaining with anti‐MAP2 and anti‐neurofilament demonstrates crossing and long axonal fibres of peripheral nerves in the palatal area which are double positive for both markers. Dermis below nasal mucosa of Sprague–Dawley rats stained with monoclonal anti‐neurofilament (green), anti‐MAP2 (orange) and DAPI (blue).Figure Sg. Positive control for neurofilament staining in the brain. Brain of Sprague–Dawley rats stained with monoclonal anti‐neurofilament (green) and DAPI (blue).Figure Sh. A: With CD3 staining, BALT tissue can be clearly divided in T‐cell (Tz) and B‐cell (Bz) areas. (Av) Alveolar tissue.Table S1. Statistical information about species, number of organs, slices and type of section.Click here for additional data file.


Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell dysfunction, production of autoantibodies directed toward cellular and nuclear components, and multiorgan damage caused by immune complex deposition and inflammation within affected tissues In healthy individuals, B cells with autoreactive receptors are selected out during B cell maturation, starting at the initial stages of B cell receptor (BCR) development in the bone marrow and continuing through to the fine tuning that occurs in activated mature B cells in secondary lymphoid tissue. Studies in lupus patients as well as mouse models indicate that these processes are altered in SLE.The etiology of the disease is complex and its phenotype is highly heterogeneous, but genetic susceptibility is thought to contribute as much as 60% of disease risk The crucial role of B cells in SLE pathogenesis is well recognized, from producing autoantibodies to abnormal regulation of immune responses lpr mice (which develop lupus‐like disease spontaneously), disease severity can be attenuated and mortality reduced by ~50% if antibody secretion is blocked, providing robust evidence that autoantibodies are more than spectators in disease etiology B cells contribute to disease mainly by producing autoantibodies targeting nuclear components including DNA (anti–double‐stranded DNA [anti‐dsDNA]), RNP particles , histones, and nonhistone chromatin proteins. These are present in >90% of patients and contribute to disease progression via immune complex formation A recent explosion in genome‐wide association studies (GWAS) has identified >80 potential risk loci across multiple immunopathologic pathways Normal B cell development starts in the bone marrow, where the first round of negative selection of autoreactive B cells  occurs. This process is summarized in Figure The molecular mechanisms by which SLE autoreactive B cells evade central tolerance have yet to be fully elucidated. There are some clues, however, from various genetic studies Outside of single‐gene mutations, GWAS have expanded our knowledge of the molecular basis of B cell developmental anomalies in SLE In early B cell development, several stages have been described which are associated with distinct genetic and molecular events. Two of these genes have been identified as risk loci and are discussed in the next section.IKZF1) and Aiolos (encoded by IKZF3), among others Commitment of the multipotent progenitor cells in the bone marrow to lymphocyte development depends on the expression of several transcription factors, including Ikaros  have been associated with increased transcription of their respective genes in whole blood Both the Ikaros and Aiolos variants IKZF1 have an early block in common lymphocyte precursor development, with reduced pro–B cell numbers and normal pre–B cell numbers Humans with germline mutations in Many abnormalities in peripheral tolerance have been identified in SLE, from problems with somatic hypermutation to memory B cell dysfunction  The other major player in this process of secondary maturation and in the maintenance of tolerance is the T cell–B cell interaction  could be induced in deficient cells after stimulation with high‐affinity antigen; however, this could not switch off autoantibody production The SLE‐associated variant rs6590330 in the promoter of the gene UBE2L3 gene  on chromosome 22 contains a risk variant  within its promoter that leads to increased expression and protein levels in B cells (but not in other immune cells such as T cells) The Variants affecting B cell and pre–B cell signaling affect both central and peripheral tolerance, and many genes involved in both BCR and pre‐BCR signaling have been identified from GWAS P = 3.4 × 10–12) for PTPN22, which encodes the tyrosine kinase Lyp, is associated with impaired BCR signaling and is thought to contribute to autoreactive B cell survival through various mechanisms The risk allele  during BCR signaling Blk, a Src family tyrosine kinase, is normally up‐regulated at the pre–B cell stage and is important in the transduction of pre‐BCR signaling –/– mice develop autoimmune disease, with anti‐dsDNA autoantibodies and immune complex–mediated glomerulonephritis, together with an increase in the proportion of B‐1 cells (a low‐affinity IgM‐producing subtype of B cells in mice) BlkCSK, located on