The diversity of processed transcripts in eukaryotic genomes poses a challenge for the classification of their biological functions. Sparse sequence conservation in non-coding sequences and the unreliable nature of RNA structure predictions further exacerbate this conundrum. Here, we describe a computational method, DotAligner, for the unsupervised discovery and classification of homologous RNA structure motifs from a set of sequences of interest. Our approach outperforms comparable algorithms at clustering known RNA structure families, both in speed and accuracy. It identifies clusters of known and novel structure motifs from ENCODE immunoprecipitation data for 44 RNA-binding proteins.The online version of this article (doi:10.1186/s13059-017-1371-3) contains supplementary material, which is available to authorized users. As genomic technologies progress, an ever-increasing number of non-protein-coding RNAs (ncRNAs) are being discovered. Long non-coding RNAs (lncRNAs) are of particular interest for functional genome annotation given their abundance throughout the genome. So far, few lncRNAs have been functionally characterised, and those that have seem to be involved in regulation of gene expression and epigenetic states , 2. UndeMany ncRNAs lack sequence conservation, in contrast to protein-coding genes. Most small ncRNAs have well characterised secondary and tertiary structures, as evidenced in Rfam, the largest collection of curated RNA families (2588 families as of version 12.2 ). In conWe, and others, hypothesise that lncRNAs act as scaffolds for the recruitment of proteins and assembly of ribonucleoproteins (RNPs), mediated by the presence of modular RNA structures, akin to the domain organisation of proteins , 9–14. Pdot plots) was first introduced by CARNA [The identification of RNAs with similar functions involves comparing both their primary sequence and higher-order structures simultaneously. However, sequence-based methods to identify common structural features perform poorly when sequence identity falls below 60 % . Hence, by CARNA , 30, whiThese pairwise RNA structure alignment algorithms can be used to identify clusters of homologous RNA structure motifs from a set of sequences of interest. Will et al. first showed that a (dis)similarity matrix can be constructed from all-vs-all pairwise RNA structure alignments with the pairwise alignment tool LocaRNA, identifying known and novel groups of homologous RNAs using hierarchical clustering . HoweverHere, we describe a computational pipeline for the identification and clustering of homologous RNA structures from a large set of query sequences. At its core lies DotAligner, a heuristic pairwise sequence alignment algorithm that considers the ensemble of base pair probabilities for each queried sequence. We benchmark the performance of DotAligner with other pairwise RNA structure alignment algorithms through several iterations of a stochastic sampling strategy across all Rfam seed alignments, highlighting the speed and accuracy of our method. We combine DotAligner with density-based clustering for the unsupervised identification of RNA structure motifs, which can identify both known Rfam families and novel RNA structure motifs from ENCODE enhanced cross-linked immunoprecipitation (eCLIP) data. Finally, we exemplify how clusters of homologous RNA structures identified by our method can be used to search for homologous structures across reference genomes and transcriptomes to generate a map of functionally related RNA structure motifs.We developed an algorithm that leverages the diversity of suboptimal solutions from a partition function of RNA alignments to identify an optimal sequence-structure alignment of two RNAs. The algorithm, termed DotAligner, overcomes the limitations of comparing unique RNA secondary structures (such as minimum free energy predictions) to yield a pairwise alignment that considers mutual base pair probabilities. A schematic of how DotAligner functions is illustrated in Fig. DotAligner was developed with an emphasis on runtime performance to facilitate all-vs-all pairwise comparisons of RNA structures on large data sets. Consequently, it uses pre-calculated RNA dot plots to perform alignments. It also uses the observation that a significant subset of stochastic sequence alignments between two RNAs will overlap the correct structure-based alignment, even though the optimal sequence alignment deviates significantly from the structural alignment . The algWe first tested DotAligner on BRAliBase 2.1 pairwise RNA structure alignments, a reference data set specifically designed for algorithm benchmarking , 35  scores above those from the BRAliBase 2.1 reference alignments . As justified below, the benefits of sampling are outweighed by other properties of the algorithm.DotAligner’s capacity to produce competitive pairwise alignments is demonstrated via a 5S-adenosyl methionine riboswitch  to control for and ascertain any sequence-dependent biases (see ‘Methods’). DotAligner alignment scores were then compared to a binary classification matrix representing the distinct Rfam families . The OPTICS (Ordering Points to Identify the Clustering Structure) algorithm  was chosIn comparison to GraphClust, the combination of DotAligner and OPTICS performs comparably well  that overlap evolutionarily conserved secondary structure predictions, 25 significant clusters of homologous RNA were detected, including all 11 positive controls . This pipeline was applied to eCLIP sequencing data from 44 RNA binding proteins from the ENCODE consortium , with 10spike-in Rfam sequences facilitate the identification of similar RNA structures, such as the homologues to SNORNA72 depicted in Fig. Indeed, the Our method also identifies RNA structure families impartially, as exemplified by several clusters of DKC1-associated sequences, which present consensus secondary structures indicative of snoRNAs Fig. e. CloseThe increasing accessibility of next-generation sequencing and immunoprecipitation protocols provides large resources for in-depth transcriptome and interactome profiling. Elucidating the structural features of RNAs associated with RNA-binding proteins and RNP complexes, combined with the systematic classification of their genome- or transcriptome-wide occurrence, can identify recurrent functional motifs that may form components of regulatory networks. Pragmatically, the method we describe facilitates this process by enabling rapid and unsupervised clustering of RNA structure motifs with reasonable accuracy. We also show that clustering eCLIP sequences can identify new RNA structures and their homologues throughout the genome similarity matrices for cluster analysis than other pairwise structure alignment algorithms, or at least produce them with reasonable computational power. In addition to its speed, the strength of DotAligner lies in its capacity to accurately score structurally homologous RNA sequences and the suboptimal structural landscape of RNAs, reducing several dimensions of information into a single discriminative numeric value. Our results show that this can be sufficient to extract structurally and functionally related sequences from a large amount of noisy input. It is an ideal application for screening high-throughput sequencing data – such as RNA immunoprecipitation data – for common structural motifs.The algorithm generates pairwise alignments that differ qualitatively to reference structural alignments at lower ranges of sequence identity, but it performs better than more complex algorithms within ranges of sequence similarity that substantially overlap those of functionally related RNAs, as presented in Rfam. This could be a consequence of refining the runtime parameters through testing on independently and stochastically sampled Rfam sequences. It is not impossible that other algorithms could undergo comparable parameter optimisation. However, the significantly higher computational complexity of other related tools compared to our method makes it fairly difficult (and resource intensive) to perform such brute-force parameter optimisation.High-throughput CLIPseq data pose a challenge for the detection of consensus motifs since several molecules that are in close physical proximity to the target molecule can co-precipitate together. Consequently, other RNA sequences that do not directly bind to the target protein may be present. We have shown that our method is, nonetheless, suitable for such noisy biological data. For example, the UPF1 cluster we describe may be an example of an indirect binding event, as UPF1 directly interacts with STAU1, a double-stranded RNA-binding protein that has been reported to target ALU sequences . Other cDotAligner has several variables that can influence the clustering results and speed depending on the type of input data. The most influential variables are the weight between sequence and structure similarity, and the exploration depth of suboptimal alignments in the stochastic backtracking. We have shown that stochastic sampling of suboptimal string alignments improves the alignment of RNA dot plots. However, the performance increase does not outweigh the increase in runtime associated with sampling suboptimal sequence alignments. Our Rfam clustering benchmark using a binary classification strategy has shown that the best trade-off between alignment accuracy and speed comes with the abandonment of sampling, as supported by the de novo structures identified from the ENCODE eCLIP data. Future optimisation of DotAligner parameters will likely increase its usability. For example, dynamic parameters could be implemented that adjust the degree of sampling diversity and number of samples based on the sequence identity obtained from step 1 of DotAligner. This could tune the algorithm’s performance based on the nature of the input, potentially improving DotAligner’s performance across all ranges of sequence identity. Another potential enhancement could be achieved in the stochastic sampling by considering only elements of the ensemble with probabilities larger than a threshold. By doing so, we could (1) reduce the number of useless samples, (2) guarantee that cells of high probability are passed  and (3) leave time/samples to explore the ensemble space  around these suboptimals.Another great challenge lies in the accurate depiction of RNA structure motif boundaries. Whereas global structures may stabilise the RNA molecule, local structural domains are often sufficient for recognition by RNA binding proteins. A strategy to find optimal local alignments would be desirable for this. DotAligner can search for semi-local alignments by introducing penalty-free gaps at the sequence extremities . In this study, we did not investigate the optimisation of these local pairwise similarity scores, because they may miss parts of the functional units (RNA structure) and, hence, hinder the search for optimal clusters. Instead, we circumvented this issue by overlapping eCLIP peaks to evolutionarily conserved RNA secondary structure predictions with well-characterised flanking helices