chromosome 15, carries a risk variant (SNP rs34933034) that up‐regulates expression of Csk, a Src family tyrosine kinase important in the BCR signaling cascade P = 5.4 × 10–9) and rs2667978 in the 3′ region (P = 5.1 × 10–8) in women of European ancestry, with both variants being protective. Further transancestral case–control studies identified a third variant in American women of European ancestry, rs6983130 in the 5′‐UTR of the gene (P = 0.000111) –/– mice are susceptible to an inducible immune complex–mediated nephritis as well as to antibody‐induced arthritis Lyn, another member of the Src kinase family, has also been associated with SLE P = 2 × 10–9) has been associated with SLE in several GWAS Patients deficient in IL‐1R–associated kinase 4, myeloid differentiation factor 88, and Unc‐93 homolog B  also have high levels of autoreactive B cells, suggesting that impaired signaling via these pathways results in survival of autoreactive BCRs, possibly by failing to pass the signaling threshold for clonal deletion in the bone marrow Although abnormalities in both central and peripheral tolerance are pivotal to pathology, memory B cells and long‐lived plasma cells contribute substantially to disease propagation The endurance of autoreactive long‐lived plasma cells and their resistance to treatments such as B cell depletion and chemotherapy demonstrate the importance of this plasma cell subtype in disease persistence. In lupus‐prone mice, long‐lived plasma cells develop early and continue to expand throughout their lifetime Development and survival of long‐lived plasma cells and memory cells is complex and depends on a functioning GC Various GWAS in SLE have identified an association with the gene encoding the protein OX40L/TNF ligand superfamily member 4 BACH2, PRDM1, and IKZF3–/– mice also develop a lupus‐like disease in old age, with typical autoantibodies and immune complex deposition GWAS data sets also show disease association with many of the genes associated with the development of immunologic memory including BANK1 have been identified as associated with risk BANK1 encodes a scaffolding protein that acts during BCR signaling by promoting phosphorylation of Src tyrosine kinases such as Lyn through enabling protein–protein interactions between them and inositol 1,4,5‐trisphosphate receptor Two polymorphisms in Failure of tolerance in SLE is due in large part to the intrinsic defects discussed above, but it is important to note that B cells develop in an abnormal milieu in lupus patients compared to healthy controls. Analysis of bone marrow aspirates from patients with SLE demonstrates some interesting findings IKZF1 variant has also been shown to affect expression of complement components and several IFN response genes, while Lyp is required for T cell receptor signaling Although epigenetic annotation of risk variants identifies the B cell as the main cell of interest for the disease, risk variants affecting many aspects of the immune system from innate to adaptive have been described PRDM1, which encodes for B lymphocyte–induced maturation protein 1 (BLIMP‐1), a transcription factor crucial for plasma cell differentiation. The effect of the risk variant rs548324, however, is demonstrable in dendritic cells (DCs), which show a reduction in expression as a result of the variant, a reduction not replicated in B cells Another good example of this is the variant affecting Although the immunologic abnormalities in SLE are complex, numerous, and not fully understood, B cells have a central role in the development of the disease, from driving immune complex production to secretion of proinflammatory cytokines. Since SLE is an archetypal complex disease, the genetic association data reflect that complexity, with many variants, affecting several areas of the immune system The advent of GWAS has identified many potential abnormal pathways relevant to the breaking of self tolerance in SLE (summarized in Table PRDM1), demonstrating how data from GWAS can inform future drug therapy Traditionally, B cell–targeted therapy has mostly been focused on depletion of numbers , but with our growing understanding, less crude approaches are being investigated. For example, Aiolos depletion therapy with the CC‐220 molecule results in down‐regulation of genes mediating B cell differentiation, reduces proliferation, and inhibits antibody secretion in B cells from SLE patients Although the results from GWAS confirm the complexity of SLE pathogenesis and show that different intrinsic B cell abnormalities are likely to drive disease in each individual case, the principles underlying the loss of central tolerance  and peripheral tolerance (dysregulated T cell–B cell interaction) are universal. Genetic information helps us to personalize therapy, whereby relevant molecules can be targeted through understanding the exact pathways involved in an individual's disease.Drs. Karrar and Cunninghame Graham drafted the article, revised it critically for important intellectual content, and approved the final version to be published.