supported by base pair covariation . While pAn efficient pairwise RNA sequence alignment heuristic, which intrinsically considers suboptimal base pairings, accurately discriminates between distinct structured RNA families. When combined with a noise-tolerant density-based clustering algorithm, this approach identifies known and novel RNA structure motifs from a set of input sequences without a priori knowledge. The resulting RNA structure motifs are subsequently used to identify homologues in the human genome, improving the annotation of lncRNAs and increasing the repertoire of functional genetic elements.k and t from 0 to 1 in increments of 0.1; o and e from 0.2 to 1 in increments of 0.2. For each set of parameter combinations, the number of alignments producing identical structural topologies to the reference alignment was determined using RNAdistance, SCI, a robust measure of RNA structural alignment integrity [predicted and reference RNA secondary structure were also calculated for all resulting alignments: The DotAligner algorithm implements several parameters that first need to be tuned before it can be applied to biological sequence analysis. All combinations of core parameters were tested on the 8976 pairwise RNA structure alignments curated in the BRAliBase 2.1 reference data set . We firsntegrity  based onThe baseline parameters were then selected via a product rank of these two metrics and subjected to refinement using a binary classification approach, described in the next section.Further refinement of the optimal parameters was performed using a binary classifier for two sets of 200 stochastically sampled Rfam entries with published structures: (i) a low pairwise sequence identity (PSI) set and (ii) a high PSI set, where any two sequences from the same family share between 0–55 % and 56–95 % PSI, respectively Fig. a, b. Thx and y have a score of 1 if they belong to the same Rfam family, or a score of 0 if they do not. The similarity matrix resulting from all-vs-all pairwise comparisons with DotAligner was tested for accuracy using the AUC of the receiving operator characteristic, as calculated by the R package pROC [θ=0.5, κ=0.3, go=1 and gext=0.05  is the weight of sequence similarity compared to the similarity of unpaired probabilities, κ (or -k) is the weight between sequence-based similarity and dot plot similarity, go (or -go) is the gap opening penalty and gext (-go) is the gap extension penalty. Sampling-specific parameters s (number of samples) and T (measure of sampling diversity) had minimal impact in the refined parameter optimisation from sampled Rfam clans, although the parameters can increase alignment scores in low and medium pairwise sequence identity ranges  variant, we ran the program with default parameters, whereas for the NoFold (filtered) variant, we reduced the feature space to 1902 CMs by removing Rfam families from our benchmark set.The following clustering performance metrics were used: https://www.encodeproject.org/search). The resulting 5,040,096 peaks were filtered to keep only those with ≥eightfold enrichment over the total input background and an associated P value ≤10−4. Furthermore, peaks were merged if they overlapped by more than 50 nt to avoid over-representing the same sequence  using the command opticsXi, xi = 0.006, minimum=T). The sequences for each cluster were then extracted and submitted to multiple structure alignment with mLocaRNA version 1.9.1 using parameters --probabilistic --iterations=10 --consistency-transformation --noLP.The normalised similarity matrix resulting from all-vs-all pairwise comparisons with DotAligner was then subjected to clustering with the dbscan 1.1-1 R package from Michael Hahsler Additional file 2Supplementary tables and figures with descriptions. (PDF 3890 kb)


As synthetic biology advances the intricacy of engineered biological systems, the importance of spatial organization within the cellular environment must not be marginalized. Increasingly, biological engineers are investigating means to control spatial organization within the cell, mimicking strategies used by natural pathways to increase flux and reduce cross-talk. A modular platform for constructing a diverse set of defined, programmable architectures would greatly assist in improving yields from introduced metabolic pathways and increasing insulation of other heterologous systems. Here, we review recent research on the shell proteins of bacterial microcompartments and discuss their potential application as “building blocks” for a range of customized intracellular scaffolds. We summarize the state of knowledge on the self-assembly of BMC shell proteins and discuss future avenues of research that will be important to realize the potential of BMC shell proteins as predictively assembling and programmable biological materials for bioengineering. Figure 1A). Because of their self-assembly properties, proteins containing the pfamdomain 00936 (pfam0936) are promising building blocks that can be repurposed to design novel protein scaffolds in vivo, distinct from their natural propensity to assemble bacterial microcompartments (BMCs). In this perspective, we discuss current research on pfam0936 proteins in the context of their potential as a biological material for the construction of custom nano-architectures and intracellular scaffolds.With the advent of synthetic biology and recent advances in protein engineering, designing, constructing, and controlling biomolecule-based materials at the nanoscale is a rapidly developing field. Currently, there is a lack of modular building blocks for predictably fabricating custom sub-cellular architectures that can be subsequently programed with precise functions  is perhaps the best developed molecular building platform . We discuss the promise and potential limitations of this strategy below.Toward this goal, engineering naturally found proteins which self-assemble into defined, nano to macromolecular architectures offers a powerful base to approach artificial scaffold construction ; the comFigure 1B) [summarized in Figure 1B; Figure 1B; Numerous crystal structures of pfam0936-containing proteins have contributed to a detailed structural understanding of BMC shell proteins and models of how they “tile” into the facets of BMCs . We find that expression of BMC-H homologs PduA, MicH, RmmH, and CcmK2 form varied macromolecular assemblies in vivo , generally in agreement with prior reports. PduA and RmmH form nanotube-like structures (Synechococcus elongatus PCC 7942), over-expressing these homologs in E. coli does not lead to the formation of prominent macromolecular structures . It is unclear if the absence of visible structures via transmission electron microscopy (TEM) thin section represents a lack of higher-order self-assembly, or if smaller assemblies are formed in the cytoplasm which are insufficiently discriminated from other cytoplasmic elements; as was previously proposed for other smaller BMC assemblies  contribute to differences in self-assembly are currently unknown.Because the methodology used to express BMC-H proteins varies among labs and studies , it is not always clear if the distinct intracellular structures generated by BMC-H homologs in separate reports are due to intrinsic self-assembly properties, or the specific experimental conditions. Nonetheless, multiple lines of evidence suggest that properties of BMC-H proteins predispose them toward specific higher-order architectures . To illuructures  and MicHructures . Despitesemblies . CollectFigure 1D), there must be subtle primary structure differences that dictate changes in higher-order assembly. One region anticipated to influence assembly dynamics surrounds residues at the inter-hexamer junction; although there is strict conservation of some sidechains at this interface, some positions exhibit variance across homologs . Supporting this hypothesis, experimental evidence generated by targeted amino acid substitutions at interface residues of PduA indicate they influence macromolecular assembly in vivo . Longer C-terminal extensions  originally were hypothesized to interfere with lateral molecular tiling through steric clash, a hypothesis partially supported by the observation of that truncation mutants form hexamers which pack more tightly in crystal lattices  is one emergent technique because of the high spatial and temporal resolution it affords. HS-AFM was used to capture the individual changes in BMC-H proteins association/dissociation rates into larger sheets . DynamicSynechocystis sp PCC 6803)  . In thesCC 6803) . In turnCC 6803) .Figures C). Employing the Thomas-Dill (Mycobacterium smegmatis) or two CcmK2 hexamers (S. elongatus 7942). The PMF was calculated as a function of the distance between the center of mass of each hexamer tile and the angle 𝜃 . CcmK2 and RmmH structures were obtained by relaxing the corresponding crystal structure in an aqueous environment . The angular dependence of PMF for CcmK2 and RmmH pairs is shown in Figure 2B; the PMF of RmmH has a clear minimum at 𝜃 ∼-45°, whereas CcmK2 is, by comparison, flatter. This minimum association angle of RmmH is consistent with the formation of regular repeating curved surface akin to the model created of in vivo/in vitro RmmH nanotubes , where the PMF of the relaxed pair lacks any clear minimum. This is notable because the backbone root mean-square deviation between the two structures only differs by 0.67 Å. The distinct PMFs are caused by differences in side-chain rotamers altered in the relaxation procedure. These small structural changes are enough to cause a difference of almost 1 KBT, indicating that subtle structural changes which may occur between crystallization and in situ conditions can have a profound impact on self-assembly; also the overall lower PMF values for CcmK2 suggests a net weaker hexamer–hexamer interaction, consistent with other predictions . It is also noteworthy to mention the synergy between crystallography and electron microscopy for solving the macromolecular interactions in whole BMC assemblies , often they do not possess defined geometry  or are nications . In a laFigure 2D). In the simplest case, scaffolding proteins to single hexamers or very small tiled arrays would be expected to concentrate up to 6 proteins per hexamer into cytosolic microdomains, akin to the earliest synthetic scaffold designs (Furthermore, the ability to construct different pfam0936 domain architectures raises the possibility that specific geometries can be chosen to tailor them for a given scaffolding application ( designs . Steppin designs . More co designs . It is w designs , the abiin vivo nanometer scale molecular engineering for the design of programmable architectures.Broadly speaking, we have outlined the promise and hurdles inherent to the use of pfam0936 domain containing proteins as building blocks for designer scaffold assemblies. One strength of using this domain to build ordered protein assemblies rests on the potential to construct a multitude of different intracellular assemblies from one common, protein domain. Even though the majority of published examples of higher-order pfam0936 assemblies are in prokaryotes, a recent report has documented engineering of several distinct higher-order structures in plant chloroplasts  indicatiEY conceived the project ideas, conducted experiments and analyzed data, and wrote and edited the manuscript. RB, JM, BS, and MF-C contributed project ideas and experiments related to the computational work, and wrote and edited the manuscript. CK and DD conceived project ideas and wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The second iteration of the Autism Brain Imaging Data Exchange (ABIDE II) aims to enhance the scope of brain connectomics research in Autism Spectrum Disorder (ASD). Consistent with the initial ABIDE effort (ABIDE I), that released 1112 datasets in 2012, this new multisite open-data resource is an aggregate of resting state functional magnetic resonance imaging (MRI) and corresponding structural MRI and phenotypic datasets. ABIDE II includes datasets from an additional 487 individuals with ASD and 557 controls previously collected across 16 international institutions. The combination of ABIDE I and ABIDE II provides investigators with 2156 unique cross-sectional datasets allowing selection of samples for discovery and/or replication. This sample size can also facilitate the identification of neurobiological subgroups, as well as preliminary examinations of sex differences in ASD. Additionally, ABIDE II includes a range of psychiatric variables to inform our understanding of the neural correlates of co-occurring psychopathology; 284 diffusion imaging datasets are also included. It is anticipated that these enhancements will contribute to unraveling key sources of ASD heterogeneity. At the macroscale, abnormal connections among brain regions have been revealed by functional and structural neuroimaging in children, adolescents and, adults with ASD. Yet, both the complexity of the brain connectome and the striking heterogeneity of ASD have hampered efforts to specify the nature of putative dysconnections. In response, open-data sharing is increasingly being encouraged to rapidly amass the large-scale datasets needed to confront heterogeneity, engage a broader range of scientific disciplines, and facilitate independent replications. To bring the open data sharing model to autism neuroimaging, the Autism Brain Imaging Data Exchange (ABIDE) was launched in 2012. The initial ABIDE initiative—now termed ABIDE I—was the first open-access brain imaging repository of resting state functional magnetic resonance imaging (R-fMRI) and corresponding structural data of individuals with ASD and typical controls  aggregated from multiple international institutions. Here, we introduce ABIDE II (Data Citation 1), a new multi-site open data resource containing 1,044 independent datasets  created to enhance the significance of the questions that can be addressed regarding the neural correlates of ASD and accelerate the pace of discovery.Multiple sources of evidence have substantiated models of abnormal neural connectivity in autism spectrum disorder (ASD)—a practice that has plagued biological psychiatry and neuroscience more broadly. Additionally, as recently demonstrated, the utility of datasets for prediction increases with sample size—even if heterogeneous data sources are used to amass large samples.The initial ABIDE I effort established the feasibility of aggregating multisite data without prior harmonization, leading to more than 55 peer-reviewed studies in the 48 months since inception. Despite its success, ABIDE I is limited in regard to sample characterization and sample size. Specifically, despite containing more than 1,000 datasets, ABIDE I was not sufficiently large to furnish optimally sized discovery and replication subsamples. By combining the ABIDE I and ABIDE II data resources, investigators can select larger samples for discovery and replication, depending on their investigative endeavors. Replication samples are needed to minimize false positives and avoid settling for ‘approximate replications’. Accordingly, ABIDE II actively encouraged investigators to provide phenotypic information regarding co-occurring illness, if assessed. The second source of heterogeneity is driven by sex-related differences. These have been generally ignored in the ASD imaging literature due to the markedly higher prevalence of males with ASD and the tendency of single sites to exclude or minimally represent females. The ABIDE II sample has increased the number of available datasets from females with ASD from 65 in ABIDE I to 138 when ABIDE I+II are combined. We believe these enhancements will allow investigators to more directly investigate pathophysiology specific to ASD, to potentially identify neurobiological subgroups and facilitate the identification of protective and risk factors.Along with increased sample size, ABIDE II provides greater phenotypic characterization than was available across the ABIDE I data collections to better address two key sources of heterogeneity. The first is psychopathology co-occurring with ASD, which has been largely overlooked in the imaging literatureN=284) with diffusion-weighted images. In order to facilitate immediate access and use of ABIDE II, the methods utilized to generate this resource, the resulting currently available data and their technical validation are described below.Finally, beyond its focus on intrinsic functional connectivity and other indices of intrinsic brain function, ABIDE II now includes a subset of datasets  measures incorporated into ABIDE II).Contributors are encouraged to share at least 20 unique datasets per diagnostic group . Data collections of only individuals with ASD are also accepted as they can be utilized for data-driven explorations addressing heterogeneity e.g., refs The availability of minimal phenotypic information essential for data analyses and sample characterization  is required for contribution. To enhance phenotypic characterization, sharing of additional measures commonly used in ASD research, information on psychiatric comorbidity, medication status, cognition and/or language are highly encouraged. Similarly, to enhance the breath of investigations about the ASD connectome, whenever available, contributions of corresponding diffusion images for each individual are welcome for aggregation.Finally, prior to data contribution, sites are required to confirm that their local Institutional Review Board (IRB) or ethics committee have approved both the initial data collection and the retrospective sharing of a fully de-identified version of the datasets .n=23 ASD and n=15 controls) previously contributed to ABIDE I. Given the relevance of developmental changes, these datasets are also included in the ABIDE II. To distinguish them from the cross-sectional aggregates, these datasets are organized into a separate set of collections focused on longitudinal data using the original ABIDE I IDs.Of note, two institutions provided longitudinal MRI scans from subsets of individuals’ datasets  for review and harmonization within and across sites. Specifically, MRI data are visually inspected and edited as needed to ensure complete removal of facial information. Additionally, to further protect personal privacy, images of ears are removed from high-resolution images. Regarding phenotypic datasets, each entry is also reviewed to identify and correct missing data, any impossible entry values , and extreme outliers (relative to each sample). To ensure uniformity across sites, all entries are recorded as needed and organized in a common template along with a legend of code keys. As a final step in preparation for release, both donating and coordinating sites jointly prepare a narrative for each data collection, documenting information on the methods utilized, funding sources, the investigators involved, whether any link with other databases  exists, along with publications related to the contributed datasets. Before open release, each donating site reviews their reorganized phenotypic records, five random images per imaging modality and their collection-specific narrative for final approval.Prior to contribution, each institution is asked to rename all data by replacing local subject identification numbers with FCP/INDI identifiers. They are also asked to remove personally identifying information (PHI) including those from images  using the FCP/INDI anonymization script available in The current ABIDE II dataset encompasses 17 collections of unique independent datasets  yielding 487 datasets classified as ASD and 557 as controls , Table 1http://fcon_1000.projects.nitrc.org/indi/abide/). The datasets are organized by data collection and stored in.tar files, each containing imaging and phenotypic data.All ABIDE II datasets can be accessed, after establishing a login and user password, through FCP/INDI at the Neuroimaging Informatics Tools and Resources Clearinghouse  files. A legend describing each phenotypic variable source is available at the website  and/or Autism Diagnostic Interview-Revised ;  or 2) by using these ‘gold standard’ diagnostic instruments only , with one exception. Specifically, in EMC_1 (n=27 datasets), which was selected from the longitudinal Generation R sample, the ASD classification was based on prior medical records documenting ASD among those individuals meeting screening cutoffs in at least one of two distinct ASD questionnaires or for whom the mother reported a diagnosis of ASD. Regarding controls , all datasets are characterized by absence of ASD diagnosis and absence of history of any other major neurodevelopmental disorders for the vast majority of the datasets . This was determined using semi-structured/unstructured in-person interviews , or parent/self- (if adults) reports/questionnaires . The remaining 11 control datasets (OHSU_1 data collection) are from individuals assigned a ‘rule out’ psychiatric disorder, but without ASD or Attention-Deficit/Hyperactivity Disorder (ADHD) diagnoses.A dummy variable indicates diagnostic group . Given the retrospective nature of this data aggregate, assessment protocols used to identify ASD and controls varied across institutions. They are documented in each data collection narrative. Briefly, ASD classification was determined by either 1) combining clinical judgment with ‘gold standard’ diagnostic instruments—Autism Diagnostic Observation ScaleOther specific inclusion/exclusion criteria used for selecting controls  or ASD  varied across collections. Each collection narrative on the ABIDE II website provides details regarding these criteria., 15% of the ASD datasets consist of females versus 31% of the control datasets —and eight enrolling only children and/or adolescents. The remaining five data collections include children, teens and young adults, which allows for cross-sectional age-related explorations . All butdatasets .P<0.0001; Full scale intelligence quotient (FIQ) and/or verbal and/or performance IQ standard scores are provided. Across collections, although variation exists with respect to the minimum FIQ, 97% of the