Organic photovoltaics (OPVs) hold great promise for next‐generation photovoltaics in renewable energy because of the potential to realize low‐cost mass production via large‐area roll‐to‐roll printing technologies on flexible substrates. To achieve high‐efficiency OPVs, one key issue is to overcome the insufficient photon absorption in organic photoactive layers, since their low carrier mobility limits the film thickness for minimized charge recombination loss. To solve the inherent trade‐off between photon absorption and charge transport in OPVs, the optical manipulation of light with novel micro/nano‐structures has become an increasingly popular strategy to boost the light harvesting efficiency. In this Review, we make an attempt to capture the recent advances in this area. A survey of light trapping schemes implemented to various functional components and interfaces in OPVs is given and discussed from the viewpoint of plasmonic and photonic resonances, addressing the external antireflection coatings, substrate geometry‐induced trapping, the role of electrode design in optical enhancement, as well as optically modifying charge extraction and photoactive layers. Due to low carrier mobility and small exciton diffusion length of the commonly used small molecules and polymers in OPVs, the film thickness of the photoactive layers is normally limited to 100–200 nm for minimum charge recombination loss and efficient charge extraction. The use of ultrathin photoactive layers in OPVs inevitably leads to insufficient photon absorption and carrier generation, exhibiting low external quantum efficiency (EQE) and PCE.FigureIn this review we concentrate on the recent advances at the intersection of light manipulation and OPVs. A schematic envelope for light trapping‐enhanced OPVs is presented in 2t). However, the presence of the MLA can generate an additional angular component, and the optical path length is increased to L = 2t/cosθ, where θ is the incident angle on a microlens relative to the normal direction (0° < θ < 90°).ηA) can be obtained according to the Beer–Lambert law, ηA = 1 – exp(–αL), where α is the wavelength‐dependent absorption coefficient.To date, a variety of optical strategies have been introduced to enhance the light trapping efficiency of OPVs. From the point of view of ray optics, altering the macroscopic configurations of planar OPVs can cause the light absorption enhancement, like the folding of two cells into a V‐groove style.FigureTo fully guide and modulate the incident light at the nanoscale for enhanced trapping and absorption in OPVs, plasmonic or biomimetic structures  in term of wave optics have been implemented Figure2, provBesides the plasmonic effect with nanostructured metallic contacts, the patterning of dielectric materials in OPVs can play a crucial role in the enhancement of light harvesting in OPVs. Nanostructuring the high‐refractive‐index dielectric layers at a subwavelength scale can effectively manipulate the light absorption of OPVs due to strong optical resonances and scattering induced by the electromagnetic mode coupling in these structures.Jsc) was generated in the inverted device , which was independent on the photoactive layer thickness.To quantificationally evaluate the photon harvest of an OPV, the optical absorption characteristics can be calculated by an 1D transfer matrix formalism (TMF). For example, Pettersson et al. modeled the photocurrent spectra of a bilayer heterojunction OPV by using the 1D TMF, which was in line with experimental results of the photocurrent and EQE spectra.3R),n0 and ns are the refractive indexes of the air and substrate, respectively. For the increase in efficiency of OPVs, the use of antireflection (AR) coatings on the substrate surface is an important consideration, which can reduce the reflected light intensity and allows more light to enter the OPVs by overcoming the mismatched optical impedance at the air/substrate interface. In addition, self‐cleaning AR coatings may benefit OPV operation in real environment by blocking the accumulation of moisture and dust particles on the cell surface over time.When light is incident on the surface of planar transparent substrates , it becomes