datasets have FIQ above 80 . For botn=244 ASD and n=327 controls). Across collections, right-handedness is more frequent in both diagnostic groups , though a significantly higher prevalence of non-right-handedness (either left or mixed handedness) occurs in ASD relative to controls  recently designed to adjust for differences in age, intellectual abilities and language skills across ADOS modules. As illustrated in  scaled total scores are available for 15 collections (n=280 ASD datasets). Additionally, data from parent or self-report questionnaires commonly used in the field to quantify severity on multiple ASD domains collected across both diagnostic groups are also available. The Social Responsiveness Scale is the most common  and/or as severity scores in one or multiple psychopathology dimensions across available for 11 collections  categorical diagnostic labels  with corresponding diagnostic code based on the International Classification of Diseases-9th editionions see . Categoriagnoses ; the mosFor each of the 17 ABIDE II cross-sectional collections, for each unique ID#, at least one structural MRI (sMRI), one corresponding R-fMRI dataset are available ; corresponding diffusion MRI (dMRI) datasets are available for six collections. One data collection (SDSU) provided field map-corrected version of its R-fMRI and DTI data. The two pilot longitudinal collections include sMRI and R-fMRI datasets collected at two time points (1–2 years apart) for 23 individuals with ASD and 15 controls.Consistent with its popularity in the imaging community and prior usage in FCP/INDI efforts, the NIFTI file format was selected for storage of the ABIDE II MRI datasets. With the exception of a single collection , all MRI data were acquired using 3 Tesla scanners. . The rationale of this decision includes the lack of consensus on optimal quality criteria in regards to specific measures or their combinations and cutoffs. Additionally, depending on the study goal, the availability of scans with a range of quality can facilitate the development of artifact correction techniques. For initiatives focusing on clinical populations like ABIDE II, the inclusion of datasets with artifacts such as motion are valuable, as they enable investigators to determine impact of such real-world confounds on reliability and reproducibility.Consistent with the established FCP/INDI policy, all data contributed to ABIDE II was made available to users regardless of data quality (http://preprocessed-connectomes-project.github.io). These encompass quantitative metrics commonly used in the imaging literature for assessing data quality, particularly for multisite projects, e.g., ref.  artifactual voxel detection as well as temporal metrics including those quantifying head motion; all metrics are summarized in To facilitate quality assessment of the ABIDE II collections and selection of datasets for analyses by individual users, we used the Preprocessed Connectome Project quality assurance protocolAs data aggregation followed independent data collections across multiple sites, various sources of heterogeneity exist between collections. They can range from inclusion/exclusion criteria, recruitment/sampling strategies, MRI scanner types, data acquisition parameters and instructions . Users must be aware of such factors when designing their research questions and selecting data for analyses accordingly. Care should be taken when attempting to draw comparisons across ABIDE I and ABIDE II, as they are independently created aggregate datasets, bringing with them both commonalities and differences. Nine institutions participated in both initiatives with either related collections in regard to both phenotypic and imaging protocols  or collections acquired through independent protocols . We suggest consideration of the commonalities and differences among contributions when attempting to combine datasets from the two ABIDE initiatives. The narratives included in the ABIDE II website should facilitate this process—see .The impact of known and unknown sources of heterogeneity between collections should also be taken in account at the analytical level. First, we encourage the use of standardization at individual- and group-level analyses e.g., refs The need for careful consideration of variation in acquisition parameters also applies to the use of the quality assurance (QA) metrics available for the ABIDE-II sample. Some QA measures may be more or less comparable across data collections. Mean FD is an example of a measure commonly used for QA in resting state fMRI studies, albeit without significant considerations on the impact of the specific acquisition protocol employed. Motion-induced fluctuations in the BOLD signal are primarily due to spin history effects and partial voluming, which are proportional to the amount of tissue displacement between subsequent excitations. From this perspective, one might expect that factors capable of impacting spin history effects or partial voluming, would in turn impact meanFD. Importantly, these relationships may not necessarily be linear or additive. As such, some caution is suggested when interpreting systematic differences in meanFD, or related motion metrics , across collections. Users may also employ this and other shared multisite datasets e.g., refs http://fcon_1000.projects.nitrc.org/indi/abide/manuscripts.html). Users are also requested to acknowledge the primary funding source for ABIDE II (NIMH 5R21MH107045) in any manuscripts using the ABIDE II data.Finally, to facilitate replications among studies using ABIDE data, we encourage users to provide the ID list utilized for their published manuscripts in the manuscript section of the ABIDE website .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Elephas maximus and Loxodonta africana) diagnosed with a variety of inflammatory conditions. Twenty-five of 27 elephants had morphological changes in their leukocytes, although only 16 of these had a concurrent inflammatory leukogram. Morphological changes included heterophil left-shifting with or without concurrent dysgranulopoiesis, toxicity, or hypersegmentation, reactive lymphocytes, plasma cells, and/or vacuolated monocytes. Although the observed leukocyte morphological changes are non-specific, their early recognition upon blood film evaluation may provide important, clinically-relevant information, particularly if the leukogram is normal. This case series is the first description of qualitative morphological changes in the leukocytes of elephants in association with inflammation.Although the hematology of healthy elephants has been well-described, published information on hematological changes during disease is limited. The objective of this study was to describe qualitative morphological changes in the leukocytes of Asian and African elephants ( Elephas maximus) and African (Loxodonta africana) elephants managed in human care. Both hematological reference intervals and normal leukocyte morphology of healthy elephants have been reported. Elephants, like other members of the Afrotheria, especially the well-studied hyraxes and manatees possess leukocytes with several unique features. These include having heterophils instead of neutrophils and a unique monocyte type with a bilobed—but sometimes tri-lobed—nucleus and peroxidase-positive cytoplasmic staining. This unique monocyte is observed in addition to the typical monocyte type of other mammals [The complete blood count (CBC) is an essential part of the minimum baseline diagnostics in Asian -coated vacuum tubes from which two blood films were prepared within one to two hr of blood collection. The blood films were air-dried, placed in cardboard or plastic slide containers, then shipped overnight to a clinical pathology laboratory along with the EDTA-preserved blood tubes on cold packs. Upon arrival at the lab, samples were kept at refrigerated temperatures (between 0 and 4°C) until processing within two hours of arrival. For comparative purposes, in a few cases, an additional set of blood films was made at the laboratory, more than 24 hr after the blood was drawn. An automated analyzer  performed a routine CBC on each sample including a WBC count. The WBC count from the analyzer was used for the calculation of absolute WBC numbers based on a 200 WBC differential by blood film evaluation. An inflammatory leukogram was defined as a WBC count greater than >13.6 K/μl, a heterophil bands greater than 0 K/μl, segmented heterophils greater than 3.2 K/μl, lymphocytes greater than 2.7 K/μl, and/or monocytes greater than 7.1 K/μl. These numbers are the mean from previously published reference intervals for captive elephants [All blood films were stained with Wright–Giemsa  prior to a full blood film evaluation . The WBCTwenty-seven elephants, consisting of 16 females and 11 males, were identified with the following clinical diagnoses: non-specific gastrointestinal disease (n = 12), salmonellosis (n = 2), chronic foot disorders (n = 5), elephant endotheliotropic herpesvirus (EEHV) infection (n = 4), dental issues (n = 2), a retained fetus causing metritis (n = 1), and metastatic ovarian adenocarcinoma (n = 1). Twenty-two of these elephants had multiple samples submitted during the course of their disease. Elephant ages ranged from 5 mo to 63 yr. Elephants included 17 Asian and 10 African elephants. Twenty-four elephants fully recovered from their clinical conditions. Three elephants died of their medical problems and included an African elephant that developed metritis then sepsis due to a retained fetus, an Asian elephant with metastatic ovarian adenocarcinoma, and an Asian elephant with EEHV .Twenty-five of the 27 elephants had morphological changes in their leukocytes. Two adult Asian elephants with mild chronic foot disorders did not have morphological changes in their leukocytes although both had an inflammatory leukogram characterized by a mild monocytosis. In elephants with leukocyte morphological changes, one or more of the following were observed: heterophil left-shifting with or without concurrent dysgranulopoiesis, heterophil toxicity, hypersegmented heterophils, reactive lymphocytes, presence of plasma cells, and vacuolated/activated monocytes Figs  and 2. TThe most frequently observed leukocyte change was heterophil left-shifting. Heterophil left-shifting was characterized in most cases by the presence of band heterophils. Five elephants exhibited left-shifting that also included heterophil metamyelocytes and myelocytes, accompanied in four elephants by secondary dysgranulopoiesis  characterized by large heterophils. Earlier heterophil precursors such as promyelocytes or myeloblasts were not present in any cases. Heterophil left-shifting often occurred concurrently with toxicity, which was characterized by the presence of Doehle bodies and variable cytoplasmic basophilia on a scale of 1+ to 3+ per standard hematology practice . Figs 3 ToxicityReactive lymphocytes were identified based on the presence of increased amounts of cytoplasm, increased cytoplasmic basophilia, and/or increases in cell size. Plasma cells were present in two elephants and were similar in morphology as described in other species. Activated monocytes were characterized by the presence of variable degrees of vacuolation. Rare trinucleated macrophages were also observed.At time of recovery, 22 elephants with initial WBC changes no longer had leukocyte morphological changes in their follow-up blood film evaluations. In contrast, the three elephants that died of their medical problems exhibited increasingly severe heterophil toxicity and left-shifting as their conditions progressed. In the two elephants with chronic foot problems that did not have leukocyte changes initially, no morphological changes were noted in follow-up blood evaluations either.Leukocytes showed several morphological changes after 24 hr of storage that included cell and/or nuclear swelling, cytoplasmic vacuolation, karyorrhexis, and/or pyknosis. Heterophil cell swelling could be clearly identified as storage artifact if it occurred alone and without toxic cytoplasmic changes.Mycobacterium tuberculosis [This is the first description of qualitative morphological changes in Asian and African elephant leukocytes associated with various inflammatory conditions. The observed morphological leukocyte changes were non-specific for the underlying conditions and there were no obvious species differences in response to inflammation. Leukogram variations in an individual animal, especially subtle changes, require accurate identification of cells to be made during the WBC differential by blood film evaluation. This is especially important with regard to the differentiation of elephant monocytes and lymphocytes, and correct identification of left-shifted heterophils. Quantitative leukogram changes provide important clinical information and can occur with various infectious diseases. In several reports, elephant endotheliotropic herpesvirus (EEHV) viremia in Asian elephants correlated with significant leukopenia and thrombocytopenia, whereas resolution of viremia was associated with leukocytosis and thrombocytosis ,6. Otherrculosis .As this descriptive case series shows, even elephants with normal leukograms may have morphological changes in their leukocytes indicative of inflammation. Although low numbers of band neutrophils are considered normal in other mammalian species, band heterophils or cytoplasmic toxic changes in heterophils should not be present in blood films from healthy Asian or African elephants ,2. Left-Abnormal lymphocyte morphology observed in elephants was very similar to typical morphological changes described in other mammals with inflammation. A reactive lymphoid population is characterized by a heterogenous mixture of lymphocytes exhibiting morphological features such as variation in size, increased cytoplasmic basophilia, and/or increased amount of cytoplasm .In contrast to reactive lymphocytes, activated monocytes are defined by the presence of variable numbers of cytoplasmic vacuoles. Since the unique bilobed elephant monocyte may present in various stages of maturation, i.e. slight indentation of a round to oval nucleus, it is necessary to emphasize the importance of the proper differentiation of lymphocytes and monocytes as a basis for an accurate WBC differential.While very low numbers of reactive lymphocytes and/or activated monocytes in healthy animals of other mammalian species are considered normal , their pWhy two elephants with chronic foot problems did not have changes in their leukocyte morphology remains unclear, although both had a mild monocytosis. Possibly some types of foot problems, especially those that are mild and chronic as in these two elephants, eventually cease to cause a significant enough level of an inflammatory response to result in leukocyte morphological changes. Inflammation, in general, refers to a series of responses made by the immune system in response to multiple stimuli that may include microorganisms, trauma, radiation, or chemicals ,10. AlthThe morphological changes noted in blood films prepared 24 hr after blood draw emphasizes the importance of timeliness in making blood films. Morphological changes due to artifact can resemble those resulting from mild toxicity  or may hinder proper cell identification. The degree of change may reflect the amount of elapsed time between blood draw and blood film manufacture or temperature variations during shipment to a laboratory. Although the time course of such changes is unknown, delays in processing should be noted before interpreting toxic changes on a blood film, and facilities should strive to make blood films as soon as possible after blood collection for best sample quality.This report highlights the importance of blood film evaluation as part of the CBC for routine wellness examinations as well as for diagnosing sick elephant patients. Quantitative leukogram changes may be absent in sick elephants until advanced stages of disease. Thus, evaluation of qualitative changes in appropriately prepared blood films using standard methods of evaluation, which include describing the severity of toxicity on a scale of 1+ to 3+ and providing a subjective estimate of affected heterophils   may yielFresh blood films can also be helpful in monitoring patients during the course of disease as evidenced by the majority of elephants in which leukocyte abnormalities disappeared after recovery and by the three elephants whose heterophil toxicity worsened as their conditions declined. In EEHV cases, confirmation of diagnosis requires at least 24 hr for completion of PCR, and measurements of viral loads are not always possible. Additionally, some infected young elephants show variable and vague signs that confound even recognition of whether they are actually sick ,17. In tThe leukocytes of elephants affected by various inflammatory conditions can exhibit subtle to significant morphological changes with or without an abnormal leukogram. The proper recognition of these morphological changes by blood film evalution provides clinically-relevant diagnostic information early in the disease and is useful for monitoring patient progress. Blood films prepared within one to two hrs of collection provide optimal quality to detect morphological changes in elephant leukocytes.


Frequency-dependent, neurogenic contractions in intact strips were reduced by irradiation across the force-frequency graph. The radiation-difference persisted in atropine (1μM); subsequent addition of PPADs (100μM) blocked the radiation effect at higher stimulation frequencies and decreased the force-frequency plot. Conversely, neurogenic contractions in mucosa-free strips were radiation-insensitive. Radiation did not affect agonist-evoked contractions  in intact or mucosa-free strips. Interestingly, agonist-evoked contractions were larger in irradiated mucosa-free strips vs irradiated intact strips suggesting that radiation may have unmasked an inhibitory mucosal element. Spontaneous activity was larger in control intact vs mucosa-free preparations; this difference was absent in irradiated strips. Spontaneous Ca2+-transients in smooth muscle cells within tissue preparations were reduced by radiation. Radiation affected neurogenic and agonist-evoked bladder contractions and also reduced Ca2+-signalling events in smooth muscle cells when the mucosal layer was present. Radiation eliminated a positive modulatory effect on spontaneous activity by the mucosa layer. Overall, the findings suggest that radiation impairs contractility via mucosal regulatory mechanisms independent of the development of radiation cystitis.Radiation-induced bladder toxicity is associated with radiation therapy for pelvic malignancies, arising from unavoidable irradiation of neighbouring normal bladder tissue. This study aimed to investigate the acute impact of ionizing radiation on the contractility of bladder strips and identify the radiation-sensitivity of the mucosa vs the detrusor. Guinea-pig bladder strips (intact or mucosa-free) received  Radiation-induced bladder toxicity (RIBT) is a common and poorly managed clinical condition which negatively impacts the health of patients undergoing radiation therapy for pelvic malignancies ,2. RIBT Acute RIBT affects 23–80% of patients, manifested as lower urinary tract symptoms (LUTS) including urgency, dysuria, frequency and haematuria  with conRadiation damage occurs in cells of the bladder wall in animal models and patients such that the normal physiological barrier function of the urothelium is impaired, exposing underlying nerves, interstitial cells, smooth muscle, and the microvasculature to urine contents which has an irritative effect on detrusor smooth muscle . SignifiOur knowledge of the impact of radiation on bladder contractility and subsequent changes to physiological mechanisms is limited. Early studies demonstrated an immediate tetrodotoxin-resistant, contractile response in excised bladder strips during irradiation which either developed slowly or exhibited tachyphylaxis . While tex vivo irradiation directly affects bladder strip contractility through targeting mucosal modulation of detrusor contraction’. Experiments were designed to compare neurogenic, agonist-evoked and spontaneous contractions and Ca2+-signalling in non-irradiated and irradiated bladder strips. The findings demonstrate that irradiation reduced neurogenic contractions when the mucosal layer was present through a purinergic-sensitive mechanism. Cholinergic and purinergic evoked contractions were larger in irradiated mucosa-free strips indicating possible loss of an inhibitory mucosal component. Finally, irradiation eliminated mucosal-modulation of spontaneous contractions and spontaneous Ca2+-signalling was reduced, showing effects on bladder smooth muscle physiology.The present study tested our hypothesis that ‘Bladders were removed from male Dunkin Hartley guinea-pigs  that had been killed by cervical dislocation in accordance with Schedule 1  and with institutional AWERB  committee approval. Female animals were not included as we wanted to exclude the effect of oestrogen levels on bladder contractility during the oestrous cycle which is known to affect the parameters being measured in the present study . Bladderin vivo and ex vivo tissue irradiation studies of radiation-induced bladder damage; 20Gy has been determined as the ED50 in studies measuring functional outputs including reduced bladder capacity )+ (high-K+) to evoke contractions in the absence of receptor-mediated stimulation (+ contractions were unaffected by irradiation (P>0.05) indicating that the intracellular contractile mechanisms of bladder smooth muscle were not damaged by radiation. High-K+ contractions (AUC) were also not affected by mucosal layer removal (P>0.05).To test whether the ability of bladder smooth muscle to contract was affected by irradiation, tissues were challenged with 60mM Kmulation . NotablyGuinea-pig bladder strips typically exhibited spontaneous contractions that were maintained up to 4hr during experiments, and occurred in both intact and mucosa-free strips . Radiati2+ signalling was then investigated in irradiated