partially reflected due to the large difference in refractive indices between air and substrates. Even for normal incidence, the light suffers from the Fresnel loss with a reflection coefficient , the light harvesting efficiency of OPVs with the similar dual‐side biomimetic moth's eye nanostructures was further increased, yielding an enhanced PCE of 9.33% without sacrificing the charge transport properties.Regardless of microscale textures, several designs of the nanostructures were conduced in OPVs as an AR coating, such as nano‐replicated periodic moth's eye structures, 1D or 2D diffraction gratings at the light incident surface.Hyun et al. proposed to directly attach a soft elastomeric stamp with a 2D hexagonal array of nanopillars onto the glass substrate to enhance light in‐coupling for OPVs.2) nanomaterials have attracted great interest in fabricating large‐area, low‐cost AR films. For example, Yun and co‐workers produced the SiO2 NPs‐based light scattering layer on the substrate surface in bendable OPVs with enhanced light absorption  technique with controlled deposition thickness. By optimizing the dimensional parameter of SiO2 NPs, the flexible OPV yielded a PCE of 7.4% with an improvement ratio of 13% compared to the reference device without a SiO2 NP layer. Alternatively, a nanostructured AR coating could be simply fabricated on the back surface of an ITO‐coated glass substrate by self‐assembling a monolayer of SiO2 nanospheres with the immersion into the negatively charged monodispersed nano‐SiO2 colloidal solution (≈126 nm in diameter).Recently, silica .3 layer with additional synergetic improvement in this hybrid electrode nanostructure. The corresponding PCE for top‐illuminated OPVs was enhanced by ≈34%, with an efficiency recovery up to 68%.For top‐illuminated OPVs, a transparent metal‐dielectric electrode is typically used as the top electrode.Taking advantage of conversion and manipulation of light via luminescent down‐shifting (LDS), Nam et al. developed a multifunctional transparent and luminescent LDS platform bearing sub‐wavelength nanopatterns in OPVs.2 NP‐based) techniques.Regardless of the superior AR capabilities from both microscale textures and biomimetic nanostructures, the following factors should be taken into account. First, different mechanisms are invovled for the AR process when using microscale textures or biomimetic nanostructures. The ray‐optical schemes using microtextures lead to multiple reflection of incident light on the organic photoactive layers, whereas the biomimetic nanostructures utilize the wave‐optical property of light to interact with the subwavelength structures. Second, the microscale AR coatings generally have the advantages of intuitive geometries, broadband response, and manufacture scalability. However, they are still limited by narrow angular tolerance and reflection loss induced by multiplied optical bounces. In contrast, the biomimetic AR coatings such as moth's eye nanostructures can simultaneously achieve broadband and omnidirectional anti‐reflectivity with polarization insensitivity in spite of the stringent specification of nanoscale structural design and fabrication. Besides inherent superiorities in thin form factor and self‐cleaning characteristics, these biomimetic nanostructures offer a great promise in producing large scale OPV panels at low cost through soft NIL  and solution processing ‐polarized light due to the large plasmonic field enhancement in the vicinity of the grating strips.Another style of plasmonic metal electrodes used in OPVs is depicted in Figure 5.25.2.11D PCs or dielectric multilayers are known as a distributed Bragg reflector (DBR), providing a way to manipulate the photon propagation. Photons with energies lying in the photonic band gap cannot propagate through the DBR.Figure71BM photoactive layer exhibited a PCE of 5.6% as well as 30% visible light transmission. In addition, modifying the layer structure can tune the device color without significantly altering cell performance.To enhance the trapping of the electromagnetic field in semitransparent OPVs over a wide wavelength range, 1D photonic structures are usually integrated above the rear semitransparent electrode. As shown in 3)/lithium fluoride (LiF) DBR system /LiF,According to the theoretical mapping of the electric field distribution in semitransparent OPVs with a DBR, the light trapping effect can be evaluated. In the case of molybdenum trioxide  and SiO2 units between ultrathin metal transparent electrode and glass substrate.In addition to the use of DBRs for rear metal electrodes, light trapping can be realized by modifying the transparent front electrodes by virtue of the DBR concept.5.2.2In the past few years, the emerging field of plasmonics has received extensive explorations in OPVs for light trapping enhancement at the nanoscale, well below the scale of the wavelength of light in free space.FigureCommonly, SPPs in the style of propagating light waves along the interface between a metal and a semiconductor material can be excited in OPVs by employing a periodically nanopatterned metal film, resulting in near field enhancement and improved optical absorption. The OPV structure with metallic gratings at the rear electrodes represents the typical light trapping scheme by the excitation of SPPs Figure8a.113 A comparison of 1D gratings and 2D patterns indicated that the patterned OPVs with 2D nanostructured metal rear electrodes will exhibit superior light harvesting capacity due to a polarization independent plasmonic response.3 layer. This pattern was subsequently translated to the Ag layer. After the PS beads were removed by a residue‐free adhesive tape, a nanostructured Ag rear electrode with protrusions that filled in the holes in the MoO3 layer was realized, showing the dependency on the diameter of the PS beads and the MoO3 thickness. Cheng et al. provided an alternative route to realize the plasmonic Ag rear electrodes with the self‐assembly feature of thermally deposited Ag at the sub‐monolayer thickness, which resulted in the formation of Ag NPs on top of the MoO3 layer with the excitation of localized SPRs under the solar illumination.In addition to SPPs, localized SPRs excited by randomly nanostructured metal films have been utilized to assist light harvesting in OPVs. To fabricate nonperiodic nanopatterns, Rand et al. introduced PS nanosphere‐templated colloidal lithography for patterning the Ag rear electrode of the nanostructured devices Figure b.125 A sAn alternative route to achieving SPP or localized SPR enhancement in OPVs is the incorporation of quasi‐random or quasi‐periodic nanostructures into metal electrodes. Unlike the case of perfectly periodic or totally random nanostructures, both broadband absorption enhancement and customizable spectral response can be offered for different photovoltaic semiconductors by quasi‐random nanostructures.5.2.3FigureN‐9′‐heptadecanyl‐2,7‐carbazole‐alt‐5,5‐]:‐phenyl C70‐butyric acid methyl ester (PCDTBT:PC70BM) BHJ. Instead of the near field effect commonly used in SPR‐enhanced OPVs, the dominant contribution of such a structure to the absorption enhancement was claimed from the fact that the metamaterial mirror enabled the device to operate closer to resonance by providing the desired reflection phase. As a result, an improved resonant recirculation of the light was achieved, which in turn increased the overall field intensity and the light absorption.Very recently, metasurfaces built up from 2D sub‐wavelength metallic building blocks have demonstrated unique responses to the incoming light that transcend those of natural materials.Large‐area metasurfaces composed of Ag NP arrays on Ag thin films coated with various photovoltaic absorber layers were investigated using dark‐field scattering spectroscopy Figure b.135 The5.35.3.1FigurePatterning both transparent front electrode and reflective rear electrode with novel optical structures will realize the synergistic effect on light trapping for optimizing the OPV performance. Recently, a series of nanostructured metal grating electrodes for light trapping enhanced OPVs were explored experimentally and theoretically. Nalwa et al. realized high‐efficiency corrugated OPVs based on 1D diffraction ITO gratings, where the grating dimensions with sub‐micrometer height topographies enabled a uniformly thick photoactive layer and a conformally structured metal grating rear