smooth muscle cells (SMC) within mucosal preparations, similar to Gray et al [2+-transients which appeared to be somewhat synchronized with neighbouring SMC  was reduced in irradiated  compared to non-irradiated SMC ; this reduction was reflected by lower amplitude (P<0.001), less frequent  activity  strips were performed so that the impact of the mucosa could be assessed.The present study was designed to assess irradiation impact on the contractile properties of bladder tissue and has demonstrated negative effects on neurogenic contractions, complex effects on agonist-evoked contractions and reduced SMC Cacapacity ,18–20. HFrequency-dependent neurogenic contractions in intact bladder strips were negatively impacted by irradiation. Interestingly, this effect was absent in mucosa-free strips, consistent with the findings of Vale et al  in rat dNeurogenic contractions in mucosa-free detrusor strips were similar to those in intact strips, indicating that normally, there is little input from mucosal cells to neurogenic contractions. Radiation may therefore be activating a mucosal mechanism that does not normally affect detrusor neurogenic contractions. Further work will determine whether irradiated tissues are more likely to release inhibitory factor(s) from mucosal cells such as the urothelium e.g. urothelium derived inhibitory factor  or inter+) were not affected by radiation nor by removal of the mucosal layer demonstrates that the ability of bladder smooth muscle to contract was radiation-resistant. Radiation may therefore have activated inhibitory mechanisms in the mucosal layer [The radiation-effect on neurogenic contractions in intact strips could also be explained by changes to pre-synaptic or post-synaptic mechanisms however, carbachol-evoked and ATP-evoked responses in intact or mucosa-free strips were unaffected by irradiation. This suggests that receptor expression and activity was not impaired by the radiation dose over the experimental time course. Intriguingly, irradiated mucosa-free strips exhibited larger agonist-evoked contractions than irradiated intact strips showing that the presence of the mucosal layer may have acted in some way to dampen the response. Given that carbachol- or ATP-contractions were similar in non-irradiated intact and mucosa-free strips, it seems likely that radiation activated an as yet unidentified mucosal mechanism that then modified the overall response to carbachol or ATP. Vale et al  reportedal layer  that actSpontaneous activity in intact or mucosa-free bladder strips was not significantly impacted by irradiation. In non-irradiated strips, absence of the mucosa significantly reduced spontaneous activity, consistent with a positive mucosal effect, similar to the findings of others ,28. Seve2+-transients in SMC were observed in guinea-pig bladder preparations consistent with other reports [2+-activity was reduced by irradiation, but this was apparently insufficient to significantly impact spontaneous contractions. While Ca2+-activity, particularly high-magnitude, long-duration and whole smooth muscle bundle Ca2+-flashes [2+-events observed here in irradiated SMC here may have been sufficient to drive spontaneous activity. Gap junctions mediate intercellular communication in bladder smooth muscle; interestingly gap junction signaling is reportedly enhanced after radiation and is a primary mediator of radiation-bystander responses [2+-signaling within the acute timeframe of the present study.Spontaneous Ca reports . SMC Ca2-flashes  is normaesponses . Furtherin vivo research models of acute RIBT where additional mechanisms relating to urothelium loss and an inflammatory microenvironment complicate interpretation of contractility data. The findings of the present study demonstrate direct effects of irradiation on bladder strip contractility and this should now be studied in a longitudinal in vivo study to correlate early and late changes in physiology with development of inflammation and fibrosis. A limitation of interpretation is that this design does not directly model bladder irradiation in patients which is typically delivered as fractionated doses via external beams in intensity modulated radiation therapy (IMRT); however, an important consideration is brachytherapy where radiation emitting seeds are implanted e.g. to the prostate or cervix and are in close proximity to the bladder tissue.A key strength of the study lies in the experimental design, enabling us to extend the early work on sub-acute radiation-effects and informing future 2+-transients in SMC. Furthermore, the important modulatory influence of the mucosal layer on spontaneous contractions in normal guinea-pig bladder tissues was confirmed and this was apparently eliminated by irradiation. Whether these changes persist through the acute phase of RIBT remains to be seen, however, the present results suggest that relief of RIBT symptoms and enhancement of the health-related quality-of-life of patients may result from the development of mucosal specific radioprotectant agents.Our novel findings demonstrate that radiation targets the regulatory signalling mechanisms of the bladder mucosal layer, resulting in impaired neurogenic and enhanced agonist-evoked contractions and a concomitant reduction of Ca


Prion diseases are a unique group of transmissible, typically sub-acute, neurodegenerative disorders. During central nervous system (CNS) prion disease, the microglia become activated and are thought to provide a protective response by scavenging and clearing prions. The mammalian intestine is host to a large burden of commensal micro-organisms, especially bacteria, termed the microbiota. The commensal microbiota has beneficial effects on host health, including through the metabolism of essential nutrients, regulation of host development and protection against pathogens. The commensal gut microbiota also constitutively regulates the functional maturation of microglia in the CNS, and microglial function is impaired when it is absent in germ-free mice. In the current study, we determined whether the absence of the commensal gut microbiota might also affect prion disease pathogenesis. Our data clearly show that the absence of the commensal microbiota in germ-free mice did not affect prion disease duration or susceptibility after exposure to prions by intraperitoneal or intracerebral injection. Furthermore, the magnitude and distribution of the characteristic neuropathological hallmarks of terminal prion disease in the CNS, including the development of spongiform pathology, accumulation of prion disease-specific protein (PrP), astrogliosis and microglial activation, were similar in conventionally housed and germ-free mice. Thus, although the commensal gut microbiota constitutively promotes the maintenance of the microglia in the CNS under steady-state conditions in naïve mice, our data suggest that dramatic changes to the abundance or complexity of the commensal gut microbiota are unlikely to influence CNS prion disease pathogenesis. BSE, bovine spongiform encephalopathy; CNS, central nervous system; FDC, follicular dendritic cell; GALT, gut-associated lymphoid tissues; GFAP, glial fibrillary acidic protein; IC, intracerebral; IHC, immunohistochemistry; ILF, isolated lymphoid follicle; IP, intraperitoneal; PrP, prion protein; SCFA, short chain fatty acid; SPF, specific pathogen free; vCJD, variant Creutzfeldt-Jakob disease.Sc, abnormally folded isoforms of the host-encoded cellular prion protein, PrPC [Sc is the major, if not sole, component of the infectious prion agent [Sc, and neurodegeneration [Prion diseases, or transmissible spongiform encephalopathies, are typically sub-acute neurodegenerative diseases that affect humans and a number of captive and free-ranging animal species. During prion disease progression, affected tissues accumulate abundant aggregations of PrPin, PrPC . Cumulaton agent . Once thneration .Microglia are the tissue macrophages of the CNS and play important roles in maintaining neuronal homeostasis, synaptic remodelling, clearing dead and dying cells, and providing a first line of defence against pathogens . A changThe mammalian gastrointestinal tract is home to a vast community of commensal micro-organisms, termed the microbiota . For exaA bidirectional neurohumoral communication system, termed the gut–brain axis, can integrate neural, hormonal and immunological signalling between these two tissues. This enables the brain to influence a variety of physiological activities in the intestine including motility and secretion, and the actions of the mucosal immune system . Gut- ann=10/group) or intraperitoneal  routes and survival times and disease incidence compared. As anticipated, all the conventionally housed mice succumbed to clinical prion disease with a mean survival time of 174±2 days after IC exposure (median 175 days), and 248±3 days after IP exposure . These data clearly demonstrate that the absence of the commensal microbiota in germ-free mice does not influence disease susceptibility or duration when infected with prions directly into the CNS (IC) or after peripheral exposure via the peritoneal cavity (IP).Groups of conventionally housed mice (specific pathogen free control) and germ-free mice were injected with prions by either intracerebral  accumulate in affected tissues. Immunohistochemistry (IHC) analysis suggested that the abundances of prion disease-specific PrPd within the brains of clinically affected conventionally housed or germ-free mice were similar  and hippocampus (G6) regions. Our data clearly show that the density and morphology of the microglia within each region were similar in brains from clinically affected conventionally housed or germ-free mice . Our IHC analysis showed that the abundance of GFAP disease . These dree mice .The commensal gut microbiota can constitutively regulate the functional maturation of the microglia in the CNS and influence their responses to LPS-stimulation or CNS virus infection . DisturbData presented in the current study appear to contradict data reported in two independent studies reported more than 20 years ago . HoweverThe study by Erny and colleagues  showed tMany of the actions of the commensal microbiota on the host immune system have been shown to be mediated through the ability of some bacterial species to break down indigestible dietary fibre into short chain fatty acids (SCFAs) . FurtherThe replication of certain prion strains on FDCs in the gut-associated lymphoid tissue (GALT) in the small intestine  is essential for efficient neuroinvasion after oral exposure . AlthougSalmonellatyphimurium, can also increase prion disease susceptibility [Although changes to the abundance or complexity of the commensal gut microbiota are unlikely to influence prion disease duration or susceptibility, concurrent gastrointestinal infection with pathogenic bacteria or systemic exposure to their pro-inflammatory components can exacerbate prion disease pathogenesis. For example, systemic treatment with bacterial LPS can induce an aggressive pro-inflammatory phenotype in the microglia in the