electrode Figure10a.136Except for the periodic corrugation of the electrode pairs in OPV structures, another effective route is the use of self‐organizing wrinkles and deep folds as photonic structures that were formed on polymer surfaces induced by mechanical stress Figure b.30 The In addition, surface plasmon‐mediated absorption enhancement was demonstrated in bottom‐up corrugated small‐molecule OPVs with 1D, 2D periodic gratings, or dual‐periodic grating.5.3.23, TeO2, WO3) were utilized to manipulate the local electromagnetic field distribution in the microcavity cell.2 film in resonance with the Ag‐Ag microcavity, the total reflection of an ultrathin Ag film was theoretically reduced and an increased electric field was thus observed within the active layer  typically having a few tens of nanometers play an important role in OPVs, which can favor the efficient extraction of generated carriers from organic photoactive layers into the electrodes.FigureTo realize a plasmonic‐mediated layer for the light harvesting enhancement, the incorporation of metal NPs with different materials, sizes, shapes, concentrations or distributions into charge extraction layers (CELs) has recently been extensively investigated.e = 0), slight oblate spheroid (e = 0.75), and optimized spheroid (e = 0.89). It is found that increasing the spheroid eccentricity can provide an overall increase of scattering power across the entire spectral range with a broadened, red‐shifted localized SPR.Jsc were attributed to an increase in exciton generation induced by the strong local electric field and the scattering generated by localized SPRs of the hexagonal nanotriangle arrays. Similarly, self‐assembled Au nanopyramid arrays fabricated through the PS spheres template were demonstrated by Ren et al. to greatly enhance the photocurrent of OPVs due to the plasmonic near field effect, yielding an increase of up to 200% in PCE.Jsc by 23.2% was then obtained by optimizing the spacer layer thickness for extending the evanescent field into the photoactive layer and varying the geometry of the Au nanowires bands for favoring the enhanced scattering. The geometry effects of Au NPs with different morphologies  in the PEDOT:PSS HEL were compared, showing 29% increase in PCE for Au nanostars, 14% for Au nanorods, and 11% for Au nanospheres, respectively.The impact of NP shapes on the directionality of scattering and localized SPRs of metallic spheroidal nanostructures was investigated by Park et al.2 could preserve the localized SPR effect of Ag NPs by preventing oxidation of the Ag core under ambient conditions  as multifunctional HEL to facilitate hole extraction at the anode and light trapping enhancement with coupled plasmonic near field.Apart from dielectric shells, Kim et al. designed carbon dot‐supported Ag (CD‐Ag) NPs using the carbon dots both as a reducing agent and a template to realize versatile localized SPR Figure d.31 The 3‐covered Ag NPs adjacent to the photoactive layer, where the NPs with precisely controlled concentration and size were generated by atmospheric evaporation and mobility classification methods and the MoO3 layer was used to enclose Ag NPs via vacuum thermal evaporation to isolate the undulated active layer , leading to 20.7% enhancement in Jsc and a maximum PCE of 7.52% with an optimized concentration of Ag NPs.Different from the mixture of metal NPs and PEDOT:PSS, Jung and co‐workers constructed a nanobump assembly of MoOr Figure e.173 The2 EEL of OPVs was achieved with a PCE improvement ratio of 18%  as the plasmonic mirror enabled positive electrical effects and broadband absorption enhancement by the collective excitation and hybridization of localized SPR, SPP, and Floquet modes Figure d.204 Enh2 nanorods into the photoactive layer led to superior broadband absorption improvement in a solution‐processed small molecule OPV with 31% enhancement in PCE up to 8.72%.Instead of the utilization of both localized SPR and SPP modes, dual localized SPR enhancements totally by metal NPs have been demonstrated in enhancing light harvesting for OPVs. For example, simultaneously incorporating Au nanospheres into the HEL and Au@SiO11Ag89 alloy NPs embedded in the photoactive layer