prion disease affected brain, leading to increased neuropathology and accelerated disease progression . Patholotibility , most litibility .Sc accumulation in archived human GALT specimens suggest the prevalence of vCJD infection may be higher than the clinical cases suggest [Sc within specific regions of the brains of prion-affected hosts is insufficient on its own to trigger neurodegeneration [Sc accumulation was associated with an upregulated innate immune response by the microglia within the same brain regions. This indicates that factors manipulating the activation status of the microglial may have a significant impact on the magnitude of the neurodegeneration exhibited during CNS prion disease. For example, factors that enhance clearance of prions by microglia may reduce disease pathogenesis. Conversely, factors that enhance the destruction of neurones by microglia may enhance the progression of CNS prion disease [Despite the apparent widespread exposure of the UK human population to bovine spongiform encephalopathy (BSE) prions through the food chain , it is f suggest . This imneration . Neurode disease . HoweverC3H/LacDk mice (6–10 weeks old) were used throughout this study. Where indicated, some mice were conventionally housed and maintained under standard specific pathogen free (SPF) conditions. Others were bred and maintained under strict germ-free conditions in sterile isolators. SPF mice and germ free mice were each housed in groups of four to six in conventional cages with sterilized Aspen Chip bedding, under a 12 h light/12 h dark cycle, and provided with unrestricted access to sterile water and food . The germ-free status of the colony was continually maintained over many years and confirmed by regular microbial testing of sentinel mice from the same isolators. Indeed, in separate studies our analyses confirmed that germ-free mice from our colony had the disturbed development of gut-associated lymphoid tissues in their intestines and substantially enlarged caeca  reported50 units). The brains used to prepare the homogenate used in this study were washed in absolute alcohol and γ-irradiated before use to eliminate potential contamination from commensal micro-organisms. Following prion exposure, mice were coded and assessed weekly for signs of clinical disease and culled at a standard clinical endpoint. The clinical endpoint of disease was determined by rating the severity of the clinical signs of prion disease exhibited by the mice. Mice were clinically scored as ‘unaffected’, ‘possibly affected’ and ‘definitely affected’ according to the standard clinical characteristics typically present in mice with terminal scrapie prion disease. Clinical signs following infection of mice with scrapie prions may include: weight loss, ruffled or starry/spiky fur, hunched posture, jumpy behaviour (at early onset) progressing to limited movement, upright tail, wet genitals, decreased awareness, discharge from eyes/blinking eyes and ataxia of hind legs. The clinical endpoint of disease was defined in one of the following ways: (i) the day on which a mouse received a second consecutive ‘definite’ rating; (ii) the day on which a mouse received a third ‘definite’ rating within four consecutive weeks; (iii) the day on which a mouse was culled in extremis. Survival times were recorded for mice that did not develop clinical signs of disease during the observation period. Prion diagnosis was confirmed by histopathological assessment of vacuolation in the brain. For the construction of lesion profiles, vacuolar changes were scored in nine distinct grey-matter regions of the brain as described previously [Where indicated, mice were injected by either the intracerebral (IC) or intraperitoneal (IP) routes with 20 µl of a 1.0 % (w/v) dilution of scrapie brain homogenate prepared from mice terminally affected with 22C scrapie prions (containing approximately 4.5 log IC infectious doseeviously .d), brains were fixed in paraformaldehyde fixative and embedded in paraffin wax. Sections  were deparaffinized and pre-treated to enhance the detection of PrPd by hydrated autoclaving  and subsequent immersion in formic acid (98 %) for 5 min. Sections were then immunostained with PrP-specific mAb BH1 [For the detection of prion disease-specific PrP (PrP mAb BH1 . For thehttp://rsb.info.nih.gov/ij/) as described on coded sections [For morphometric analysis, images were analysed using ImageJ software  according to a previously established protocol [+ microglia within the dorsal medulla (G1) and hippocampus (G6) regions of their brains. Microglial morphology was compared by measuring dendrite length, numbers of segments, branching and terminal points. Thus, from each mouse group, data were collected from at least 150 individual Iba1+ microglia.The density and morphology of Iba1protocol . Six indse and significant differences between groups were sought by using Student’s t-test. Values of P<0.05 were accepted as significant.Unless indicated otherwise, data are presented as mean±


Tracheal intubation remains a life-saving procedure that is typically not difficult for experienced providers in routine conditions. Unfortunately, difficult intubation remains challenging to predict and intubation conditions may make the event life threatening. Recent technological advances aim to further improve the ease, speed, safety, and success of intubation but have not been fully investigated. Video laryngoscopy, though proven effective in the difficult airway, may result in different intubation success rates in various settings and in different providers’ hands. The rescue surgical airway remains a rarely used but critical skill, and research continues to investigate optimal techniques. This review highlights some of the new thoughts and research on these important topics. Fortunately, recent advances in airway management have facilitated improved intubation conditions by augmenting oxygenation during laryngoscopy and providing indirect video laryngeal views. This review will briefly discuss recent research in these areas, highlighting that all devices may not be appropriate in all health-care settings. Further emphasis is placed on recent research and recommendations on the definitive surgical airway in the CICO scenario.Airway management remains a difficult skill to master, requiring hands-on training with human patients and extensive clinical experience. Fortunately, most intubations are not challenging in experienced hands. Current prediction models to anticipate the difficult intubation are poor and have limited application to new tools other than the traditional direct laryngoscopy (DL). Recent reviews of cannot-intubate cannot-oxygenate (CICO) events have continued to highlight the low rate of success for rescue despite the application by experienced anesthesiologists and surgeons2. These populations with increased oxygen consumption and large shunts may require higher delivery of apneic oxygenation than standard nasal cannula can provide.Apneic oxygenation is not a new concept, but new research has begun to emphasize its potential value during airway management. Oxygenation via standard nasal cannula can be limited by patient comfort and maximum flow rates. A recent study demonstrated that high-flow nasal cannula at 15 mL/min does not increase time to desaturation in the critically ill population3, transnasal humidified rapid-insufflation ventilatory exchange (THRIVE) devices5, and dual-use laryngoscopes6. These techniques show potential to substantially prolong apnea time. The THRIVE was recently found to be as effective as face-mask pre-oxygenation at maintaining oxygenation during rapid sequence induction7.Alternative devices offer higher concentrations of inspired oxygenation as well as increased comfort, including humidification. Various techniques for this enhanced apneic oxygenation include buccal RAE (Ring-Adair-Elwyn) tubes10. Accordingly, recent literature has focused on extending the use of VL to various settings, including pre-hospital, emergency department, and intensive care unit (ICU). These settings often present a high-stress scenario with an actively decompensating patient with widely varying provider experience. Current data suggest that the success seen with VL may not automatically translate to other settings outside the operating room.Video laryngoscopy (VL) has been shown to increase the rate of first-attempt success in patients with the predicted difficult airway in operating roomsVL has clear benefits and disadvantages in the pre-hospital setting. VL may have a special role for intubations in the field where patients are challenged with spine immobilization precautions, suboptimal positioning, and altered facial anatomy or trauma. However, secretions, blood, and vomitus may obscure VL cameras, and camera lenses are difficult to clear of contamination. In addition, first responders may have different training and comfort levels with new devices.11. A retrospective study in a pre-hospital paramedic helicopter system observed increased first-pass success and a decrease in overall attempts with C-MAC laryngoscopy12. A prospective observational study of out-of-hospital intubations showed improved first-pass success with rapid sequence intubations but did not significantly change overall first-pass success when compared with DL13.The Airtraq , C-MAC , and GlideScope  devices have been directly compared with DL in the pre-hospital emergency intubation setting. Initial observational studies have shown an association with decreased number of intubation attempts when using a GlideScope versus DL15. In another study, VL was significantly more successful in patients with a grade II–IV DL view, underlying the importance and success of indirect VL in patients with difficult DL views16.Further randomized research has questioned these benefits. Two prospective randomized trials of pre-hospital VL observed decreased rates for first-pass success when compared with DL17. In contrast, pre-hospital practitioners have a wide variety of training, skill, and experience that may limit their comfort with new devices. Given the challenges seen with indirect VL in the pre-hospital setting, we believe that devices with both DL and VL capabilities may be best in these environments, but this hypothesis is currently untested in the literature.VL may have a unique role in the pre-hospital setting where certain patient and environmental factors limit an adequate DL view19. However, a prospective randomized study showed no significant increase in first-pass success with C-MAC versus DL blades when used by senior residents in emergency resident training20. A similar study of more experienced anesthesia providers showed no difference in first-pass success despite improved glottic views21. Overall, VL may not change initial intubation success in the emergency department but may be beneficial for subsequent attempts, but prospective randomized data are limited.VL has also been studied in the emergency department setting where experience and a controlled hospital setting differ from pre-hospital emergency airways. Observational studies have demonstrated higher first-attempt success rates with VL over DL in the emergency department23. Furthermore, in prospective randomized trials, the rate of successful first intubation was significantly higher with GlideScope VL than with DL in the hands of fellow-trained physicians, a finding also found in a study of resident physicians25. However, a different trial with fellow physicians showed no significant differences in first-pass intubation success comparing a variety of VL and DL devices26. A recent multicenter study including the McGrath MAC video laryngoscope  versus Macintosh laryngoscope (MACMAN) trial clarified optimal laryngoscopy technique in the ICU setting, but results again suggest that there is no difference in success rate between VL and DL in the ICU environment27. The utility of VL in this environment, like that in other emergency environments, may be limited by the presence of blood in the airway28.The intensive care setting often presents urgent or emergent airways in an actively decompensating patient. Observational data demonstrate a higher intubation success rate for VL over DL29. However, interruptions in chest compressions were minimized with VL in two different studies30. In addition, VL was noted to have higher success rates when used by novice physicians during ongoing CPR30.Intubation during cardiopulmonary resuscitation (CPR) provides definitive airway control and essential information about ongoing resuscitation via expired carbon dioxide. Unfortunately, successful intubation is often hampered by patient movement due to chest compressions. During CPR events, there was no difference in successful intubations or speed of intubation when prospectively comparing the GlideScope VL with DLAwake intubations present a high-stress challenge with an anticipated difficult airway with a need for smooth, comfortable, and rapid intubation. In contrast to the scenarios discussed above, awake intubations are typically performed by providers with specific training in awake airway management techniques. Flexible bronchoscopic techniques are traditionally used for awake intubations because of their flexibility and comfort, but recent studies have investigated VL as an alternative technique.31. Average patient comfort scores after topical and regional anesthesia and sedation were similar for the two techniques. However, a high dropout rate in the VL group due to failed regional transtracheal block may limit interpretation of this study. Another randomized trial compared flexible bronchoscopic intubation with C-MAC D-blade VL for nasal awake intubations under topical anesthesia. It also showed no difference in the success of intubations32. These findings were validated in healthy volunteers in the awake upright (sitting) position34. GlideScope VL was compared with fiber-optic techniques for the awake intubation of morbidly obese patients, a population with a higher rate of difficulty airway, with similar rates of successful intubation on first attempt35. These cumulative findings suggest that VL may be a useful alternative to flexible bronchoscopic intubation in select patient populations with anticipated difficult airways.A randomized trial of oral awake intubations with the flexible bronchoscope versus McGrath Series 5 VL  found no change in time to intubation or overall intubation success1. In contrast, only 9 out of 25 (36%) anesthesiologists were successful in rescuing the airway with a surgical technique. Review of these cases underlined the importance of a simple rapid technique with an optimal chance of success in unpracticed and stressed providers.The CICO scenario requiring surgical airway is a rare and stressful occurrence. Despite ongoing research and education in this area, the overall success of anesthesiologist-placed emergency surgical airway appears to be very low. The 4th National Audit Project (NAP4) recorded surgical airway management across the United Kingdom during a 1-year period. Most surgical airways were performed by a surgeon with 100% success36. In contrast, cannula techniques such as percutaneous cricothyrotomies are still included in similar guidelines, including the Australian and New Zealand College of Anaesthetists (ANZCA) Guidelines on Equipment to Manage a Difficult Airway During Anaesthesia37.Guidelines are currently divided between two techniques: scalpel or cannula airways. The 2015 Difficult Airway Society guidelines strongly favor scalpel techniques over cannula techniques, a change from the 2004 guidelines1. An observational study of success rates of trauma intubation and airway rescue techniques in both anesthesiologists and other physicians showed a 100% success rate of traditional scalpel approaches38. However, the overall rate of surgical airways was lower than previously reported. These studies, though limited by their observational approach and low denominators, add to a growing amount of evidence that surgical cricothyrotomies may have a higher success rate when compared with cannula techniques40.Scalpel airways are thought to be faster, simpler, and overall more successful than cannula techniques. In the NAP4, 100% of first-choice surgical cricothyrotomies (3 out of 3) were successful, but the low number of first-choice scalpel airways may limit study interpretation1. The cannula may provide a conduit for oxygenation via jet ventilation to temporize airway management, but barotrauma remains a significant risk in the obstructed airway. A systematic review of emergency transtracheal jet ventilation, including 90 out of 132 CICO scenarios (68%), showed higher rates of device failure, barotrauma, and overall complications in CICO emergency scenarios when compared with non-CICO emergencies41.Advocates of cannula techniques note that the needle-wire movements are more familiar to anesthesiologists, but cannula techniques may be vulnerable to a high rate of complications in the CICO scenario. The NAP4 analysis found that 90% of cricothyrotomies (26 out of 29) were first attempted with cannula techniques, potentially reflecting provider preference. However, 58% of cannula cricothyrotomies (15 out of 26) performed by anesthesiologists failed and required rescue36. Many techniques have similar steps  with variations including the number of incisions and methods to keep the incision open. The 2015 Difficult Airway Society guidelines recommend a scalpel-bougie technique for tracheal cannulation with a gum-elastic bougie to keep the incision open36. Other techniques, including the “4-step technique”, include devices such as tracheal hooks that may not be immediately available42. Training models in humans and animal cadaveric models have not favored one technique over another44.There is a lack of randomized data supporting any scalpel technique45. Recent studies of needle versus scalpel techniques in animal cadaveric models show that senior anesthesiologists also achieve a higher rate of success with surgical scalpel techniques46. However, more research is required before any definitive conclusions can be made about ease of learning with the different techniques.Given the challenges in surgical airway training, recent studies have investigated which techniques have higher success in both inexperienced and experienced learners. A recent study in surgical airway-naïve trainees showed a higher rate of success when using scalpel techniques versus cannula techniques on cadaversOne of the more recent innovations in airway management is ultrasonography for the identification of landmarks for the surgical airway. The thought is that airway structures can be identified before induction of anesthesia so that emergency surgical airway management is performed with sound knowledge of the underlying airway anatomy. The growing number of obese patients significantly increases the likelihood of a difficult-to-assess surface anatomy. In addition, patients with previous head-and-neck trauma, surgery, or radiation (or a combination of these) may have difficult or impossible anatomy to identify by surface structure.47. In contrast, a study of women in labor showed that only 39% of anesthesiologists correctly identified the cricothyroid membrane in obese patients compared with 71% of non-obese patients48. A study of overweight volunteers also showed a significant increase in ability to identify the cricothyroid membrane with ultrasound versus palpation 49. A study in human cadavers showed that naïve trainees were over five times more successful in cannulating the trachea with ultrasound guidance than palpation in difficult or impossible anatomy50. So it appears that ultrasound is a useful tool for the identification of surgical airway structures in the obese patient population or others with difficult-to-identify surface airway structures.Patient selection for ultrasound identification of airway structures impacts its utility. A study of emergency medicine physicians and senior residents showed no significant difference in the ability to identify the cricothyroid membrane with palpation or ultrasound in healthy volunteers51. The ANZCA provides standards for CICO training sessions but does not clarify which algorithm is preferred52. In addition, such training programs are not often used by anesthesiologists or others who manage the airway, regardless of practice settings.Given the low incidence of CICO airways, most providers should be considered novice regardless of years in practice. Despite the known benefits of both cognitive and procedural skills training in the CICO scenario, there are few standardized training programs. The current US Accreditation Council for Graduate Medical Education (ACGME) anesthesiology requirements do not require programs to train graduates in the surgical airwayAs laryngoscopy devices continue to evolve, the literature continues to investigate whether these new devices improve intubation success and patient outcomes. Currently, initial data suggest that new techniques such as VL are not universally superior to traditional DL. Variations in setting, patient characteristics, situational stress, and provider training likely contribute to overall device success and are confounders that cannot all be modified.A recent focus on CICO events and surgical airway management has highlighted the importance of simple, rapid techniques with ongoing training throughout one’s career. Ultrasonography provides a helpful adjunct for the identification of surgical airway access anatomy when surface landmarks are unclear and should be introduced in surgical airway training.Regardless of the technique, quick decision-making, familiarity with equipment, proper training, and effective communication remain the most important factors in a successful airway intervention. Fortunately, these characteristics can be improved with educational interventions and simulation training. As intubation techniques, devices, and evidence grow, airway education must continue to do so as well. The greatest opportunity for airway success remains in a straightforward plan with familiar equipment and practiced techniques.