of OPVs resulted in 31% PCE improvement due to the enhancement of both light‐trapping and charge transport in the active layer  NPs held the potential to yield significantly greater plasmonic enhancement than Ag or Au, since much higher plasma frequency of Al ensured a better overlap between plasmonic resonance and absorption band of organic semiconductors.6.22 NPs into photoactive layer was also reported to result in 13% increase in PCE, while PCE was increased by 20% when SiO2 NPs were incorporated into charge extracting layers.In spite of the incorporation of metal NPs, dielectric nanostructures provide an alternative route to enhance the light scattering capacity for efficient OPVs.FigureFor the application of large‐area OPVs, the implementation of photonic structures directly patterned on the CELs and photoactive layers through continuous and scalable fabrication techniques are highly desirable. Photonic structure designs in photoactive layers of OPVs have attracted considerable attention, and theoretical considerations of photonic geometries suggested light harvesting efficiency enhancement with a periodic nanostructured polymer absorber film due to the strongly enhanced light‐matter interaction with maximized density of Bloch modes.Wei et al. constructed bilayer OPV by using electrochemical deposition (with PS beads as the template) to fabricate a PEDOT:PSS HEL with nanobowl array structures on ITO substrate Figure b.222 By FigureInstead of the necessity to precisely engineer the nanoscale periodic structures with pre‐specified spacings or at appropriate interfaces, the deterministic aperiodic nanostructures (DANs) suggest an interesting alternative to broadband, wide‐angle light manipulation, which offer unique advantages such as richer Fourier spectra.In addition, the implementation of nanostructuring the photoactive layer in OPVs may cause the change of polymer morphology and chain orientation.7We have comprehensively reviewed recent progress in the optical manipulation of light in OPVs at multiple scale from the perspective of plasmonic scattering and photonic resonances. Addressing the critical challenge of insufficient absorption efficiency in ultrathin photoactive films, a variety of light trapping schemes with the implementation of photonic structures have been demonstrated to contribute to improved OPV device performance through increased trapping and enhanced absorption in photoactive layers. It involves a brief comparison of light manipulation between microstructures in ray optics domain and nanostructures of wave optics. A major discussion on light trapping schemes corresponds to the different functional components and their interfaces in thin film OPVs. Antireflection coatings on the external cell surface are implemented at both microscale and nanoscale, without interference with electrical properties and complications in OPV fabrication. Some investigations on altering substrate geometries of OPVs are presented for inspirations of novel, effective cell architectures. Photonic designs on transparent conductors and reflective metal mirrors exhibit optical enhancement by plasmonic scattering and microcavity effects. Optically modified charge extraction and photoactive layers via metallic nanoparticles and photonic structures show strong plasmonic scattering and optical resonant enhancement.Recent noteworthy achievements in the use of efficient organic absorbers and the implementation of photonic elements are convincing and promising for OPV technology allowing the generation of electrical power at low cost on a very large scale. Taking into account different options and various degrees of freedom in device design, a holistic optical approach to highly efficient OPVs is realizing the best possible light harvesting efficiency without affecting device stability, or compatibility with flexible substrates. We anticipate that the confluence of the plasmonics, nanophotonics, and micro/nano fabrication will achieve new organic‐based photovoltaics. In particular, the promising strategy using non‐fullerene acceptors for low‐cost manufacturing has recently demonstrated high‐performance polymer BHJ OPVs with over 11% PCE and excellent thermal stability.


In this review, we discuss the emergence of the fourth‐generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution, thus enabling back mapping of sequencing reads to the original histological context. This information is used, for example, in two current large‐scale projects that aim to unravel the function of the brain. Also in cancer research, fourth‐generation sequencing has the potential to revolutionize the field. Cancer Research UK has named “Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis” one of the grand challenges in tumor biology. We discuss the advantages of sequencing nucleic acids directly in fixed cells over traditional next‐generation sequencing (NGS) methods, the limitations and challenges that these new methods have to face to become broadly applicable, and the impact that the information generated by the combination of in situ sequencing and NGS methods will have in research and diagnostics. Tissue is constituted of a complex organization of different cell types that are tightly regulated by the interplay of individual cells within it. Thus, to better understand the physiological and pathological status of normal or diseased tissue, decomposition of the complexity by single cell analysis is necessary. In recent years, next‐generation sequencing (NGS)‐based single cell RNA sequencing (scRNA‐seq) technology has been proven to be a powerful tool for different applications, for example, classifying cell subpopulations . In this new technology, a fresh‐frozen tissue section is deposited onto a chip containing an array of 100 μm features of unique sequence‐barcoded oligo‐dT capture probes equipped with sequencing adaptors. After imaging the tissue to record the positions of the cells relatively to the array, the sample is permeabilized and the mRNA diffuse onto the array of capture probes. The probes are then used as primers for cDNA synthesis ”on‐chip,” generating a sequencing library that can subsequently be retrieved and analyzed by NGS. Each read can then be mapped back to a feature based on its spatial barcode. While not offering single cell resolution in its current state, spatially defined regions can be analyzed transcriptome wide at a high throughput.Several sequential and combinatorial labeling and imaging approaches based on single molecule fluorescence ISH (smFISH) have been developed in order to expand the throughput of in situ RNA detection. Sequential staining methods can rely on one or multiple fluorophores. In the approach developed by Lubeck et al. , sets ofChen et al.  developeThe above‐described smFISH methods require high‐resolution optical imaging setups and are consequently limited in throughput. Therefore, they have only been demonstrated in single cells and not in whole tissue sections.Two pioneering methods have been developed that utilize second‐generation NGS chemistry to sequence single RNA molecules directly in fixed cells and tissues [Ke et al., ISS is potentially adaptable to diagnostics applications, which typically rely on tissue material that has been preserved by extensive cross‐linking followed by paraffin embedding: the formalin‐fixed, paraffin‐embedded (FFPE) tissues. Cross‐linking leads to covalent modification of nucleic acid bases as well as strand cleavage [Evers et al., β‐actin (ACTB) mRNA in cultured cells. We also sequenced short fragments of ACTB and HER2 transcripts in a breast cancer tissue, and sequenced codon 12 and 13 of the KRAS transcript to detect a KRAS mutation in rare  KRAS mutation‐positive cancer cells spiked into a background of KRAS‐negative cells.Our group has developed methods for in situ single RNA molecule detection with single nucleotide resolution by using padlock probes combined with rolling circle amplification (RCA) [Larsson et al., 4) different transcripts. We demonstrated this multiplexed mRNA detection approach targeting 39 different genes in frozen tissue sections of one ER‐negative and two ER‐positive breast tumors. The set of genes include the Oncotype DX 21 gene expression panel used to predict distant tumor recurrence in breast cancer patients [Sparano and Paik, Sequencing a short RNA fragment of four to six bases is good for the detection of SNVs and small deletions, but is not needed for the purpose of just detecting and quantifying a large number of transcripts. Therefore, in a second approach, we used our ISS protocol to sequence a molecular barcode of four bases in the nontarget hybridization part of the padlock probes, providing a coding capacity of up to 256 . According to recent scRNA‐seq studies, the brain consists of hundreds of subtypes of the different cell types. Understanding how they are connected and where they are located will, without doubt, enhance our understanding of neurological disorders and how the mind works in general. Cancer Research UK has named “Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis” one of the grand challenges in tumor biology. Our laboratory engages in several projects that ultimately aim at relating treatment response to spatial transcription profiles of tumors.Although current ISS fails to provide whole transcriptome profiles, mainly due to its limitation in sensitivity and molecular crowding, it has a great potential to become a tool that synergistically can be combined with scRNA‐seq technology. For instance, scRNA‐seq can identify and define cell‐types among a population of cells isolated from a tissue. The expression profile from these molecularly defined cell types can then be compared and matched to expression profiling generated by ISS on the same type of tissue or, even better, on a consecutive section of the same tissue. In this way, the cell types and states defined by scRNA‐seq can be mapped to their positions in the tissue, providing a more complete picture of spatial gene expression, whereas the ISS provides positional information for millions of cells based on a selection of biomarkers identified by scRNA‐seq, whereas the scRNA‐seq provides deep information about the transcriptional state of the molecularly defined cell types Fig. .In conclusion, fourth‐generation ISS provides a different and complementary paradigm to the analysis of genome and transcriptome using other NGS technologies. It may be possible to become a standard method for the sequencing of tissue samples if technical obstacles are overcome through further development of the technologies [Crosetto et al.,